WO2009125804A1 - Screening method - Google Patents

Screening method Download PDF

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Publication number
WO2009125804A1
WO2009125804A1 PCT/JP2009/057229 JP2009057229W WO2009125804A1 WO 2009125804 A1 WO2009125804 A1 WO 2009125804A1 JP 2009057229 W JP2009057229 W JP 2009057229W WO 2009125804 A1 WO2009125804 A1 WO 2009125804A1
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Prior art keywords
protein
gpr120
cells
partial peptide
epc
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PCT/JP2009/057229
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French (fr)
Japanese (ja)
Inventor
信津 塚田
俊文 喜多
孝之 浅原
治史 増田
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武田薬品工業株式会社
学校法人東海大学
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Publication of WO2009125804A1 publication Critical patent/WO2009125804A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1796—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5064—Endothelial cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a novel screening method for substances involved in the regulation of differentiation / proliferation of vascular endothelial precursor cells.
  • Endothelial progenitor cells are bone marrow-derived cells that are mobilized from the bone marrow to peripheral blood and circulate in the body as part of the mononuclear cell fraction to form blood vessels. It is involved in new vasculogenesis through engraftment at the site and proliferation / differentiation / migration at the site (see Non-Patent Documents 1 and 2). Normally, EPC is always mobilized from the bone marrow to the periphery at a constant rate, and the peripheral blood EPC and bone marrow EPC are numerically balanced unless there is any stimulus in the living body.
  • EPC is mobilized from the bone marrow by the action of various cytokines, growth factors, hormones, etc., and the number of EPCs in peripheral blood increases. Utilizing this mechanism, clinical trials of autologous EPC transplantation treatment for severe ischemic diseases including coronary artery disease and limb ischemia disease have been started and good results are being reported. On the other hand, it has been pointed out that the amount and quality of EPC of patients are reduced due to basic diseases such as diabetes, and a sufficient therapeutic effect cannot be obtained even after transplantation.
  • Non-Patent Document 6 In patients with ischemic heart disease, the number of EPCs and angiogenic ability are decreased (see Non-Patent Document 6), and the mortality after a cardiovascular event is increased when the blood EPC number is decreased (Non-Patent Document 7). It is known that a decrease in bone marrow cells and progenitor cells contributes to the progression of arteriosclerosis (see Non-Patent Document 8). Furthermore, the number of EPCs in blood is reduced in patients with cerebral infarction (see Non-Patent Document 9), the survival rate increases when EPC is transplanted into a cirrhosis model animal (see Non-Patent Document 10), and bone marrow cells are transplanted. It has been reported that the fibrosis of the liver is suppressed (see Non-Patent Document 11).
  • GPR120 is a G protein-coupled receptor (GPCR) that is highly expressed in the pituitary gland, intestinal tract, adipose tissue, and the like.
  • GPCR G protein-coupled receptor
  • Patent Document 1 describes that the ligand of GPR120 is a fatty acid, and also screens for agonists / antagonists using GPR120 and the ligand, agonists, etc. (GPR120, DNA, ligand) / antagonists, etc.
  • Patent Document 2 discloses a compound having agonist activity with respect to GPR120, and describes that it is useful for the prevention and treatment of arteriosclerosis, angina pectoris, myocardial infarction, cardiovascular disease and the like.
  • Non-patent Document 12 When GPR120 is knocked down by siRNA in enteroendocrine cells (STC-1), GLP-1 production induced by free fatty acids decreases, so in the intestine, GPR120 agonists stimulate GLP-1 production. It has been suggested that it exhibits an obesity-suppressing action (Non-patent Document 13).
  • the present inventors first made various receptors (GPCR, tyrosine kinase type receptor, nuclear receptor) in two types of EPCs having different differentiation stages (large EPC and small EPC) in mice.
  • GPCR tyrosine kinase type receptor
  • nuclear receptor tyrosine kinase type receptor
  • GPR120 the expression of which was most increased in large EPC, and further studied.
  • GPR120 is specifically expressed in small and large EPCs derived from mouse bone marrow cells and peripheral blood mononuclear cells, and small and large EPCs derived from human bone marrow cells, but not in bone marrow cells or endothelial cells. I found out.
  • the present invention [1] Use of a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof, or a cell producing the protein or the partial peptide.
  • a method for regulating differentiation / proliferation of vascular endothelial progenitor cells or a method for screening a substance for increasing vascular endothelial cells in peripheral blood [2] A nucleic acid encoding a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial polynucleotide thereof, or an antibody against the protein or the partial peptide is further used.
  • the method according to [1] above characterized in that [3] The method according to [1] above, further comprising using a fatty acid or a low molecular weight compound that changes the binding property between the fatty acid and the protein, or a salt thereof. [4] The method according to [1] above, wherein the cells are vascular endothelial progenitor cells or progenitor cells thereof, [5] The method according to [1] above, for selecting a prophylactic / therapeutic substance for a disease whose regulation of angiogenic ability can exhibit a prophylactic / therapeutic effect [6] An agent for promoting differentiation / proliferation of vascular endothelial progenitor cells, comprising any of the following substances: (A) a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof (b) a base sequence encoding the protein of (a) or a partial peptide thereof (C) a compound that enhances the expression of the
  • GPR120 is involved in differentiation from bone marrow cells to EPC and proliferation of EPC, GPR120 or cells producing it are brought into contact with a test substance, and the expression or activity of the receptor (eg, ligand binding activity, signal transduction activity, etc.) ) Can be screened for drugs that modulate EPC differentiation / proliferation. Moreover, since suppression of GPR120 promotes the mobilization of EPC to peripheral blood, drugs that increase EPC in peripheral blood can also be screened by the same technique. Since GPR120 is highly expressed specifically in EPC in hematopoietic cells and peripheral blood cells, it can be used as a marker for EPC in the diagnosis of diseases associated with EPC selection / quantification and abnormal EPC amount.
  • the receptor eg, ligand binding activity, signal transduction activity, etc.
  • FIG. 4 shows changes in (A) body weight (BW), (B) plasma glucose concentration (PG), and (C) glycated hemoglobin value (GHB) in C57BL / 6J mice and GPR120 KO mice.
  • BW body weight
  • PG plasma glucose concentration
  • mice glycated hemoglobin value
  • EPC differentiation / proliferation regulation or screening method for EPC increasing substance in peripheral blood uses GPR120 protein or a partial peptide thereof, or a cell that produces the protein or a partial peptide thereof.
  • screening method of the present invention a method for screening an EPC-increasing substance in peripheral blood.
  • GPR120 used in the screening method of the present invention is a receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.
  • GPR120 protein is a cell of humans or other warm-blooded animals (eg, monkeys, cows, horses, pigs, sheep, goats, rabbits, mice, rats, guinea pigs, hamsters, chickens, etc.) [eg, hepatocytes, spleen cells, Nerve cells, glial cells, pancreatic ⁇ cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells (eg, vascular endothelial cells), goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibers Cells, muscle cells, adipocytes, immune cells (eg macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils,
  • it may be a chemically synthesized protein or a biochemically synthesized protein using a cell-free protein synthesis system.
  • transduced may be sufficient.
  • amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 is about 60% or more, preferably about 70% or more, More preferred is an amino acid sequence having a homology of about 80% or more, more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97% or more.
  • homology refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm uses a sequence of sequences for optimal alignment). The ratio of the same amino acid residue and similar amino acid residues to all overlapping amino acid residues in the case of introducing a gap into one or both).
  • Similar amino acids means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn). ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution).
  • the amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 is about 60% or more, preferably about 70%, with the amino acid sequence shown in SEQ ID NO: 2, 4 or 6. More preferably, it is an amino acid sequence having an identity of about 80% or more, more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97% or more.
  • Protein comprising an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 means the above-mentioned “amino acid substantially identical to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6”. It means a protein having an activity substantially equivalent to that of a protein comprising the “sequence” and comprising the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.
  • the substantially homogeneous activity includes, for example, ligand (eg, physiological ligand such as fatty acid, agonist / antagonist (eg, see Patent Document 2)) binding activity, signal transduction activity (eg, activation of phospholipase C ⁇ (PLC ⁇ )) , Intracellular Ca 2+ concentration increase, protein kinase C (PKC) activation, etc.), EPC differentiation promoting activity, EPC proliferation promoting activity, and the like.
  • ligand eg, physiological ligand such as fatty acid, agonist / antagonist (eg, see Patent Document 2) binding activity
  • signal transduction activity eg, activation of phospholipase C ⁇ (PLC ⁇ )
  • PLC protein kinase C
  • EPC differentiation promoting activity e.g, EPC proliferation promoting activity
  • EPC proliferation promoting activity e.g, EPC proliferation promoting activity, and the like.
  • the activities such as ligand binding activity, signal transduction activity, and EPC differentiation / proliferation promoting activity are equivalent (for example, about 0.5 to about 2 times), but the amount of these activities and the amount of protein such as molecular weight
  • the target elements may be different.
  • Ligand binding activity, signal transduction activity, and EPC differentiation / proliferation can be measured according to a method known per se.
  • the GPR120 in the present invention includes, for example, (1) one or more of the amino acid sequences represented by SEQ ID NO: 2, 4 or 6 (for example, about 1 to 100, preferably about 1 to 50, More preferably, about 1 to 30, more preferably about 1 to 10, particularly preferably 1 to several (2, 3, 4, or 5) amino acid sequences, (2) SEQ ID NO: 2 1 or 2 or more (for example, about 1 to 100, preferably about 1 to 50, more preferably about 1 to 30, more preferably about 1 to 10) in the amino acid sequence shown in 4 or 6; Particularly preferred is an amino acid sequence to which 1 to several (2, 3, 4 or 5) amino acids are added, (3) one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 (for example, 1 About 50, preferably about 1-30, more preferably about 1-10, and even more preferably 1-number ( 2, 3, 4 or 5) amino acid sequence inserted, (4) one or more of the amino acid sequences shown in SEQ ID NO: 2, 4 or 6 (for example, about 1
  • the GPR120 in the present invention is preferably a human GPR120 protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (GenBank accession number: NP_859529) and a mouse GPR120 protein consisting of the amino acid sequence shown in SEQ ID NO: 4 (GenBank accession number: NP_861413), rat GPR120 protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (GenBank accession number: NP_001040553), or an ortholog thereof in other mammals (for example, a dog ortholog registered in GenBank as accession number XP_534968) (Mouse, rat and dog GPR120 have about 83%, about 81% and about 85% amino acid identity to human GPR120, respectively).
  • proteins and peptides are described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation.
  • the GPR120 of the present invention including a protein comprising the amino acid sequence shown in SEQ ID NO: 2, has a C-terminal carboxyl group (—COOH), carboxylate (—COO ⁇ ), amide (—CONH 2 ) or ester (— COOR).
  • R in the ester for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; C 6-12 aryl groups such as ⁇ -naphthyl; phenyl-C 1-2 alkyl groups such as benzyl and phenethyl; C 7- such as ⁇ -naphthyl-C 1-2 alkyl groups such as ⁇ -naphthylmethyl; 14 aralkyl group; pivaloyloxymethyl group is used.
  • a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl
  • a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl
  • GPR120 has a carboxyl group (or carboxylate) other than the C-terminus
  • those in which the carboxyl group is amidated or esterified are also included in the GPR120 of the present invention.
  • the ester in this case, for example, the above C-terminal ester or the like is used.
  • the amino group of the N-terminal amino acid residue is protected with a protecting group (for example, a C 1-6 acyl group such as C 1-6 alkanoyl such as formyl group, acetyl group, etc.).
  • N-terminal glutamine residue that can be cleaved in vivo is pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (eg, —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.) a suitable protecting group (e.g., formyl group, those protected by C 1-6 an acyl group) such as C 1-6 alkanoyl group such as acetyl group, or a sugar chain-binding Also included are complex proteins such as so-called glycoproteins.
  • a substituent on the side chain of an amino acid in the molecule eg, —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.
  • a suitable protecting group e.g., formyl group, those protected by C 1-6 an acyl group
  • C 1-6 alkanoyl group such as acetyl
  • partial peptide of GPR120 is a peptide containing the partial amino acid sequence of GPR120 described above and has substantially the same activity as GPR120, Any one may be used.
  • substantially the same quality of activity has the same meaning as described above. Further, “substantially the same quality of activity” can be measured in the same manner as in the case of GPR120.
  • a region involved in binding to a fatty acid that is a ligand for example, in the case of human GPR120, for example, SEQ ID NO: 2.
  • a region involved in signal transduction through interaction with the ligand for example, three Those having a partial amino acid sequence containing a region (hereinafter also referred to as “G ⁇ activation domain”) having the activity of binding to the ⁇ subunit of the monomeric G protein and promoting its GDP / GTP exchange reaction are used. It is done.
  • the partial peptide of the present invention is not particularly limited in size as long as it includes a region involved in binding to the above ligand and a region involved in signal transduction, but preferably contains a partial amino acid sequence of 100 or more. Preferably, those containing 200 or more partial amino acid sequences are included.
  • the partial amino acid sequence may be a single continuous partial amino acid sequence, or may be a concatenation of a plurality of discontinuous partial amino acid sequences.
  • the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁇ ), an amide (—CONH 2 ), or an ester (—COOR).
  • R in the ester include the same as those described above for GPR120.
  • the partial peptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention.
  • the ester in this case, for example, the same ester as the C-terminal ester is used.
  • the amino group of the N-terminal amino acid residue is protected with a protecting group
  • the N-terminal glutamine residue is pyroglutamine oxidized
  • Examples include those in which a substituent on the side chain of the amino acid is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound.
  • GPR120 or a partial peptide thereof used in the present invention may be in the form of a salt.
  • a salt with a physiologically acceptable acid eg, inorganic acid, organic acid
  • base eg, alkali metal
  • a physiologically acceptable acid addition salt is particularly preferable.
  • Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
  • inorganic acids eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • GPR120 can be produced from the aforementioned mammalian cells or tissues by a known protein purification method. Specifically, after homogenizing mammalian tissue or cells and removing cell debris by low-speed centrifugation, the supernatant is centrifuged at high speed to precipitate a cell membrane-containing fraction (if necessary, cell membrane fractionation by density gradient centrifugation or the like). GPR120 or a salt thereof can be prepared by subjecting the fraction to chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography, and the like.
  • chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography, and the like.
  • GPR120 or a partial peptide thereof can also be produced according to known peptide synthesis methods.
  • the peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method.
  • the target protein can be produced by removing the protecting group.
  • the condensation and the removal of the protecting group are carried out according to a method known per se, for example, the method described in the following (1) to (5). (1) M.
  • the GPR120 thus obtained can be isolated and purified by a known purification method.
  • the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
  • GPR120 obtained by the above method is a free form
  • the free form can be converted to an appropriate salt by a known method or a method according thereto, and conversely, when GPR120 is obtained as a salt.
  • the salt can be converted into a free form or other salt by a known method or a method analogous thereto.
  • resins for protein synthesis include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl.
  • an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the intended GPR120 according to various condensation methods known per se.
  • proteins and the like are cut out from the resin, and at the same time, various protecting groups are removed, and an intramolecular disulfide bond forming reaction is carried out in a highly diluted solution to obtain the target GPR120 or its amide.
  • carbodiimides are particularly preferable.
  • the carbodiimide DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used.
  • a protected amino acid is added directly to the resin together with a racemization inhibitor (eg, HOBt, HOOBt), or the protected amino acid is activated in advance as a symmetric anhydride, HOBt ester or HOOBt ester. Can then be added to the resin.
  • a racemization inhibitor eg, HOBt, HOOBt
  • the solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc.
  • Examples include sulfoxides, amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof.
  • the reaction temperature is appropriately selected from a range that is known to be usable for protein bond forming reaction, and is usually selected from the range of about -20 ° C to 50 ° C.
  • the activated amino acid derivative is usually used in an excess of 1.5 to 4 times.
  • the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.
  • the protection of the functional group that should not be involved in the reaction of the raw material, the protection group, the removal of the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.
  • the protective group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc, etc. are used.
  • the carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl).
  • alkyl esterified eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl.
  • Aralkyl esterification eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification
  • phenacyl esterification eg, benzyloxycarbonyl hydrazide, tertiary butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • groups suitable for esterification include groups derived from carbonic acid such as lower alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethoxycarbonyl groups.
  • groups suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
  • Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl 2 -Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like.
  • Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like.
  • Examples of methods for removing (eliminating) protecting groups include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, and anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro.
  • a catalyst such as Pd-black or Pd-carbon
  • anhydrous hydrogen fluoride methanesulfonic acid, trifluoro.
  • Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used.
  • the elimination reaction by the acid treatment is generally performed at a temperature of about ⁇ 20 ° C. to 40 ° C.
  • Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like.
  • active ester alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like.
  • active ester alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide,
  • an amide form of GPR120 for example, first, the ⁇ -carboxyl group of the carboxy terminal amino acid is amidated and protected, and then the peptide chain is extended to the desired chain length on the amino group side and then the peptide A protein (peptide) from which only the protecting group of the ⁇ -amino group at the N-terminus of the chain is removed and a protein (peptide) from which only the protecting group of the carboxyl group at the C-terminal has been removed are prepared. Condensation in a mixed solvent as described above. The details of the condensation reaction are the same as described above.
  • the ester form of GPR120 can be obtained in the same manner as in the case of the amide form of GPR120, for example, after condensing the ⁇ -carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester.
  • the partial peptide of the present invention can also be produced by cleaving the full-length protein of GPR120 with an appropriate peptidase.
  • GPR120 can also be produced by culturing a transformant containing the nucleic acid encoding it, and separating and purifying GPR120 from the resulting culture.
  • the nucleic acid encoding GPR120 or a partial peptide thereof may be DNA or RNA, or may be a DNA / RNA chimera.
  • DNA is used.
  • the nucleic acid may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid.
  • a single strand it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
  • DNA encoding GPR120 or a partial peptide thereof genomic DNA, human or other warm-blooded animals (eg, monkeys, cows, horses, pigs, sheep, goats, rabbits, mice, rats, guinea pigs, hamsters, chickens, etc.) Cells [eg, hepatocytes, spleen cells, neurons, glial cells, pancreatic ⁇ cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells (eg, vascular endothelial cells), goblet cells, endothelium Cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg macrophages, T cells, B cells
  • Genomic DNA and cDNA encoding GPR120 or its partial peptides are prepared using Polymerase Chain Reaction (hereinafter referred to as “PCR method”) using the genomic DNA fraction and total RNA or mRNA fraction prepared from the cells and tissues as templates. (Abbreviated) and Reverse Transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”).
  • PCR method Polymerase Chain Reaction
  • RT-PCR method Reverse Transcriptase-PCR
  • genomic DNA and cDNA encoding GPR120 or a partial peptide thereof can be prepared by inserting a genomic DNA prepared from the cells and tissues described above and a total RNA or mRNA fragment into an appropriate vector and It can also be cloned from a cDNA library by colony or plaque hybridization method or PCR method, respectively.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
  • DNA encoding GPR120 for example, DNA containing the base sequence shown in SEQ ID NO: 1, 3 or 5 or hybridized with the base sequence shown in SEQ ID NO: 1, 3 or 5 under highly stringent conditions And a DNA encoding a protein having substantially the same quality as GPR120 described above (eg, ligand binding activity, signal transduction activity, EPC differentiation / proliferation promoting activity, etc.).
  • DNA capable of hybridizing under high stringency conditions with the base sequence shown in SEQ ID NO: 1, 3 or 5 include, for example, about 60% or more, preferably about A DNA containing a base sequence having a homology of 70% or more, more preferably about 80% or more, further preferably about 90% or more, particularly preferably about 95% or more is used.
  • NCBI BLAST National Center for Biotechnology Information Basic Local Alignment Search Tool
  • the above-mentioned algorithm for calculating homology of amino acid sequences is also preferably exemplified.
  • Hybridization is carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can do.
  • hybridization can be performed according to the method described in the attached instruction manual.
  • Hybridization can be preferably performed according to stringent conditions. High stringent conditions include, for example, a hybridization reaction at 45 ° C. in 6 ⁇ SSC (sodium chloride / sodium citrate), followed by one or more washings at 65 ° C. in 0.2 ⁇ SSC / 0.1% SDS. Can be mentioned.
  • a person skilled in the art may appropriately change the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc.
  • the desired stringency can be easily adjusted.
  • the DNA encoding GPR120 preferably encodes a mouse GPR120 protein shown in SEQ ID NO: 3, which is a DNA containing the base sequence encoding human GPR120 protein shown in SEQ ID NO: 1 (GenBank accession number: NM_181745) DNA containing the nucleotide sequence (GenBank accession number: NM_181748), DNA containing the nucleotide sequence encoding the rat GPR120 protein shown in SEQ ID NO: 5 (GenBank accession number: NM_001047088), or its ortholog in other mammals ( For example, a dog ortholog registered as an accession number XM_534968 in GenBank).
  • Orthologs in other mammals can be searched using BLAST or FASTA, for example, by querying the genome and / or cDNA database of mammals other than humans by querying the base sequence shown in SEQ ID NO: 1.
  • Mouse Genome Informatics provided by Jackson Laboratories (http://www.informatics.jax.org/) is searched using the accession number or gene symbol / gene name as a keyword, and Mammalian Orthology of the hit data
  • the array information can be acquired by accessing the information.
  • the DNA encoding the partial peptide of the present invention includes a base sequence encoding a peptide having the same or substantially the same amino acid sequence as part of the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 It can be anything.
  • the DNA encoding the partial peptide of the present invention for example, (1) DNA having a partial base sequence of the base sequence shown in SEQ ID NO: 1, 3 or 5, or (2) SEQ ID NO: 1, It has a base sequence that hybridizes with DNA having the base sequence shown in 3 or 5 under highly stringent conditions, and has substantially the same activity as GPR120 described above (eg, ligand binding activity, signal transduction activity, EPC DNA encoding a peptide having differentiation / proliferation promoting activity) is used.
  • a DNA encoding GPR120 or a partial peptide thereof is amplified by PCR using a synthetic DNA primer having a part of a base sequence encoding the GPR120 or a partial peptide thereof, or DNA incorporated into an appropriate expression vector is used.
  • Cloning can be carried out by hybridization with a DNA fragment encoding a part or the entire region of GPR120 protein or a labeled synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning Second Edition (described above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.
  • the DNA base sequence is determined using a known kit such as Mutan TM -super Express Km (Takara Shuzo), Mutan TM -K (Takara Shuzo), etc., using the ODA-LA PCR method, the Gapped duplex method, Conversion can be performed according to a method known per se such as the Kunkel method or a method analogous thereto.
  • the cloned DNA can be used as it is depending on the purpose, or after digestion with a restriction enzyme or addition of a linker as desired.
  • the DNA may have ATG as a translation initiation codon on the 5 'end side and TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
  • An expression vector containing DNA encoding GPR120 or a partial peptide thereof is produced, for example, by excising a target DNA fragment from DNA encoding GPR120 and ligating the DNA fragment downstream of a promoter in an appropriate expression vector. can do.
  • Expression vectors include plasmids derived from E.
  • coli eg, pBR322, pBR325, pUC12, pUC13
  • plasmids derived from Bacillus subtilis eg, pUB110, pTP5, pC194
  • yeast-derived plasmids eg, pSH19, pSH15
  • insect cell expression Plasmid eg, pFast-Bac
  • animal cell expression plasmid eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo
  • bacteriophage such as ⁇ phage
  • insect virus vector such as baculovirus ( Examples: BmNPV, AcNPV); animal virus vectors such as retroviruses, lentiviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, herpes viruses, etc.
  • the promoter may be any promoter as long as it is appropriate for the host used for gene expression.
  • SR ⁇ promoter SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex) Virus thymidine kinase) promoter or the like is used.
  • CMV promoter, SR ⁇ promoter and the like are preferable.
  • trp promoter When the host is Escherichia, trp promoter, lac promoter, recA promoter, ⁇ P L promoter, lpp promoter, T7 promoter and the like are preferable.
  • SPO1 promoter When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable.
  • yeast When the host is yeast, the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable.
  • the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
  • an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), etc. is used as desired.
  • the selection marker include a dihydrofolate reductase gene (hereinafter abbreviated as dhfr, methotrexate (MTX) resistance), an ampicillin resistance gene (hereinafter abbreviated as amp r ), a neomycin resistance gene ( hereinafter sometimes abbreviated as neo r, include G418 resistance) and the like.
  • the target gene can also be selected using a medium that does not contain thymidine. If necessary, a base sequence (signal codon) encoding a signal sequence suitable for the host is added (or replaced with a native signal codon) to the 5 ′ end of DNA encoding GPR120 or a partial peptide thereof. May be.
  • insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule / signal sequence, etc. are used respectively.
  • GPR120 or a partial peptide thereof can be produced by transforming a host with an expression vector containing a DNA encoding GPR120 or a partial peptide thereof and culturing the resulting transformant.
  • a host for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
  • the genus Escherichia include, for example, Escherichia coli K12 • DH1 [Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA).
  • Bacillus examples include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], Bacillus subtilis 207-21 [Journal of Biochemistry, 95, 87 (1984)].
  • yeast examples include Saccharomyces cerevisiae AH22, AH22R ⁇ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris (Pichia pastoris (Pichia pastoris) KM71 etc. are used.
  • Insect cells include, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five TM cells derived from eggs of Trichoplusia ni , Cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used.
  • larvae-derived cell lines Spodoptera frugiperda cells; Sf cells
  • MG1 cells derived from the midgut of Trichoplusia ni
  • High Five TM cells derived from eggs of Trichoplusia ni
  • Cells derived from Mamestra brassicae cells derived from Estigmena acrea, and the like are used.
  • BmNPV moth-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used as insect cells.
  • Sf cells examples include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like.
  • insects examples include silkworm larvae [Maeda et al., Nature, 315, 592 (1985)].
  • animal cells examples include monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO cells (hereinafter abbreviated as CHO (dhfr ⁇ ) cells), mouse L Cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells, HeLa cells, HepG2 cells, HEK293 cells and the like are used.
  • CHO cells Chinese hamster ovary cells
  • CHO (dhfr ⁇ ) cells) cells examples include monkey L Cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells, HeLa cells, HepG2 cells, HEK293 cells and the like are used.
  • Transformation can be performed according to a known method depending on the type of host.
  • Escherichia bacteria for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972) and Gene ( Gene), 17, 107 (1982), and the like.
  • Bacillus can be transformed according to the method described in, for example, Molecular & General Genetics, 168, 111 (1979).
  • Yeast is, for example, Methods in Enzymology, 194, 182-287 (1991), Proceedings of the National Academy of Sciences of USA ( Proc. Natl. Acad. Sci. USA), 75, 1929 (1978).
  • Insect cells and insects can be transformed according to the method described in, for example, Bio / Technology, Vol. 6, 47-55 (1988).
  • Animal cells should be transformed according to the method described in, for example, Cell Engineering Annex 8 New Cell Engineering Experimental Protocol, 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). Can do.
  • the culture of the transformant can be performed according to a known method depending on the type of the host. For example, when culturing a transformant whose host is an Escherichia or Bacillus genus, a liquid medium is preferable as a medium used for the culture. Moreover, it is preferable that a culture medium contains a carbon source, a nitrogen source, an inorganic substance, etc. which are required for the growth of a transformant.
  • the carbon source for example, glucose, dextrin, soluble starch, sucrose, etc .
  • the nitrogen source for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, Inorganic or organic substances such as potato extract; examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like.
  • yeast extract, vitamins, growth promoting factors and the like may be added to the medium.
  • the pH of the medium is preferably about 5 to about 8.
  • a medium for culturing a transformant whose host is an Escherichia bacterium for example, an M9 medium containing glucose and casamino acids [Miller, Journal of Experiments in Molecular Genetics ( Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972].
  • an agent such as 3 ⁇ -indolylacrylic acid may be added to the medium in order to make the promoter work efficiently.
  • Culturing of a transformant whose host is an Escherichia bacterium is usually performed at about 15 to about 43 ° C. for about 3 to about 24 hours. If necessary, aeration or agitation may be performed.
  • Culturing of the transformant whose host is Bacillus is usually performed at about 30 to about 40 ° C. for about 6 to about 24 hours. If necessary, aeration or agitation may be performed.
  • a medium for culturing a transformant whose host is yeast for example, a Burkholder minimum medium [Bostian, KL et al., Proceedings of the National Academy of Sciences of Science The USA (Proc. Natl. Acad. Sci. USA), 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Bitter, GA, et al., Proceedings of the National Academy ⁇ Of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)].
  • the pH of the medium is preferably about 5 to about 8.
  • the culture is usually performed at about 20 ° C. to about 35 ° C. for about 24 to about 72 hours. Aeration and agitation may be performed as necessary.
  • a medium for culturing a transformant whose host is an insect cell or an insect for example, 10% bovine serum deactivated in Grace's Insect Medium [Grace, TCC, Nature, 195, 788 (1962)] The thing etc. which added additives, such as these, etc. suitably are used.
  • the pH of the medium is preferably about 6.2 to about 6.4.
  • the culture is usually performed at about 27 ° C. for about 3 to about 5 days. You may perform ventilation
  • a medium for culturing a transformant whose host is an animal cell for example, a minimum essential medium (MEM) containing about 5 to about 20% fetal bovine serum [Science, 122, 501 (1952 ], Dulbecco's Modified Eagle Medium (DMEM) [Virology, Vol. 8, 396 (1959)], RPMI 1640 Medium [The Journal of the American Medical Association, 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] Used.
  • the pH of the medium is preferably about 6 to about 8.
  • the culture is usually performed at about 30 ° C. to about 40 ° C. for about 15 to about 60 hours. You may perform ventilation
  • GPR120 or a partial peptide thereof can be produced inside or outside the transformant.
  • GPR120 or a partial peptide thereof can be separated and purified from a culture obtained by culturing the transformant according to a method known per se.
  • a method known per se For example, when GPR120 or a partial peptide thereof is extracted from cultured cells or cytoplasm of cells, the cells or cells collected from the culture by a known method are suspended in an appropriate buffer, and are subjected to ultrasound, lysozyme and / or freezing.
  • a method of obtaining a crude extract of soluble protein by centrifugation or filtration after disrupting cells or cells by thawing or the like is appropriately used.
  • the buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride and a surfactant such as Triton X-100 TM .
  • a protein denaturant such as urea or guanidine hydrochloride
  • a surfactant such as Triton X-100 TM .
  • the cell debris is precipitated and removed by low-speed centrifugation, and the supernatant is centrifuged at high speed to obtain the cell membrane-containing fraction.
  • a method such as precipitation (purifying a cell membrane fraction by density gradient centrifugation or the like as required) is used.
  • GPR120 or a partial peptide thereof When GPR120 or a partial peptide thereof is secreted outside the cells (cells), a method of separating the culture supernatant from the culture by centrifugation or filtration is used. Isolation and purification of GPR120 or a partial peptide thereof contained in the thus obtained soluble fraction, membrane fraction or culture supernatant can be performed according to a method known per se. Examples of such methods include the use of solubilities such as salting out and solvent precipitation; mainly the differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • a method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography; A method using a difference in isoelectric point, such as point electrophoresis, is used. These methods can be combined as appropriate.
  • the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein or peptide is obtained as a salt, The salt can be converted into a free form or other salt by a method known per se or a method analogous thereto.
  • the GPR120 produced by the transformant or a partial peptide thereof can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification.
  • the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
  • the presence of GPR120 or a partial peptide thereof thus obtained can be confirmed by enzyme immunoassay or Western blotting using an antibody against GPR120.
  • GPR120 or a partial peptide thereof uses a cell-free protein translation system comprising a rabbit reticulocyte lysate, a wheat germ lysate, an Escherichia coli lysate, etc., using an RNA corresponding to the DNA encoding the GPR120 or the partial peptide as a template. It can be synthesized in vitro.
  • a cell-free transcription / translation system further containing RNA polymerase can be used to synthesize a DNA encoding GPR120 or a partial peptide thereof as a template.
  • the cell-free protein transcription / translation system can be a commercially available one, or a known method, specifically, Escherichia coli extract is Pratt JM et al., “Transcription and Tranlation”, Hames BD and Higgins SJ, IRL It can also be prepared according to the method described in Press, Oxford 179-209 (1984).
  • Examples of commercially available cell lysates include E. coli S30 extract system (Promega) and RTS 500 Rapid Tranlation System (Roche) derived from E. coli, and Rabbit Reticulocyte Lysate System derived from rabbit reticulocytes. Examples of those derived from wheat germ include Proteios TM (manufactured by TOYOBO).
  • wheat germ lysate those using wheat germ lysate are preferred.
  • a method for producing wheat germ lysate for example, the method described in Johnston FB et al., Nature, 179, 160-161 (1957) or Erickson AH et al., Meth. Enzymol., 96, 38-50 (1996), etc. should be used. Can do.
  • a system or apparatus for protein synthesis a batch method (Pratt, JM et al. (1984) mentioned above) or a continuous cell-free protein synthesis system (Spirin AS et al.
  • cells producing GPR120 or a partial peptide thereof include cells that naturally express GPR120 (eg, EPC (large EPC, small EPC), adipocytes, enteroendocrine cells, etc.) and progenitor cells thereof ( For example, bone marrow cells, preadipocytes, etc.), and transformed cells into which a nucleic acid encoding GPR120 or a partial peptide thereof has been introduced.
  • EPC large EPC, small EPC
  • progenitor cells thereof For example, bone marrow cells, preadipocytes, etc.
  • the promoter and / or enhancer / suppressor that regulates the expression of the nucleic acid encoding GPR120 or the partial peptide is used as an endogenous GPR120 gene.
  • promoters and / or enhancer / suppressor sequences are preferred to use.
  • Such a regulatory sequence of GPR120 gene can be isolated and cloned from genomic DNA extracted from mammalian cells and tissues by a method known per se.
  • the cells that produce GPR120 or a partial peptide thereof preferably include EPC (large EPC, small EPC) and its precursor cells (bone marrow cells). Using these cells, not only can EPC differentiation / proliferation regulation or EPC increase in peripheral blood be screened using the activity or expression of endogenous GPR120 as an index, but differentiation from bone marrow cells to EPC, It is also possible to verify whether the growth rate can be adjusted.
  • bone marrow cells include all EPC progenitor cells present in bone marrow unless otherwise specified.
  • typical EPC progenitor cells can be obtained from fractions characterized by c-kit + , Sca-1 + , and lineage marker ⁇ for the expression of various cell surface antigen markers.
  • Such compounds include (a) GPR120 binding to cell stimulating activity (eg, GTP-GDP exchange reaction acceleration in conjugated G ⁇ , PLC ⁇ activation, regulation of inositol phosphate production, intracellular Ca 2+ uptake, PKC activity) , EPC differentiation promotion, EPC proliferation promotion, insulin secretion promotion, hypoglycemia, ACTH secretion suppression, growth hormone secretion regulation, etc.) (b) A compound that binds to GPR120 but exhibits cell-stimulating activity A compound that does not have (antagonist), (c) a compound that enhances the binding force between GPR120 and the ligand, or (d) a compound that decreases the binding force between GPR120 and the ligand.
  • cell stimulating activity eg, GTP-GDP exchange reaction acceleration in conjugated G ⁇ , PLC ⁇ activation, regulation of inositol phosphate production, intracellular Ca 2+ uptake, PKC activity
  • EPC differentiation promotion EPC proliferation promotion
  • insulin secretion promotion hypogly
  • GPR120 has an action of promoting differentiation of bone marrow cells to EPC (in the present specification, “differentiation of EPC” simply means differentiation of bone marrow cells to EPC) and proliferation of EPC
  • a compound that enhances the binding force between an agonist or GPR120 and a ligand can promote differentiation and proliferation of EPC.
  • an antagonist of GPR120 or a compound that decreases the binding force between GPR120 and a ligand can suppress the differentiation / proliferation of EPC.
  • an antagonist of GPR120 or a compound that decreases the binding force between GPR120 and a ligand is also useful for increasing EPC in peripheral blood.
  • the present invention includes GPR120 or a partial peptide thereof and a known low-molecular compound (hereinafter sometimes referred to as “surrogate ligand”) that changes the binding property of fatty acid that is a physiological ligand or fatty acid and GPR120.
  • EPC differentiation / proliferation regulation (promotion or suppression) or EPC in peripheral blood characterized by contacting in the presence and absence of a test compound and comparing the activity of GPR120 or its partial peptide under both conditions
  • a method for screening increased substances is provided. In the screening method described above, the activity of GPR120 or a partial peptide thereof is measured using the binding amount of a ligand (fatty acid or surrogate ligand) to the protein or peptide, or the cell stimulating activity as an index.
  • the present invention provides: (1) Binding amount of labeled fatty acid or surrogate ligand to GPR120 or its partial peptide when the labeled fatty acid or surrogate ligand is contacted with GPR120 or its partial peptide in the presence or absence of the test compound EPC differentiation / proliferation regulation or screening method for EPC increasing substance in peripheral blood, characterized by measuring and comparing (2) A labeled fatty acid or surrogate ligand when the labeled fatty acid or surrogate ligand is contacted with a cell producing GPR120 or a partial peptide thereof or a membrane fraction thereof in the presence and absence of a test compound A method of screening for a substance that regulates differentiation / proliferation of EPC or an EPC-increasing substance in peripheral blood, which comprises measuring and comparing the binding amount of the cells to the cell or membrane fraction, (3) A labeled fatty acid or surrogate ligand and the protein or peptide expressed on the cell membrane by culturing a transformant containing
  • GPR120 or a partial peptide thereof used in the screening method of the present invention may be any as long as it contains GPR120 or the partial peptide described above, but physiological conformation of the extracellular region. Is preferably a cell membrane fraction of a mammalian organ that produces GPR120 or a partial peptide thereof. However, since human-derived organs are particularly difficult to obtain, human-derived GPR120 or a partial peptide thereof expressed in large quantities using a recombinant is suitable for use in screening.
  • GPR120 or a partial peptide thereof In order to produce GPR120 or a partial peptide thereof, the above-mentioned method is used, but it is preferable to carry out expression in mammalian cells or insect cells of DNA encoding GPR120 or a partial peptide thereof.
  • CDNA is used as a DNA fragment encoding a target protein portion, but is not necessarily limited thereto. For example, gene fragments or synthetic DNA may be used.
  • the DNA fragment can be expressed using an SV40-derived promoter, a retroviral promoter, a metallothionein promoter, human It is preferably incorporated downstream of a heat shock promoter, a cytomegalovirus promoter, an SR ⁇ promoter, a polyhedrin promoter of a nuclear polyhedrosis virus (NPV) belonging to baculoviruses with insect hosts.
  • SV40-derived promoter a retroviral promoter, a metallothionein promoter, human It is preferably incorporated downstream of a heat shock promoter, a cytomegalovirus promoter, an SR ⁇ promoter, a polyhedrin promoter of a nuclear polyhedrosis virus (NPV) belonging to baculoviruses with insect hosts.
  • NPV nuclear polyhedrosis virus
  • the cells when cells that produce GPR120 or a partial peptide thereof are used, the cells may be fixed with glutaraldehyde, formalin or the like.
  • the immobilization method can be performed according to a method known per se.
  • a cell that produces GPR120 or a partial peptide thereof refers to a host cell that expresses GPR120 or a partial peptide thereof. Examples of the host cell include animal cells and insect cells.
  • the cell membrane fraction refers to a fraction containing a lot of cell membranes obtained by a method known per se after disrupting cells.
  • Cell disruption methods include crushing cells with a Potter-Elvehjem homogenizer, disrupting with a Waring blender or polytron (manufactured by Kinematica), disrupting with ultrasonic waves, and pressurizing with a French press while ejecting cells from a thin nozzle. Crushing by things.
  • a fractionation method using centrifugal force such as a fractional centrifugation method or a density gradient centrifugation method is mainly used.
  • the cell lysate is centrifuged at low speed (500 rpm to 3000 rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further centrifuged at high speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours.
  • the membrane fraction is rich in the expressed GPR120 or a partial peptide thereof and membrane components such as cell-derived phospholipids and membrane proteins.
  • GPR120 obtained as described above in an artificial lipid bilayer prepared by a conventional method from a solution in which various lipids are mixed at an appropriate ratio, preferably a ratio close to that in the cell membrane of a eukaryotic cell.
  • the reconstituted partial peptide can be used as the cell membrane fraction.
  • the lipid constituting the artificial lipid bilayer include phosphatidylcholine (PC), phosphatidylserine (PS), cholesterol (Ch), phosphatidylinositol (PI), phosphatidylethanolamine (PE), etc., one or two of these What mixed the above by the appropriate ratio is used preferably.
  • an artificial lipid bilayer (proteoliposome) incorporating GPR120 or a partial peptide thereof can be prepared by the following method.
  • An appropriate amount of the purified fusion protein is added thereto, incubated for about 20 to 30 minutes with occasional stirring in ice, and then dialyzed against an appropriate buffer.
  • the desired proteoliposome can be obtained by collecting the sediment by centrifugation at about 100,000 ⁇ g for 30 to 60 minutes.
  • the amount of GPR120 or its partial peptide in a cell producing GPR120 or its partial peptide or its membrane fraction is preferably 10 3 to 10 8 molecules, more preferably 10 5 to 10 7 molecules per cell. More preferred.
  • the higher the expression level the higher the ligand binding activity (specific activity) per membrane fraction, making it possible not only to construct a highly sensitive screening system, but also to measure a large amount of samples in the same lot. .
  • an appropriate GPR120 or a partial peptide-containing membrane fraction thereof is used.
  • a labeled fatty acid or surrogate ligand is desirable.
  • GPR120 or a partial peptide-containing membrane fraction thereof a natural GPR120 or a partial peptide-containing membrane fraction thereof, or a recombinant GPR120 having a similar activity or a partial peptide-containing membrane fraction thereof is desirable.
  • the equivalent activity indicates an equivalent ligand binding activity and the like.
  • the fatty acid may be any fatty acid as long as it can serve as a ligand for GPR120 or a partial peptide thereof. Acid) and the like. Among them, palmitoleic acid, linoleic acid, and ⁇ -linolenic acid are preferably used.
  • the fatty acid may be a free form or a salt form.
  • Preferred examples of the fatty acid salts include salts with bases (eg, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, ammonium salts and alkanolamine salts). However, it is not limited to them.
  • Examples of the “low molecular weight compound that changes the binding property between fatty acid and GPR120”, that is, the surrogate ligand include, for example, the formula:
  • ring A represents an aromatic ring which may have a substituent
  • ring B represents an aromatic ring which may further have a substituent other than —Y—COOH
  • X and Y each represent a spacer.
  • -Y-COOH is substituted at any position on ring B.
  • the fatty acid or surrogate ligand can be labeled with, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like according to a conventional method.
  • a radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • the enzyme those which are stable and have high specific activity are preferable.
  • ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • GPR120 or a partial peptide preparation thereof is prepared by suspending in a buffer.
  • the buffer may be any buffer that does not inhibit the binding of GPR120 or a partial peptide thereof and a fatty acid (or surrogate ligand), such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a Tris-HCl buffer.
  • a surfactant such as CHAPS, Tween-80 TM (Kao-Atlas), digitonin, deoxycholate and the like can be added to the buffer.
  • a surfactant such as CHAPS, Tween-80 TM (Kao-Atlas), digitonin, deoxycholate and the like
  • protease inhibitors such as PMSF, leupeptin, E-64 (manufactured by Peptide Institute) and pepstatin can be added for the purpose of suppressing the degradation of the receptor by the protease.
  • the solution is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and then the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or ⁇ -counter.
  • the specific binding amount (B-NSB) is, for example, Therefore, a test compound that is 50% or less can be selected as a candidate compound for EPC differentiation / proliferation regulation or an EPC increasing substance in peripheral blood.
  • an appropriate buffer that is not toxic to fresh medium or cells in advance eg, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetic acid
  • the solution is exchanged with a buffer solution, etc., and a ligand, a test compound, etc. are added and incubated for a certain period of time.
  • the cells are extracted or the supernatant is collected, and the produced product is quantified according to each method.
  • an assay may be performed with an inhibitor for the degrading enzyme added.
  • test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like, and these compounds may be novel compounds. It may be a known compound.
  • a screening kit for a compound or a salt thereof that alters the binding property between GPR120 or a partial peptide thereof and a fatty acid or a surrogate ligand contains GPR120 or a partial peptide thereof, a cell that produces GPR120 or a partial peptide thereof, a membrane fraction thereof, or the like Is.
  • Examples of the screening kit of the present invention include the following. 1. Screening reagents (a) Buffer for measurement and buffer for washing Hanks' Balanced Salt Solution (Gibco) plus 0.05% bovine serum albumin (Sigma). The solution is sterilized by filtration through a 0.45 ⁇ m filter and stored at 4 ° C. or may be prepared at the time of use.
  • GPR120 or its partial peptide preparation CHO cells expressing GPR120 or its partial peptide are passaged to a 12-well plate at 5 ⁇ 10 5 cells / well, 37 ° C., 5% CO 2 , 95% air Cultured for 2 days.
  • Labeled fatty acid or surrogate ligand A commercially available fatty acid or surrogate ligand aqueous solution labeled with [ 3 H], [ 125 I], [ 14 C] or the like is stored at 4 ° C. or ⁇ 20 ° C., Dilute to 1 ⁇ M with buffer for measurement.
  • Fatty acid or surrogate ligand standard solution Fatty acid or surrogate ligand is dissolved to 1 mM in PBS containing 0.1% bovine serum albumin (manufactured by Sigma) and stored at -20 ° C.
  • cells that express GPR120 or a partial peptide thereof on the cell membrane further express conjugated G ⁇ .
  • the family of G ⁇ conjugated to GPR120 or its partial peptide is G ⁇ q / 11 .
  • the cells expressing GPR120 or a partial peptide thereof and its conjugated G ⁇ include transformed cells co-transfected with DNA encoding GPR120 or its partial peptide and DNA encoding G ⁇ q / 11 .
  • G ⁇ activation domain of the receptor interacts with the receptor binding region of conjugated G ⁇ , resulting in a conformational change of G ⁇ , and a GTP / GDP binding region GDP dissociates from GTP and quickly binds GTP.
  • G ⁇ -GTP acts on the effector PLC ⁇ to promote its activity.
  • the antagonist binds, the conformation of the receptor does not occur and the G ⁇ activation domain is in an inactivated state, so that the activated G ⁇ -GTP level is decreased and the action on PLC ⁇ is inhibited.
  • GTP for example, if a GTP analog that does not undergo hydrolysis due to GTPase activity of G ⁇ such as 35 S-labeled GTP ⁇ S is added to the system, the membrane in the presence and absence of the test compound can be used. By measuring and comparing the radioactivity bound to G ⁇ , the effect of the test compound on the GDP-GTP exchange reaction in G ⁇ can be evaluated, and screening for substances having agonist / antagonist activity of GPR120 or its partial peptides Can do.
  • GTP for example, if a GTP analog that does not undergo hydrolysis due to GTPase activity of G ⁇ such as 35 S-labeled GTP ⁇ S is added to the system, the membrane in the presence and absence of the test compound can be used. By measuring and comparing the radioactivity bound to G ⁇ , the effect of the test compound on the GDP-GTP exchange reaction in G ⁇ can be evaluated, and screening for substances having agonist / antagonist activity of GPR120 or its partial peptides Can do.
  • the test compound has an agonist activity against GPR120 or a partial peptide thereof, and if the radioactivity decreases, the test compound has an antagonist activity against GPR120 or a partial peptide thereof.
  • the activity of PLC ⁇ that interacts with G ⁇ is compared in the presence and absence of a test compound.
  • a screening method is provided. Therefore, cells expressing GPR120 or its partial peptide and its conjugated G ⁇ also express PLC ⁇ that receives signals from the G ⁇ .
  • the PLC ⁇ activity is determined, for example, by adding [ 3 H] -labeled phosphatidylinositol-4,5-diphosphate to the PLC ⁇ -containing membrane fraction, and determining the amount of inositol-1,4,5-triphosphate produced by a known amount. It can evaluate by measuring using a method.
  • PLC ⁇ activity is measured and compared in the presence and absence of the test compound, and if the enzyme activity increases in the presence of the test compound, the test compound has agonist activity against GPR120 or its partial peptide, and conversely PLC ⁇ If the activity decreases, the test compound is evaluated as having antagonist activity against GPR120 or a partial peptide thereof.
  • the action of G ⁇ on PLC ⁇ is measured by adding [ 3 H] -labeled inositol to the cells and measuring the radioactivity of the generated [ 3 H] -labeled inositol phosphate, It can also be evaluated by measuring the amount of intracellular Ca 2+ .
  • intracellular Ca 2+ levels incubate the cells for an appropriate time in the presence and absence of the test compound, and then use a fluorescent probe (fura-2, indo-1, fluor-3, Calcium-Green I, etc.) It can be measured spectroscopically or using aequorin, which is a calcium-sensitive photoprotein, but any other known method may be used.
  • An apparatus suitable for spectroscopic measurement using a fluorescent probe is a FLIPR (Molecular Devices) system.
  • a reporter gene under the control of a TPA (12-O-tetradecanoylphorbol-13-acetate) response element (TRE) that is up-regulated by Ca 2+
  • TRE TPA (12-O-tetradecanoylphorbol-13-acetate) response element
  • a method for evaluating the amount of Ca 2+ That is, eukaryotic cells into which a vector containing an expression cassette linked with a DNA encoding a reporter protein (eg, luciferase, GFP, peroxidase, alkaline phosphatase, etc.) is introduced downstream of a promoter containing TRE in the presence of a test compound.
  • a reporter protein eg, luciferase, GFP, peroxidase, alkaline phosphatase, etc.
  • the amount of Ca 2+ in the cell is evaluated by measuring and comparing the expression of the reporter gene in the extract obtained by culturing for an appropriate time in the absence and disrupting the cells using a known method. Is. Therefore, if the intracellular Ca 2+ level (or the expression level of the reporter gene under TRE control) increases in the presence of the test compound, the test compound has agonist activity for GPR120 or its partial peptide, and conversely If the intracellular Ca 2+ level (or reporter gene expression level) decreases, the test compound has antagonist activity against GPR120 or a partial peptide thereof.
  • GPR120 or GPR120 when cells producing GPR120 or a partial peptide thereof are cultured in the presence and absence of a test compound without contacting with a fatty acid or a surrogate ligand.
  • a compound that promotes or suppresses the activity of GPR120 can be selected regardless of whether or not the binding property to the ligand is changed.
  • a fusion protein in which conjugated G ⁇ is linked to the C-terminal side of GPR120 or a partial peptide thereof is used in mammalian cells, insect cells, and the like.
  • GPR120 or a partial peptide thereof can be constitutively activated.
  • yeast eg, baker's yeast
  • Yeast has STE2, which is the receptor for mating pheromone alpha factor, as the only GPCR.
  • GPA1 is a conjugated G ⁇
  • G ⁇ dissociates from G ⁇ / G ⁇ .
  • Kinase cascade signals from dissociated G ⁇ / G ⁇ induce FUS1 expression and conjugation occurs.
  • GPR120 or a partial peptide thereof is expressed in yeast instead, and GPR120 or a conjugated G ⁇ of the partial peptide is co-expressed under the control of the GPA1 promoter, a fatty acid (or an agonist of GPR120) GPR120 or a partial peptide thereof mediates intracellular signal transduction as if by STE2.
  • G ⁇ coupled to GPR120 or its partial peptide is co-expressed under the control of the GPA1 promoter, and a gene supplementing an auxotrophic mutation or a reporter gene (for example, LacZ) is fused to FUS1. Signal activation can be detected. Details of this method are described in, for example, Yeast, 16: 11-22 (2000).
  • the cell producing GPR120 or a partial peptide thereof is provided in the form of a non-human mammal individual.
  • the state of the individual animal is not particularly limited.
  • it is a model animal of a disease (obesity, diabetes, hypertension, arteriosclerosis, etc.) such as db / db mouse, ob / ob mouse, KKAy mouse, Zuckerfatty rat, and WHHL rabbit. May be.
  • a disease ovale.g., diabetes, hypertension, arteriosclerosis, etc.
  • db / db mouse ob / ob mouse
  • KKAy mouse KKAy mouse
  • Zuckerfatty rat and WHHL rabbit.
  • WHHL rabbit WHHL rabbit
  • the administration route is not particularly limited, and examples thereof include intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, intraperitoneal administration, oral administration, intratracheal administration, and rectal administration.
  • the dose is not particularly limited. For example, a dose of about 0.5 to 20 mg / kg can be administered 1 to 5 times a day, preferably 1 to 3 times a day for 1 to 14 days.
  • the contact of the ligand with the cell can be replaced by the action of a fatty acid that is a physiological ligand inherent in the animal individual.
  • the agonist for GPR120 or its partial peptide obtained by using the above screening method has the same action as the physiological activity of fatty acid for GPR120 or its partial peptide. It is useful as a safe and low-toxic drug that enhances differentiation / proliferation promoting action. Since antagonists to GPR120 or its partial peptide can suppress the physiological activity of fatty acids for GPR120 or its partial peptide, it suppresses GPR120-mediated signal transduction and EPC differentiation / proliferation-promoting effects, as well as peripheral blood. It is useful as a safe and low-toxic drug that exhibits an increase in EPC.
  • a compound that enhances the binding force between GPR120 or a partial peptide thereof and a fatty acid is useful as a safe and low toxic pharmaceutical for enhancing the physiological activity of the fatty acid for GPR120 or a partial peptide thereof.
  • a compound that decreases the binding force between GPR120 or a partial peptide thereof and a fatty acid is useful as a safe and low-toxic pharmaceutical for reducing the physiological activity of fatty acids for GPR120 or a partial peptide thereof.
  • GPR120 has an action of promoting differentiation and proliferation of EPC, and suppression of GPR120 promotes mobilization of EPC to peripheral blood.
  • a compound that changes the expression level of GPR120 can regulate differentiation and proliferation of EPC and increase EPC in peripheral blood. Therefore, the present invention is also characterized in that the expression of the protein or peptide (or gene encoding it) in cells producing GPR120 or a partial peptide thereof is compared in the presence and absence of the test compound.
  • the present invention provides a screening method for EPC differentiation / proliferation regulation or EPC increasing substance in peripheral blood.
  • the cells used in this method, the type of test compound, the mode of contact between the test compound and the cells, and the like are the same as those described above using the activity of GPR120 or a partial peptide thereof as an index.
  • the expression level of GPR120 or a partial peptide thereof is a nucleic acid that can hybridize with the above-described DNA encoding GPR120 or the partial peptide under high stringency conditions, that is, the nucleotide sequence represented by SEQ ID NO: 1, 3, or 5 or GPR120 or a partial peptide thereof using a nucleic acid capable of hybridizing under high stringency conditions with a DNA comprising the partial base sequence or the complementary strand sequence (hereinafter sometimes referred to as “the nucleic acid for detection of the present invention”) Can be measured at the RNA level.
  • the expression level is measured at the protein level by detecting the protein or peptide using an antibody against GPR120 or a partial peptide thereof (hereinafter sometimes referred to as “the detection antibody of the present invention”). You can also. Therefore, more specifically, the present invention (A) Cells that produce GPR120 or a partial peptide thereof are cultured in the presence and absence of a test compound, and the amount of mRNA encoding the protein or peptide under both conditions is determined using the nucleic acid for detection of the present invention. A method of screening for EPC differentiation / proliferation or screening for an EPC-increasing substance in peripheral blood, and (b) cells that produce GPR120 or a partial peptide thereof in the presence or absence of a test compound. EPC differentiation / proliferation regulation or increase of EPC in peripheral blood, characterized by measuring and comparing the amount of the protein or peptide under both conditions using the detection antibody of the present invention. A method for screening a substance is provided.
  • the measurement of the amount of mRNA or protein (peptide) of GPR120 or a partial peptide thereof can be specifically performed as follows.
  • Normal or disease model non-human mammal eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.
  • obese mouse, diabetic mouse, hypertensive rat, arteriosclerosis Rabbits, etc. are given drugs (eg, anti-obesity drugs, anti-diabetic drugs, antihypertensive drugs, vasoactive drugs, etc.) or physical stress (eg, water stress, electric shock, light / dark, low temperature, etc.)
  • drugs eg, anti-obesity drugs, anti-diabetic drugs, antihypertensive drugs, vasoactive drugs, etc.
  • physical stress eg, water stress, electric shock, light / dark, low temperature, etc.
  • peripheral blood or a specific organ for example, bone marrow, organ lesion local area, etc.
  • tissue for example, bone marrow tissue, diseased tissue, etc.
  • the mRNA of GPR120 contained in the obtained cells and the like can be quantified by, for example, extracting mRNA from cells and the like by a normal method, for example, using a technique such as RT-PCR, or a known Northern per se It can also be quantified by blot analysis.
  • the amount of GPR120 protein can be quantified using Western blot analysis or various immunoassay methods described in detail below.
  • a transformant into which a nucleic acid encoding GPR120 or a partial peptide thereof has been introduced is prepared according to the method described above, and GPR120 or a partial peptide thereof or mRNA encoding the transformant contained in the transformant is prepared as described in (i) Quantitative analysis can be performed in the same manner.
  • the amount of mRNA encoding GPR120 contained in cells isolated from the animal After 30 minutes (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the amount of mRNA encoding GPR120 contained in cells isolated from the animal, Alternatively, by quantifying and analyzing the amount of GPR120 protein, or (ii) when culturing a transformant according to a conventional method, a test compound is added to a medium or a buffer, and incubated for a certain period of time (after 1 to 7 days, Preferably after 1 to 3 days, more preferably 2 days 1-3 days after), mRNA quantity encoding GPR120 or its partial peptide contained in the transformant, or protein (peptide) amount quantitation can be carried out by analyzing.
  • the detection antibody of the present invention used in the screening method (b) is not particularly limited as long as it is an antibody that specifically recognizes GPR120 or a partial peptide thereof, and may be either a polyclonal antibody or a monoclonal antibody. .
  • the isotype of the antibody is not particularly limited, but preferably IgG, IgM or IgA, particularly preferably IgG.
  • the antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding a target antigen.
  • CDR complementarity determining region
  • Fab, Fab ′, F (ab ') 2 such as fragments, scFv, scFv-Fc, conjugation molecules prepared by genetic engineering such as minibodies and diabodies, or molecules having protein stabilizing action such as polyethylene glycol (PEG) They may be modified derivatives thereof.
  • An antibody against GPR120 or a partial peptide thereof can be produced according to a known method for producing an antibody or antiserum using GPR120 or a partial peptide thereof as an antigen.
  • a method for preparing an immunogen of the antibody of the present invention and a method for producing the antibody will be described.
  • Antigen used for preparing the antibody includes GPR120 or a partial peptide thereof (in the following description relating to the production of antibody, unless otherwise specified, these are simply referred to as “GPR120”). Or a (synthetic) peptide having one or more of the same antigenic determinants (hereinafter, these may be simply referred to as the antigen of the present invention).
  • GPR120 is prepared by, for example, (a) preparing from a mammalian tissue or cell using a known method or a method equivalent thereto, and (b) chemically using a known peptide synthesis method using a peptide synthesizer or the like.
  • GPR120 can be used as an immunogen as it is, or a partial peptide can be prepared by limited degradation using a peptidase or the like and used as an immunogen.
  • the synthetic peptide is, for example, one having the same structure as GPR120 purified from a natural material using the method (a) described above, specifically, In the GPR120 amino acid sequence, a peptide containing one or two or more amino acid sequences identical to the amino acid sequence of any position consisting of at least 3 amino acids, preferably 6 amino acids or more, is used.
  • C When the antigen of the present invention is produced using a transformant containing DNA, the DNA can be obtained by known cloning methods [for example, Molecular Cloning 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab The method described in Press, 1989)].
  • the cloning method includes (1) isolating DNA encoding the antigen from a human cDNA library by hybridization using a DNA probe designed based on the gene sequence encoding GPR120, or (2) Examples include a method of preparing a DNA encoding the antigen by a PCR method using a cDNA primer designed based on the gene sequence to be encoded as a template and inserting the DNA into an expression vector suitable for the host. It is done.
  • a desired antigen can be obtained by culturing a transformant obtained by transforming a host with the expression vector in an appropriate medium.
  • GPR120 or a partial peptide thereof, or a peptide having a shorter partial amino acid sequence can be used as an immunogen.
  • the partial amino acid sequence include those consisting of 3 or more consecutive amino acid residues, preferably 4 or more, more preferably 5 or more, and even more preferably 6 or more consecutive amino acid residues. It is done.
  • the amino acid sequence is composed of, for example, 20 or less consecutive amino acid residues, preferably 18 or less, more preferably 15 or less, and even more preferably 12 or less consecutive amino acid residues. Is mentioned. Some of these amino acid residues (eg, 1 to several) may be substituted with a substitutable group (eg, Cys, hydroxyl group, etc.).
  • the peptide used as an immunogen has an amino acid sequence containing 1 to several such partial amino acid sequences.
  • mammalian cells that express GPR120 can also be used directly as the antigen of the present invention.
  • mammalian cells natural cells as described in the above section (a), cells transformed by the method as described in the above section (c), and the like can be used.
  • the host used for transformation may be any cell collected from humans, monkeys, rats, mice, hamsters, chickens, etc., HEK293, COS7, CHO-K1, NIH3T3, Balb3T3, FM3A, L929, SP2 / 0, P3U1, B16, or P388 is preferably used.
  • Natural mammalian cells expressing GPR120 or transformed eukaryotic cells are suspended in a medium used for tissue culture (eg, RPMI1640) or buffer (eg, Hanks' Balanced Salt Solution). Can be injected into immunized animals.
  • tissue culture eg, RPMI1640
  • buffer eg, Hanks' Balanced Salt Solution
  • Any method can be used as long as it can promote antibody production, and intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, and the like are preferably used.
  • the antigen of the present invention can be directly immunized as long as it has immunogenicity, but it has a low molecular weight (for example, a molecular weight of about 3,000 or less) having only one or several antigenic determinants in the molecule.
  • Antigens ie, partial peptides of GPR120
  • these antigens are usually hapten molecules with low immunogenicity, they can be immunized as a complex bound or adsorbed to a suitable carrier (carrier).
  • carrier can.
  • a natural or synthetic polymer can be used as the carrier.
  • Examples of natural polymers include serum albumin of mammals such as cows, rabbits and humans, and thyroglobulin of mammals such as cows and rabbits, such as chicken ovalbumin, such as mammals such as cows, rabbits, humans and sheep. Hemoglobin, keyhole limpet hemocyanin (KLH), etc. are used.
  • Examples of the synthetic polymer include various latexes such as polymers or copolymers such as polyamino acids, polystyrenes, polyacryls, polyvinyls, and polypropylenes.
  • the mixing ratio of the carrier and the hapten is such that any antibody can be bound or adsorbed at any ratio as long as an antibody against the antigen bound or adsorbed to the carrier is efficiently produced.
  • the above-mentioned natural or synthetic polymer carrier which is commonly used in the production of the above, can be bound or adsorbed at a ratio of 0.1 to 100 with respect to hapten 1 by weight.
  • various condensing agents can be used for coupling of the hapten and the carrier.
  • diazonium compounds such as bisdiazotized benzidine that crosslinks tyrosine, histidine, and tryptophan, dialdehyde compounds such as glutaraldehyde that crosslink amino groups, diisocyanate compounds such as toluene-2,4-diisocyanate, and thiol groups
  • dimaleimide compound such as N, N′-o-phenylene dimaleimide, a maleimide active ester compound that crosslinks an amino group and a thiol group, a carbodiimide compound that crosslinks an amino group and a carboxyl group, and the like are advantageously used.
  • an active ester reagent having a dithiopyridyl group on one amino group for example, N-succinimidyl (SPDP) 3- (2-pyridyldithio) propionate
  • SPDP N-succinimidyl
  • a maleimide group into the other amino group with a maleimide active ester reagent, and then react both.
  • the antigen of the present invention is administered to a warm-blooded animal by itself at a site where antibody production is possible by administration methods such as intraperitoneal injection, intravenous injection, subcutaneous injection, and intradermal injection. Or, it is administered together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually once every 1 to 6 weeks, for a total of 2 to 10 times. Examples of warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, hamsters, sheep, goats, donkeys, and chickens. In order to avoid the problem of anti-Ig antibody production, it is preferable to use a mammal of the same species as that to be administered, but in general, mice and rats are preferably used for production of monoclonal antibodies.
  • antibody-producing hybridomas can be prepared by isolating antibody-producing cells and fusing them with myeloma cells.
  • the antibody titer in serum can be measured, for example, by reacting a labeled antigen with antiserum and then measuring the activity of the labeling agent bound to the antibody.
  • the myeloma cell is not particularly limited as long as it can produce a hybridoma that secretes a large amount of antibody, but it does not itself produce or secrete an antibody, and more preferably has high cell fusion efficiency.
  • a HAT hyperxanthine, aminopterin, thymidine
  • mouse myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, etc.
  • rat myeloma cells include R210.RCY3, Y3-Ag 1.2.3
  • human myeloma cells include SKO- 007, GM 1500-6TG-2, LICR-LON-HMy2, UC729-6 and the like.
  • the fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus.
  • PEG polyethylene glycol
  • the molecular weight of PEG is not particularly limited, but PEG1000 to PEG6000 having low toxicity and relatively low viscosity are preferable.
  • the PEG concentration include about 10 to 80%, preferably about 30 to 50%.
  • various buffer solutions such as serum-free medium (eg RPMI1640), complete medium containing about 5 to 20% serum, phosphate buffered saline (PBS), Tris buffer, etc. may be used. it can.
  • DMSO eg, about 10 to 20%
  • the pH of the fusion solution is, for example, about 4 to 10, preferably about 6 to 8.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of bone marrow cells is usually about 1: 1 to 20: 1, and is usually incubated at 20 to 40 ° C., preferably 30 to 37 ° C. for usually 1 to 10 minutes. Cell fusion can be carried out efficiently.
  • Monoclonal antibody-producing hybridomas are prepared, for example, by adding the hybridoma culture supernatant to a solid phase (eg, microplate) on which an antigen is adsorbed directly or with a carrier, and then anti-immunoglobulin antibodies (cells) labeled with radioactive substances or enzymes.
  • a solid phase eg, microplate
  • an antigen is adsorbed directly or with a carrier
  • anti-immunoglobulin antibodies cells labeled with radioactive substances or enzymes.
  • anti-mouse immunoglobulin antibody When mouse used for fusion is mouse, anti-mouse immunoglobulin antibody is used) or protein A is added to detect monoclonal antibody bound to the solid phase; solid phase adsorbed with anti-immunoglobulin antibody or protein A
  • the method can be screened by adding a hybridoma culture supernatant, adding a protein labeled with a radioactive substance or an enzyme, and detecting a monoclonal antibody bound to the solid phase.
  • the selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto.
  • the selection of the monoclonal antibody can be usually performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. Any medium may be used as the monoclonal antibody selection and breeding medium as long as the hybridoma can grow.
  • HAT hyperxanthine, aminopterin, thymidine
  • a medium for example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal calf serum, GIT medium containing 1 to 10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) Or a serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like.
  • the culture temperature is usually 20-40 ° C, preferably about 37 ° C.
  • the culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.
  • the monoclonal antibody thus obtained can be obtained by a method known per se, for example, an immunoglobulin separation and purification method [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, , DEAE) adsorption / desorption method, ultracentrifugation, gel filtration, antigen-binding solid phase or specific purification method to obtain antibody by dissociating the binding using active adsorbent such as protein A or protein G ] Can be separated and purified according to the above.
  • an immunoglobulin separation and purification method eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, , DEAE) adsorption / desorption method, ultracentrifugation, gel filtration, antigen-binding solid phase or specific purification method to obtain antibody by dissociating the binding using active adsorbent such as protein A or protein
  • Polyclonal antibodies for example, immunize warm-blooded animals in the same manner as the above-mentioned monoclonal antibody production method, by creating an immune antigen (protein or peptide antigen) itself or a complex thereof with a carrier protein. From the immunized animal, an antibody-containing product against GPR120 or a partial peptide thereof, such as blood, ascites, milk, egg, etc., can be preferably collected from blood. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as that of the monoclonal antibody.
  • the measurement of the amount of GPR120 or a partial peptide thereof in the screening method of (b) above is, for example, (I) A sample solution by competitively reacting the detection antibody of the present invention with the sample solution and labeled GPR120 or a partial peptide thereof, and detecting the labeled protein or peptide bound to the antibody A method for quantifying GPR120 or a partial peptide thereof, (Ii) The sample solution, the detection antibody of the present invention insolubilized on the carrier, and another labeled detection antibody of the present invention are reacted simultaneously or successively, and then the labeling agent on the insolubilized carrier A method for quantifying GPR120 or a partial peptide thereof in a sample solution by measuring the amount (activity) of the above is mentioned.
  • the two types of antibodies recognize different portions of GPR120.
  • one antibody recognizes the N-terminal part of the protein
  • one that reacts with the C-terminal part of the protein can be used as the other antibody.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used.
  • the radioisotope include [ 125 I], [ 131 I], [ 3 H], [ 14 C], [ 32 P], [ 33 P], [ 35 S] and the like.
  • the enzyme is preferably stable and has a large specific activity.
  • ⁇ -galactosidase ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • fluorescent substance for example, fluorescamine, fluorescein isothiocyanate, cyanine fluorescent dye and the like are used.
  • luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • a biotin- (strept) avidin system can be used for binding of an antibody or antigen to a labeling agent.
  • the method for quantifying GPR120 or its partial peptide using the detection antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the sample solution is chemically determined.
  • Any measurement method may be used as long as it is a measurement method that is detected by a standard or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen.
  • nephrometry, competition method, immunometric method and sandwich method are preferably used.
  • the sandwich method described later is preferably used.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
  • a sample solution is reacted with the insolubilized detection antibody of the present invention (primary reaction), and another labeled detection antibody of the present invention is reacted (secondary reaction), and then on the insolubilized carrier.
  • primary reaction By measuring the amount or activity of the labeling agent, GPR120 or its partial peptide in the sample solution can be quantified.
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times.
  • the labeling agent and the insolubilization method can be the same as those described above.
  • the antibody used for the immobilized antibody or the labeled antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
  • the detection antibody of the present invention can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
  • a competitive method GPR120 or a partial peptide thereof in a sample solution and labeled GPR120 or a partial peptide thereof are reacted competitively with an antibody, and then an unreacted labeled antigen (F) and a label bound to the antibody.
  • F labeled antigen
  • B B / F separation
  • a soluble antibody is used as an antibody
  • B / F separation is performed using polyethylene glycol or a secondary antibody against the antibody (primary antibody)
  • a solid phase is used as the primary antibody.
  • GPR120 or its partial peptide in the sample solution and the immobilized GPR120 or its partial peptide are allowed to compete with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated.
  • GPR120 or its partial peptide in the sample solution is reacted with an excess amount of labeled antibody, and then immobilized GPR120 or its partial peptide is added to bind the unreacted labeled antibody to the solid phase. Thereafter, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the sample solution. In nephrometry, the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of GPR120 or its partial peptide in the sample solution is small and only a small amount of precipitate is obtained.
  • a measurement system for GPR120 or a partial peptide thereof may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and procedures in each method.
  • a measurement system for GPR120 or a partial peptide thereof may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and procedures in each method.
  • Hiroshi Irie “Radioimmunoassay” Karlsha, published in 1974
  • Hiroshi Irie “Sequel Radioimmunoassay” published in Kodansha, 1979
  • Enzyme Immunoassay edited by Eiji Ishikawa et al.
  • the test compound when the expression level of GPR120 or a partial peptide thereof (mRNA level or protein (peptide level)) in the presence of the test compound is in the absence of the test compound is increased by about 20% or more, preferably about 30% or more, more preferably about 50% or more, the test compound is selected as a candidate for GPR120 expression promoting substance, and therefore, for EPC differentiation / proliferation promoting substance. can do.
  • the expression level of the protein or peptide in the presence of the test compound was reduced by about 20% or more, preferably about 30% or more, more preferably about 50% or more, compared to the case in the absence of the test compound.
  • the test compound can be selected as a GPR120 expression-suppressing substance, and thus a candidate for EPC differentiation / proliferation inhibition and / or an EPC increasing substance in peripheral blood.
  • a substance that promotes the activity of GPR120 obtained by the screening method of (1a) (a compound that enhances the binding property between an agonist of GPR120 or GPR120 and a ligand), and the expression of GPR120 obtained by the screening method of (1b) above.
  • the substance that promotes promotes differentiation from bone marrow cells to EPC and proliferation of EPC. Therefore, prevention and / or treatment of diseases that can have preventive and / or therapeutic effects by enhancing the ability of angiogenesis through the action of EPC Useful as a medicine.
  • Such diseases include, for example, diabetes and its complications (eg, neuropathy, nephropathy, foot lesions, blood flow disorders), obesity, arteriosclerosis, obstructive arteriosclerosis, Buerger's disease, pulmonary hypertension, myocardium Examples include infarction, cerebral infarction, hepatitis, cirrhosis, chronic obstructive pulmonary disease, wound, bone fracture, osteoporosis, periodontal disease, dementia and the like. Furthermore, these substances can also be used for the purpose of increasing the number of mononuclear cells that can be collected in autologous peripheral blood mononuclear cell transplantation or promoting the differentiation of transplanted bone marrow cells in bone marrow transplant patients.
  • a substance that suppresses the activity of GPR120 obtained by the screening method of (1a) (a compound that decreases the binding property between an agonist of GPR120 or GPR120 and a ligand), and GPR120 obtained by the screening method of (1b) above. Since the substance that suppresses the expression can increase the number of EPCs in peripheral blood, prevention and / or therapeutic effect can be obtained by enhancing the ability of angiogenesis through the action of EPC. • It is equally useful as a therapeutic agent. Furthermore, these substances can also be used for the purpose of increasing the number of mononuclear cells that can be collected in autologous peripheral blood mononuclear cell transplantation.
  • a substance that suppresses the activity or expression of GPR120 suppresses differentiation from bone marrow cells to EPC and proliferation of EPC, it has a preventive and / or therapeutic effect by suppressing the ability of angiogenesis through the action of EPC. It is useful as a preventive / therapeutic agent for diseases that can be obtained, for example, chronic inflammation such as cancer, diabetic retinopathy, rheumatism, osteoarthritis and the like.
  • GPR120 modulator A drug containing a substance that regulates (enhances or suppresses) the expression or activity of GPR120 obtained by the screening method of the present invention (hereinafter sometimes referred to as “GPR120 modulator”) has low toxicity and is used as a solution as it is or Suitable pharmaceutical forms of pharmaceutical compositions include oral or parenteral (eg, intravascular) for humans or mammals (eg, mice, rats, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.) Administration, subcutaneous administration, etc.).
  • parenteral eg, intravascular
  • mammals eg, mice, rats, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.
  • Administration subcutaneous administration, etc.
  • the GPR120 modulator may be administered per se, or may be administered as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for administration may contain a GPR120 modulator and a pharmacologically acceptable carrier, diluent or excipient.
  • Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.
  • injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included.
  • Such an injection can be prepared according to a known method.
  • the GPR120 modulator can be prepared by dissolving, suspending or emulsifying in a sterile aqueous liquid or oily liquid usually used for injection.
  • an aqueous solution for injection for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)), etc. may be used in combination.
  • alcohol eg, ethanol
  • polyalcohol eg, Propylene glycol, polyethylene glycol
  • nonionic surfactants eg polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like.
  • Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field.
  • a carrier and excipient for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • the above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dosage of the GPR120 modulator.
  • Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories.
  • the GPR120 modulator is usually contained in an amount of 5 to 500 mg per dosage unit form, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.
  • the dosage of the above-mentioned pharmaceutical containing a GPR120 modulator varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for the treatment and prevention of diabetes in adults, the GPR120 modulator Is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably 1 day a day. It is convenient to administer about 3 times by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
  • GPR120 promotes EPC differentiation / proliferation, and thus includes (a) GPR120 or a partial peptide thereof, and (b) a base sequence encoding GPR120 or a partial peptide thereof.
  • Nucleic acids like (c) compounds that enhance the expression of GPR120 and (d) compounds that enhance the activity of GPR120, have 1) an EPC differentiation / proliferation promoter, 2) an angiogenic ability through the action of EPC. It can be used as a prophylactic / therapeutic agent for diseases that can have a prophylactic / therapeutic effect by promotion.
  • Such diseases include, for example, diabetes and its complications (eg, neuropathy, nephropathy, foot lesions, blood flow disorders), obesity, arteriosclerosis, obstructive arteriosclerosis, Buerger's disease, pulmonary hypertension, myocardium Examples include infarction, cerebral infarction, hepatitis, cirrhosis, chronic obstructive pulmonary disease, wound, bone fracture, osteoporosis, periodontal disease, dementia and the like.
  • the substance (a) or (b) is used for the purpose of increasing the number of mononuclear cells that can be collected in autologous peripheral blood mononuclear cell transplantation or promoting the differentiation of transplanted bone marrow cells in bone marrow transplant patients. You can also
  • GPR120 or a partial peptide thereof when used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the above-mentioned GPR120 modulator, and can be administered to humans or mammals (for example, mice, rats, etc.) with the same administration route and dosage , Rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) orally or parenterally.
  • mammals for example, mice, rats, etc.
  • nucleic acid containing a base sequence encoding GPR120 or a partial peptide thereof When a nucleic acid containing a base sequence encoding GPR120 or a partial peptide thereof is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered according to a method known per se. That is, the nucleic acid is inserted in a functional manner into an appropriate expression vector for mammalian cells such as a retrovirus vector, lentivirus vector, adenovirus vector, adeno-associated virus vector, herpes virus vector, etc. It can be formulated according to the means.
  • the nucleic acid can be administered as it is or together with an auxiliary agent for promoting intake by a gene gun or a catheter such as a hydrogel catheter.
  • the nucleic acid may be formulated (injection) alone or together with a carrier such as a liposome and administered intravenously, subcutaneously, etc. .
  • the nucleic acid containing the base sequence encoding GPR120 or a partial peptide thereof may be administered per se or as an appropriate pharmaceutical composition.
  • the pharmaceutical composition used for administration may contain the nucleic acid and a pharmacologically acceptable carrier, diluent or excipient.
  • Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.
  • injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included.
  • Such an injection can be prepared according to a known method.
  • a method for preparing an injection it can be prepared, for example, by dissolving, suspending or emulsifying the nucleic acid in a sterile aqueous liquid or oily liquid usually used for injection.
  • an aqueous solution for injection for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)), etc. may be used in combination.
  • alcohol eg, ethanol
  • polyalcohol eg, Propylene glycol, polyethylene glycol
  • nonionic surfactants eg polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like.
  • Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field.
  • a carrier and excipient for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • the above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient.
  • dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories.
  • the nucleic acid containing a base sequence encoding GPR120 or a partial peptide thereof is preferably contained, for example, usually 5 to 500 mg per dosage unit dosage form, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms. .
  • the dosage of the above-mentioned pharmaceutical containing a nucleic acid comprising a base sequence encoding GPR120 or a partial peptide thereof varies depending on the administration subject, target disease, symptom, administration route, etc., but for example for the treatment and prevention of diabetes in adults
  • the above nucleic acid is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight per day. It is convenient to administer by intravenous injection about 5 times, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
  • EPC differentiation / proliferation inhibition / EPC increasing agent in peripheral blood GPR120 promotes EPC differentiation / proliferation, and suppression of GPR120 increases the number of EPCs in peripheral blood.
  • a neutralizing antibody against GPR120 or a partial peptide thereof a nucleic acid comprising a base sequence complementary to the base sequence encoding GPR120 or a part thereof, (c) a compound that suppresses the expression of GPR120 or (d ) Similar to compounds that suppress GPR120 activity, 1) EPC differentiation / proliferation inhibitor, 2) Prophylactic / therapeutic agent for diseases that can have preventive / therapeutic effects by inhibiting angiogenic ability through the action of EPC, 3) EPC increasing agent in peripheral blood 4) It can be used as a prophylactic / therapeutic agent for diseases that can be prevented or treated by increasing EPC in peripheral blood.
  • diseases that can be prevented or treated by suppressing angiogenic ability through the action of EPC include chronic inflammation such as cancer, diabetic retinopathy, rheumatism, and osteoarthritis.
  • diseases that can be prevented or treated by increasing EPC in peripheral blood include diabetes and its complications (eg, neuropathy, nephropathy, foot lesions, blood flow disorders), obesity, arteriosclerosis , Obstructive arteriosclerosis, Buerger's disease, pulmonary hypertension, myocardial infarction, cerebral infarction, hepatitis, cirrhosis, chronic obstructive pulmonary disease, wound, fracture, osteoporosis, periodontal disease, dementia and the like.
  • the substance (a) or (b) is used for the purpose of increasing the number of mononuclear cells that can be collected in autologous peripheral blood mononuclear cell transplantation or promoting the differentiation of transplanted bone marrow cells in bone marrow transplant patients. You can also
  • a neutralizing antibody against GPR120 or a partial peptide thereof can be produced in the same manner as the detection antibody of the present invention described above.
  • the antibody preferably a monoclonal antibody
  • the antibody exhibits antigenicity when administered to a human.
  • Antibodies with reduced risk specifically, fully human antibodies, humanized antibodies, mouse-human chimeric antibodies and the like, particularly preferably fully human antibodies or humanized antibodies.
  • Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to conventional methods.
  • Fully human antibodies can be produced from human-human (or mouse) hybridomas, but in order to provide a large amount of antibodies stably and at low cost, human antibody-producing mice and phage display methods are used. It is desirable to manufacture using. Whether or not the antibody against the obtained GPR120 or a partial peptide thereof neutralizes the physiological action of GPR120, that is, the differentiation / proliferation promoting activity of EPC, is determined in the presence of the antibody using, for example, a known colony forming assay. Can be performed by culturing EPC or its progenitor cells and measuring EPC proliferation or EPC differentiation ability.
  • a neutralizing antibody against GPR120 or a partial peptide thereof When a neutralizing antibody against GPR120 or a partial peptide thereof is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the above-mentioned GPR120 modulator, and can be administered to humans or mammals (for example, Mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) orally or parenterally.
  • mammals for example, Mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.
  • base sequence complementary to the base sequence encoding GPR120 or a part thereof is a sequence that can specifically bind to the mRNA of GPR120 and can inhibit the translation of the protein from the mRNA.
  • the length and position are not particularly limited, but from the viewpoint of sequence specificity, a portion complementary or substantially complementary to the target sequence is at least 10 bases or more, preferably about 15 bases or more, more preferably about 20 It contains more than a base.
  • the nucleic acid comprising a base sequence complementary to (or substantially complementary to) the base sequence of mRNA of GPR120 or a part thereof is preferably any of the following (i) to (iii): Illustrated.
  • siRNA against GPR120 mRNA iii) Nucleic acid capable of generating siRNA for GPR120 mRNA
  • Antisense nucleic acid against GPR120 mRNA refers to a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of the mRNA or a part thereof. Thus, it has a function of suppressing protein synthesis by forming a specific and stable double strand with the target mRNA and binding.
  • Antisense nucleic acids are polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages (provided that the polymer is a base such as found in DNA or RNA) And a nucleotide having a configuration that allows attachment of a base).
  • They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.
  • proteins eg, nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lys
  • nucleoside may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles.
  • Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.
  • the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera.
  • the antisense nucleic acid is DNA
  • the RNA DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of GPR120 gene.
  • the GPR120 gene is present in the 10q23.33 region of chromosome 10, so the homology search such as BLAST and FASTA for the genomic sequence of this region and the human GPR120 cDNA base sequence shown in SEQ ID NO: 1 Intron sequences can be determined by comparison using a program.
  • the length of the target region of the antisense nucleic acid of the present invention is not particularly limited as long as the antisense nucleic acid hybridizes, and as a result, translation into the GPR120 protein is inhibited.
  • the entire sequence or partial sequence of mRNA may be a short sequence of about 10 bases, and a long sequence of mRNA or the initial transcript.
  • an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferred, but is not limited thereto.
  • 5 'end hairpin loop of GPR120 gene 5' end 6-base pair repeat, 5 'end untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region , 3 ′ end palindromic region or 3 ′ end hairpin loop, etc. may be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.
  • the antisense nucleic acid of the present invention not only hybridizes with mRNA of GPR120 and the initial transcription product and inhibits translation into proteins, but also binds to these genes that are double-stranded DNAs and binds to triplex (tri-stranded). A plex) and can inhibit transcription to RNA (antigene).
  • the nucleotide molecule constituting the antisense nucleic acid may be natural DNA or RNA, but various chemicals may be used to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA). Modifications can be included.
  • the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate etc. It can be substituted with a phosphate residue.
  • the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R is, for example, CH 3 (2′-O-Me), CH 2 CH 2 OCH 3 (2′-O-MOE), CH 2 CH 2 NHC (NH) NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN and the like may be substituted).
  • R is, for example, CH 3 (2′-O-Me), CH 2 CH 2 OCH 3 (2′-O-MOE), CH 2 CH 2 NHC (NH) NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN and the like may be substituted).
  • the base moiety pyrimidine, purine
  • BNA LNA
  • ENA ENA
  • the antisense oligonucleotide of the present invention determines the target sequence of mRNA or initial transcript based on the cDNA sequence or genomic DNA sequence of GPR120, and is a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman, etc.) ) To synthesize a sequence complementary thereto.
  • any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a method known per se.
  • siRNA against GPR120 mRNA a double-stranded RNA consisting of an oligo RNA complementary to GPR120 mRNA and its complementary strand, so-called siRNA, is also complementary or substantially complementary to the base sequence of GPR120 mRNA. Or defined as encompassed by a nucleic acid containing a portion thereof.
  • RNAi RNA interference
  • siRNA can be appropriately designed using commercially available software (eg, RNAi Designer; Invitrogen) based on the base sequence information of the target mRNA.
  • the ribonucleoside molecule constituting siRNA may also be modified in the same manner as in the above-described antisense nucleic acid in order to improve stability, specific activity and the like.
  • siRNA if all ribonucleoside molecules in natural RNA are replaced with a modified form, RNAi activity may be lost, so the introduction of the minimum modified nucleoside that allows the RISC complex to function is necessary. .
  • the siRNA is synthesized by synthesizing a sense strand and an antisense strand of a target sequence on mRNA with a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. Alternatively, it can be prepared by synthesizing a short hairpin RNA (shRNA) that is a precursor of siRNA and cleaving it with a dicer.
  • shRNA short hairpin RNA
  • nucleic acid capable of generating siRNA for GPR120 mRNA is also complementary to the base sequence of GPR120 mRNA.
  • a nucleic acid comprising a base sequence that is complementary or substantially complementary or a portion thereof.
  • examples of such a nucleic acid include the above-mentioned shRNA and an expression vector constructed so as to express it.
  • shRNA is an oligo containing a base sequence in which the sense and antisense strands of the target sequence on mRNA are linked by inserting a spacer sequence (for example, about 15 to 25 bases) long enough to form an appropriate loop structure.
  • An expression vector containing an shRNA expression cassette can be prepared by preparing a double-stranded DNA encoding the above shRNA by a conventional method and then inserting it into an appropriate expression vector.
  • an shRNA expression vector one having a Pol III promoter such as U6 or H1 can be used.
  • the shRNA transcribed in the animal cell into which the expression vector has been introduced forms a loop by itself, and then is processed by an endogenous enzyme dicer or the like to form a mature siRNA.
  • nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of GPR120 mRNA or a part thereof is a ribozyme capable of specifically cleaving the mRNA within the coding region. It is done. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acids in a narrow sense, but in this specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleavage activity. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known.
  • the hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends (about 10 bases in total) adjacent to the part having the hammerhead structure are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA.
  • This type of ribozyme has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate.
  • the target sequence should be made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase.
  • ribozymes when used in the form of expression vectors containing the DNA that encodes them, they should be hybrid ribozymes in which tRNA-modified sequences are further linked in order to promote the transfer of transcripts to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
  • Nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of GPR120 mRNA or a part thereof is provided in a special form such as liposome or microsphere, applied to gene therapy, or added Can be given in the form of
  • the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( Examples include hydrophobic ones such as phospholipid and cholesterol.
  • Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.).
  • Such can be attached to the 3 ′ or 5 ′ end of the nucleic acid and can be attached via a base, sugar, intramolecular nucleoside bond.
  • the other group include a cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the GPR120 protein expression inhibitory activity of these nucleic acids can be examined using a transformant into which the GPR120 gene has been introduced, an in vivo or in vitro GPR120 gene expression system, or an in vivo or in vitro GPR120 protein translation system.
  • nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of GPR120 mRNA or a part thereof is used as the above preventive / therapeutic agent
  • the base sequence encoding GPR120 or a partial peptide thereof is included. It can be formulated in the same way as a nucleic acid, with a similar route and dose of human or mammal (eg, mouse, rat, rabbit, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc.) Can be administered orally or parenterally.
  • GPR120 is specifically expressed in bone marrow cells and small and large EPCs derived from peripheral blood mononuclear cells, and is differentiated from bone marrow cells and EPCs that are precursor cells of EPC. It is hardly or not expressed in other vascular cells such as cells. Moreover, in mice, GPR120 is highly expressed in large EPC that is further differentiated compared to small EPC. Therefore, by using GPR120 as a surface antigen marker, EPC can be selected from a vascular cell population, and EPC can be further selected into large EPC and small EPC having different differentiation stages.
  • the substance capable of detecting and quantifying GPR120 expressed on the cell surface of EPC is preferably the above-described detection antibody of the present invention.
  • the substance capable of detecting and quantifying the expression of the GPR120 gene in EPC at the RNA level is preferably the above-described detection nucleic acid of the present invention.
  • the present invention also provides an EPC selection reagent comprising (a) an antibody against GPR120 or a partial peptide thereof, or (b) a nucleic acid containing a base sequence encoding GPR120 or a partial polynucleotide thereof.
  • the antibody can be provided in a dissolved state in water or a suitable buffer (eg, phosphate buffered saline).
  • the nucleic acid can be provided as a solid in a dry state or in a state of alcohol precipitation, or can be provided in a state dissolved in water or a suitable buffer (eg, TE buffer).
  • a suitable buffer eg, TE buffer
  • the antibody and the nucleic acid can be provided in a state of being previously labeled with a labeling substance, or can be provided separately from the labeling substance, and can be used after labeling.
  • the cell-containing sample is not particularly limited as long as it contains a vascular system cell population, and examples thereof include peripheral blood, umbilical cord blood, and bone marrow fluid.
  • the EPC can be selected by labeling the above-described selection reagent by a conventional method (eg, fluorescent labeling using FITC, etc.), and separating GPR120 positive cells and negative cells using a cell sorter. Further, large EPC and small EPC can be selected by comparing the expression levels of GPR120. The selected EPC fraction can be collected and used, for example, for EPC transplantation therapy.
  • GPR120 is specifically expressed in small and large EPCs derived from bone marrow cells and peripheral blood mononuclear cells.
  • the amount of EPC contained in the sample can be evaluated by measuring the amount of protein or mRNA of GPR120 in the sample containing.
  • the cell-containing sample derived from the subject animal as the sample is not particularly limited as long as it contains a vascular system cell population, and examples thereof include peripheral blood, umbilical cord blood, and bone marrow fluid.
  • the detection target is mRNA encoding GPR120
  • the total RNA or poly (A) + RNA fraction may be extracted and purified from the collected peripheral blood or the like using a conventional method.
  • the expression level of GPR120 in a sample can be examined by detecting the protein or mRNA encoding the same using a conventionally known method. For example, when GPR120 protein is detected, gel electrophoresis (eg, SDS-PAGE, two-dimensional gel electrophoresis, etc.) and various separation and purification methods (eg, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography).
  • gel electrophoresis eg, SDS-PAGE, two-dimensional gel electrophoresis, etc.
  • separation and purification methods eg, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography.
  • the GPR120 protein is detected and quantified by various immunoassays and Western blot analysis described in detail in the above-described screening for substances that regulate the expression of GPR120, using the detection antibody of the present invention. It is also possible to measure the number of EPCs in a sample by performing immunohistochemical staining using a fluorescently labeled antibody without homogenizing the cells in the sample and measuring the number of stained cells under a fluorescence microscope. it can.
  • the antibody is immobilized on the surface of a probe that can be adapted to a mass spectrometer described later, the test sample is brought into contact with the antibody on the probe.
  • Examples include a method in which a component captured by an antibody is subjected to mass spectrometry to detect a peak of GPR120 protein.
  • a chromatographic carrier eg, cation exchanger, anion exchanger, hydrophobic chromatographic carrier
  • TOF-MS time-of-flight mass spectrometry
  • the washing can be performed using water or a buffer solution.
  • the pH is appropriately selected according to the isoelectric point of the target protein.
  • a Tris-HCl buffer solution having a pH of 4 to 8, a phosphate buffer solution, a borate buffer solution, an acetate buffer solution, or the like is used.
  • Mass spectrometry is preferably performed by a MALDI method using an appropriate matrix (MALDI-TOFMS).
  • matrix molecules used include sinapinic acid (SPA), saturated 2,5-dihydroxybenzoic acid (DHB), and indole acrylic. Examples include acid (IAA) and cinnamic acid.
  • a nucleic acid that can hybridize with the mRNA under highly stringent conditions, a part of the mRNA, It can be carried out by Northern hybridization, RT-PCR, etc., using a set of oligonucleotides (that is, the above-described detection nucleic acid of the present invention) that can function as a primer for amplifying the whole.
  • the nucleic acid used as the probe may be DNA or RNA, or may be a DNA / RNA chimera. Preferably, DNA is used.
  • the nucleic acid may be double-stranded or single-stranded.
  • a double strand it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid.
  • the length of the nucleic acid is not particularly limited as long as it can specifically hybridize with the target mRNA, and is, for example, about 15 bases or more, preferably about 30 bases or more.
  • the nucleic acid is preferably labeled with a labeling agent in order to enable detection and quantification of the target mRNA.
  • a labeling agent for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used.
  • the radioisotope for example, [ 32 P], [ 3 H], [ 14 C] and the like are used.
  • the enzyme those which are stable and have high specific activity are preferable.
  • ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • biotin- (strept) avidin can also be used for binding between the probe and the labeling agent.
  • the set of oligonucleotides used as primers can specifically hybridize with the base sequence (sense strand) of mRNA encoding GPR120 and the base sequence complementary to it (antisense strand).
  • the DNA fragment is not particularly limited as long as it can amplify the DNA fragment.
  • each DNA fragment has a length of about 15 to about 100 bases, preferably about 15 to about 50 bases, and about 100 bp to several kbp.
  • RT-PCR is a method in which a known amount of another template nucleic acid that can be amplified by a set of primers that can amplify the target DNA is used as a competitor in the reaction solution to cause an amplification reaction competitively.
  • an amplification product that is amplified by the primer set and can be distinguished from the target DNA for example, an amplification product having a different size from the target DNA, a restriction enzyme
  • Nucleic acids that produce amplification products that exhibit different migration patterns upon processing can be further included.
  • the competitor nucleic acid may be DNA or RNA.
  • PCR may be performed by adding a competitor after synthesizing cDNA from an RNA sample by a reverse transcription reaction.
  • RT-PCR can be performed by adding it to the RNA sample from the beginning.
  • the efficiency of the reverse transcription reaction is taken into consideration, so that the absolute amount of the original mRNA can be estimated.
  • real-time RT-PCR can monitor the amount of PCR amplification in real time, electrophoresis is unnecessary and GPR120 gene expression can be analyzed more quickly. Usually, monitoring is performed using various fluorescent reagents.
  • a nucleic acid that can be used as the probe in addition to a reagent (intercalator) that emits fluorescence by binding to double-stranded DNA such as SYBR Green I and ethidium bromide, a nucleic acid that can be used as the probe (provided that the nucleic acid is an amplification region) In which both ends of the target nucleic acid are modified with a fluorescent substance (eg, FAM, HEX, TET, FITC, etc.) and a quencher (eg, TAMRA, DABCYL, etc.).
  • a fluorescent substance eg, FAM, HEX, TET, FITC, etc.
  • a quencher eg, TAMRA, DABCYL, etc.
  • the nucleic acid used as the probe may be a cDNA encoding GPR120 or a fragment thereof, or based on the base sequence information (for example, the base sequence shown in SEQ ID NO: 1 in the case of human GPR120), It may be obtained by chemically synthesizing using an automatic RNA synthesizer or the like.
  • the oligonucleotide set used as the primer may be chemically synthesized using a commercially available DNA / RNA automatic synthesizer or the like based on the base sequence information. Can be obtained by:
  • EPC Using EPC with various known cell numbers, measure the protein amount or mRNA amount of GPR120 by any of the above measurement methods, create a calibration curve, and compare the measured values in EPC-containing samples with unknown cell numbers Thus, EPC in the sample can be quantified.
  • EPC measurement method it is possible to diagnose the presence or severity of a disease associated with abnormalities in EPC amount such as diabetes, arteriosclerosis, brain / cardiovascular disease, etc. in a test animal. . That is, when the expression charge of GPR120 in a sample collected from a test animal is measured, the EPC amount is calculated, and compared with that of a normal animal, the EPC amount is significantly reduced compared to that of a normal animal.
  • EPC levels are diseases associated with abnormal EPC levels, such as diabetes and its complications (eg, neuropathy, nephropathy, foot lesions, blood flow disorders), obesity, arteriosclerosis, obstructive arteriosclerosis, Buerger's disease, It can be diagnosed that the patient is suffering from pulmonary hypertension, myocardial infarction, cerebral infarction, hepatitis, cirrhosis, chronic obstructive pulmonary disease, wound, fracture, osteoporosis, periodontal disease, dementia, etc. .
  • Bone marrow cells were isolated from mice according to the original paper (Tamarat R et al., Am J Pathol. 2004; 164: 457-466.) did. Bone marrow cells were extruded from the femur and tibia of the left lower limb of male C57BL / 6J mice with HBSS medium (Gibco) containing 1% FBS. Bone marrow cells were collected by centrifugation, resuspended in EDTA-PBS, removed the mass through a cell strainer, and centrifuged again.
  • HBSS medium Gibco
  • Hemolysis was performed by resuspension with 0.8% ammonium chloride. After centrifugation, the cells were suspended in IMDM (Gibco) and the number of cells was counted.
  • the culture method was modified from the original paper (Masuda H et al., Circ Res. 2007; 101 (6): 598-606., 71st Annual Meeting of the Japanese Circulation Society OJ-063-H). It was.
  • MethoCult SF M3236 (Stemcell technologies) containing 10% FBS and nutrient factors was used (20 ng / ml stem cell factor (SCF), 50 ng / ml vascular endothelial growth factor (VEGF), 20 ng / ml interleukin-3 (IL-3), 50 ng / ml basic fibroblast growth factor (bFGF), 50 ng / ml epidermal growth factor receptor (EGFR), 50 ng / ml insulin-like growth factor-1, (IGF-1) 2 U / ml heparin).
  • SCF stem cell factor
  • VEGF vascular endothelial growth factor
  • IL-3 interleukin-3
  • bFGF basic fibroblast growth factor
  • EGFR epidermal growth factor receptor
  • IGF-1 insulin-like growth factor-1
  • the cell suspension was placed in MethoCult SF medium and mixed well so that the cell density was 3 ⁇ 10 4 cells / dish, seeded in 1 mL each in a 35 mm dish, and cultured for 8 days.
  • Cells cultured after 8 days were reacted with 0.4 ⁇ g / mL acLDL-DiI and 2 ⁇ g / mL FITC-conjugated BS-1 lectin, fixed with 2% PFA, washed with PBS, and observed under a fluorescence microscope.
  • DiI-LDL uptake and BS-1 lectin staining were confirmed, confirming that it was EPC.
  • Reference Example 2 Culture of mouse peripheral blood mononuclear cells by EPC colony forming assay of mouse peripheral blood mononuclear cells
  • Original paper Kerka C et al., Proc Natl Acad Sci US A. 2000; 97: 3422- 3427.
  • peripheral blood mononuclear cells were isolated from mice. Blood collected from the carotid artery of male C57BL / 6J mice was diluted 2-fold with physiological saline, then double-diluted blood was layered on 1/2 vol of Histopaque 1088 (Sigma) for blood cell separation, and concentration gradient centrifugation was performed. .
  • Mononuclear cells collected at the interface were collected, suspended in PBS / EDTA, collected by centrifugation, then hemolyzed by resuspension with 0.8% ammonium chloride, centrifuged again, and the cells were suspended in IMDM. Counted the number.
  • the culture by the colony forming assay was performed according to Reference Example 1. The seeding density of mononuclear cells in peripheral blood was 7 ⁇ 10 5 cells / dish.
  • Human cell culture medium was MethoCult SF H4236 (Stemcell technologies) containing 30% FBS and nutrient factors (20 ng / ml stem cell factor (SCF), 50 ng / ml vascular endothelial growth factor (VEGF), 20 ng / ml Interleukin-3 (IL-3), 50 ng / ml basic fibroblast growth factor (bFGF), 50 ng / ml epidermal growth factor receptor (EGFR), 50 ng / ml insulin-like growth factor-1, (IGF -1) 2 U / ml heparin).
  • SCF stem cell factor
  • VEGF vascular endothelial growth factor
  • IL-3 Interleukin-3
  • bFGF basic fibroblast growth factor
  • EGFR epidermal growth factor receptor
  • IGF -1) 2 U / ml heparin
  • Human CD133 positive bone marrow cells were suspended in IMDM, mixed well in MethoCult SF medium so that the cell density would be 1 ⁇ 10 3 cells / dish, seeded in 1 mL each in a 35 mm dish, and cultured for 21 days. After 21 days, when observing under a microscope ( ⁇ 40 lens), two types of colonies are formed in the dish. A colony composed of cells having a small cell size was determined as small EPC.
  • Mouse bone marrow cells, mouse peripheral blood mononuclear cells, and human CD133-positive bone marrow derived large EPC and small EPC RNA extraction Using colony forming assay, mouse bone marrow cells or peripheral blood mononuclear cells, and Human CD133 positive bone marrow cells were cultured, and differentiated small EPCs were collected using a pipette under a microscope. Small EPC was washed with PBS and then dissolved with RLT buffer. The culture dish was washed with PBS, RLT buffer was added to large EPC that was adhered, and the cells were scraped and lysed with a cell scraper, and RNA of each cell was extracted using RNeasy Mini Kit (Qiagen).
  • Reference Example 5 Setting up an expression analysis system for GPR120 using TaqMan PCR TaqMan PCR conditions were set in order to perform detailed expression analysis of GPR120 in mouse cells.
  • the primers used were Forward Primer (5'-TCCGAGTGTCCCAACAAGACTAC-3 '; SEQ ID NO: 7) and Reverse Primer (5'-GACTCCACATGATGAAGAAGGAAA-3'; SEQ ID NO: 8), and the probe used for detection was 5'-Fam-CCGCACGCTCTTCCTGCTCATG- Tamra (SEQ ID NO: 9).
  • TaqMan PCR is mixed with TaqMan Universal Mixture and cDNA synthesized using Forward Primer 900 nM, Reverse Primer 900 nM, Probe 250 nM, and 25 ng of total RNA as a template, and 40 cycles of 95 ° C for 15 seconds and 60 ° C for 60 seconds.
  • the mouse GPR120 gene was quantified by real-time PCR. Standard calibration (CCAGATCCGAGTGTCCCAACAAGACTACCGACTCTTCCGCACGCTCTTCCTGCTCATGGTTTCCTTCTTCATCATGTGGAGTCCCATC; SEQ ID NO: 10) was used to draw a standard curve and quantified, and a typical standard curve pattern is shown in FIG. 2A.
  • TaqMan PCR conditions were set for detailed expression analysis of GPR120 in human cells.
  • the primers used were Forward Primer (5'-GCGCCGACCAGGAAATTT-3 '; SEQ ID NO: 11) and Reverse Primer (5'-CAAAAGAGACATCCCACGAGATC-3'; SEQ ID NO: 12), and the probe used for detection was 5'-Fam-TTGCACACTGATTTGGCCCACCATT- Tamra (SEQ ID NO: 13).
  • TaqMan PCR is mixed with TaqMan Universal Mixture and cDNA synthesized using Forward Primer 900 nM, Reverse Primer 900 nM, Probe 250 nM, and 25 ng of total RNA as a template, and 40 cycles of 95 ° C for 15 seconds and 60 ° C for 60 seconds.
  • the human GPR120 gene was quantified by real-time PCR.
  • a standard oligo (CCCCGGCGCCGACCAGGAAATTTCGATTTGCACACTGATTTGGCCCACCATTCCTGGAGAGATCTCGTGGGATGTCTCTTTTGTTACT; SEQ ID NO: 14) was drawn and quantified, and a typical calibration curve pattern is shown in FIG. 2B.
  • Example 1 Examination of GPR120 mRNA Expression in Mouse Bone Marrow Cell-Derived and Peripheral Blood Mononuclear Cell-Derived EPC
  • Mouse bone marrow cells were cultured by EPC colony forming assay as in Reference Examples 1, 4, and 5 and differentiated large EPC And small EPC were isolated, and the expression level of GPR120 mRNA in mouse bone marrow cell-derived EPC was quantified using the TaqMan PCR method.
  • FIG. 3 GPR120 was found to be low in mouse bone marrow cells and highly expressed in bone marrow cell-derived EPCs. In mice, higher expression of GPR120 was observed in large EPC than in small EPC.
  • Mouse peripheral blood mononuclear cells are cultured by EPC colony forming assay as in Reference Examples 2, 4 and 5, and differentiated large EPC and small EPC are isolated.
  • the expression level of GPR120 mRNA in was quantified using the TaqMan PCR method. As a result, as shown in FIG. 3, it was found that GPR120 was not expressed in mouse peripheral blood mononuclear cells, but was expressed in peripheral blood mononuclear cell-derived EPCs.
  • mouse peripheral blood mononuclear cells showed higher expression of GPR120 in large EPCs than in small EPCs.
  • Example 2 Examination of expression of GPR120 mRNA in EPC derived from human CD133-positive bone marrow cells Human CD133-positive bone marrow cells were cultured by EPC colony forming assay as in Reference Examples 3, 4 and 5, and differentiated large EPC and small EPC By isolation, the expression level of GPR120 mRNA in human CD133-positive bone marrow cell-derived EPC was quantified using the TaqMan PCR method. As a result, as shown in FIG. 4, GPR120 is not expressed in human CD133-positive bone marrow cells or aortic endothelial cells (HAEC, KURABO), and is specific only in EPC derived from human CD133-positive bone marrow cells. It was found that
  • Example 3 Examination of differentiation ability of bone marrow cells into large EPC in GPR120 KO mice GPR120 KO mice were subjected to EPC colony forming assay of bone marrow cells, and the number of differentiated large EPC colonies and small EPC colonies were counted. We evaluated the ability of bone marrow cells to differentiate into large EPCs in KO mice. Bone marrow cells were collected from 12-week-old male GPR120 KO mice as described in Reference Example 1 and subjected to EPC colony forming assay.
  • Example 4 Examination of EPC Count in Peripheral Blood in GPR120 KO Mouse Using GPR120 KO mouse, EPC differentiation assay of peripheral blood mononuclear cells was performed, and EPC count in peripheral blood mononuclear cells in GPR120 KO mice Evaluation was performed.
  • Peripheral blood mononuclear cells were collected from 16-week-old male GPR120 KO mice as described in Reference Example 2, and the cells were collected as EGM2 (EBM-2 / 5% FCS / EGM-2 supplemented factor medium, Cambrex). And suspended in a fibronectin-coated 96-well plate at 1 ⁇ 10 5 cells / well.
  • GHB was significantly increased in the GPR120 KO mice although there was no change in body weight as compared to the wild type C57BL / 6J. Plasma glucose concentration tended to increase in GPR120 KO mice, and a significant increase was observed at 12 and 18 weeks of age.
  • a substance that regulates EPC differentiation and proliferation and a substance that increases the number of EPCs in peripheral blood can be selected. Therefore, prevention and treatment can be achieved by regulating angiogenic ability via EPC. It is useful for searching for candidate compounds for prophylactic / therapeutic agents for various diseases that can be effective. Since GPR120 can be used as a surface antigen marker for EPC, it is useful for EPC selection / quantification and diagnosis of diseases associated with abnormal EPC levels.

Abstract

Disclosed are: the identification of a molecule which can be expressed specifically in an endothelial progenitor cell (EPC) and can influence on the differentiation/proliferation of an EPC, the recruitment of an EPC into a peripheral blood or the like; a method for the screening of a modulator for the molecule; a means for the prevention/treatment of a disease associated with the dysfunction of an EPC by using the modulator; a method for the selection/quantification of an EPC by employing the molecule as an indicator; and a method for the diagnosis of an EPC-related disease. Specifically disclosed are: a method for the screening of a substance capable of modulating the differentiation/proliferation of an EPC or increasing the quantity of an EPC in a peripheral blood, which is characterized by using GPR120 or a partial peptide thereof or a cell capable of producing the protein or the partial peptide thereof; and a method for determining the quantity of an EPC in a cell-containing sample collected from a mammal, which is characterized by measuring GPR120 protein or the expression of a gene for GPR120 protein in the sample.

Description

スクリーニング方法Screening method
　本発明は、血管内皮前駆細胞の分化・増殖調節に関与する物質の新規スクリーニング方法等に関する。 The present invention relates to a novel screening method for substances involved in the regulation of differentiation / proliferation of vascular endothelial precursor cells.
（発明の背景）
　血管内皮前駆細胞（Endothelial Progenitor Cell：以下、「EPC」ともいう）は骨髄由来の細胞であって、骨髄から末梢血へ動員されて単核球分画の一部として体内を循環し、血管形成部位への生着、当該部位での増殖・分化・遊走を経て、新規な血管発生（vasculogenesis）に関与している（非特許文献１および２参照）。通常、EPCは常に一定の割合で骨髄から末梢へと動員されており、生体に何らかの刺激がない限り、末梢血EPCと骨髄EPCは数的に平衡状態を保っている。しかし、組織が虚血に陥ると、種々のサイトカインや成長因子、ホルモンなどの作用によってEPCが骨髄から動員され、末梢血中のEPC数が増大する。この機序を利用して、冠動脈疾患や下肢虚血疾患を含む重症虚血性疾患に対する自己EPC移植治療の臨床試験が開始され、良好な成績が報告されつつある。その反面、糖尿病に代表される基礎疾患により患者のEPCの量及び質が低下し、移植しても十分な治療効果が得られないという問題も指摘されている。 (Background of the Invention)
Endothelial progenitor cells (hereinafter also referred to as “EPC”) are bone marrow-derived cells that are mobilized from the bone marrow to peripheral blood and circulate in the body as part of the mononuclear cell fraction to form blood vessels. It is involved in new vasculogenesis through engraftment at the site and proliferation / differentiation / migration at the site (see Non-Patent Documents 1 and 2). Normally, EPC is always mobilized from the bone marrow to the periphery at a constant rate, and the peripheral blood EPC and bone marrow EPC are numerically balanced unless there is any stimulus in the living body. However, when a tissue falls into ischemia, EPC is mobilized from the bone marrow by the action of various cytokines, growth factors, hormones, etc., and the number of EPCs in peripheral blood increases. Utilizing this mechanism, clinical trials of autologous EPC transplantation treatment for severe ischemic diseases including coronary artery disease and limb ischemia disease have been started and good results are being reported. On the other hand, it has been pointed out that the amount and quality of EPC of patients are reduced due to basic diseases such as diabetes, and a sufficient therapeutic effect cannot be obtained even after transplantation.
　骨髄でのEPCへの分化能の低下や、末梢血へのEPCの動員能・病変部位でのEPCの定着・増殖能の低下が、種々の疾患と関連することが報告されている。たとえば、糖尿病マウスでは、骨髄細胞からEPCへの分化能が低下していること（非特許文献３参照）、糖尿病患者由来の末梢血中EPC数は健常人に比して減少しており、EPC数とHbA1cが逆相関すること（非特許文献４および５参照）が知られている。また、虚血性心疾患の患者ではEPC数や血管新生能が低下すること（非特許文献６参照）、血中EPC数が減少すると心血管イベント後の死亡率が上昇すること（非特許文献７参照）、骨髄細胞や前駆細胞の低下が、動脈硬化症の進行に寄与すること（非特許文献８参照）などが知られている。さらに、脳梗塞患者において血中EPC数が減少していること（非特許文献９参照）、肝硬変モデル動物にEPCを移植すると生存率が上昇すること（非特許文献１０参照）、骨髄細胞を移植することにより肝臓の繊維化が抑制されること（非特許文献１１参照）などが報告されている。 It has been reported that a decrease in the ability to differentiate into EPC in the bone marrow, an ability to mobilize EPC to peripheral blood, and a decrease in the ability to establish and proliferate EPC at the lesion site are associated with various diseases. For example, in diabetic mice, the ability to differentiate from bone marrow cells into EPC is reduced (see Non-Patent Document 3), and the number of EPCs in peripheral blood derived from diabetic patients is reduced compared to healthy individuals. It is known that the number and HbA1c are inversely correlated (see Non-Patent Documents 4 and 5). In patients with ischemic heart disease, the number of EPCs and angiogenic ability are decreased (see Non-Patent Document 6), and the mortality after a cardiovascular event is increased when the blood EPC number is decreased (Non-Patent Document 7). It is known that a decrease in bone marrow cells and progenitor cells contributes to the progression of arteriosclerosis (see Non-Patent Document 8). Furthermore, the number of EPCs in blood is reduced in patients with cerebral infarction (see Non-Patent Document 9), the survival rate increases when EPC is transplanted into a cirrhosis model animal (see Non-Patent Document 10), and bone marrow cells are transplanted. It has been reported that the fibrosis of the liver is suppressed (see Non-Patent Document 11).
　したがって、いったん低下した、骨髄由来のEPCの機能を回復させることができれば、糖尿病による臓器障害や、循環障害、創傷、肝硬変などの種々の疾患を改善させ得ることが期待されている。しかしながらEPCの機能を改善しうる因子（例えば、骨髄細胞からEPCへの分化を促進する因子や、骨髄から末梢血へのEPCの動員を促進する因子など）については、ほとんど知られていないというのが現状である。 Therefore, it is expected that once the decreased function of EPC derived from bone marrow can be recovered, various diseases such as organ damage due to diabetes, circulatory disorder, wound, and cirrhosis can be improved. However, little is known about factors that can improve EPC function (for example, factors that promote differentiation of bone marrow cells into EPC and factors that promote the recruitment of EPC from bone marrow to peripheral blood). Is the current situation.
　GPR120は、下垂体、腸管、脂肪組織などで高発現するＧ蛋白質共役型受容体（G protein-coupled receptor：GPCR）である。特許文献１には、GPR120のリガンドが脂肪酸であることが記載され、また、GPR120と当該リガンドとを用いたアゴニスト／アンタゴニストのスクリーニング法、アゴニスト等（GPR120、ＤＮＡ、リガンド）／アンタゴニスト等（中和抗体、アンチセンス、発現抑制化合物）の医薬用途（脂肪細胞からのグリセロール生成抑制／促進、血中グリセロール低下／上昇、脂肪分解抑制／促進、インスリン抵抗抑制／促進、成長ホルモン分泌抑制／促進、ストレス調節、副腎皮質刺激ホルモン（ACTH）分泌抑制／促進）等が記載されている。特許文献２には、GPR120に対してアゴニスト活性を有する化合物が開示され、動脈硬化、狭心症、心筋梗塞、循環器疾患などの予防・治療に有用であることが記載されている。また、脂肪前駆細胞（3T3-L1）において、GPR120の発現が分化に伴って上昇し、ｓｉＲＮＡを用いてGPR120遺伝子をノックダウンすると脂肪細胞への分化が抑制されることから、脂肪細胞の成熟・分化へのGPR120の関与が示唆されている（非特許文献１２）。
　一方、腸内分泌細胞（STC-1）においてGPR120をsiRNAでノックダウンすると、遊離脂肪酸により誘発されるGLP-1産生が低下することから、腸管においては、GPR120アゴニストは、GLP-1の産生を刺激して肥満抑制作用を示すことが示唆されている（非特許文献１３）。 GPR120 is a G protein-coupled receptor (GPCR) that is highly expressed in the pituitary gland, intestinal tract, adipose tissue, and the like. Patent Document 1 describes that the ligand of GPR120 is a fatty acid, and also screens for agonists / antagonists using GPR120 and the ligand, agonists, etc. (GPR120, DNA, ligand) / antagonists, etc. (neutralization) Antibody, antisense, expression-inhibiting compound) for pharmaceutical use (suppression / promotion of glycerol production from adipocytes, reduction / increase of blood glycerol, suppression / promotion of lipolysis, suppression / promotion of insulin resistance, suppression / promotion of growth hormone secretion, stress Regulation, adrenocorticotropic hormone (ACTH) secretion suppression / promotion) and the like. Patent Document 2 discloses a compound having agonist activity with respect to GPR120, and describes that it is useful for the prevention and treatment of arteriosclerosis, angina pectoris, myocardial infarction, cardiovascular disease and the like. In addition, GPR120 expression increases with differentiation in adipose precursor cells (3T3-L1), and when GPR120 gene is knocked down using siRNA, differentiation into adipocytes is suppressed. Involvement of GPR120 in differentiation has been suggested (Non-patent Document 12).
On the other hand, when GPR120 is knocked down by siRNA in enteroendocrine cells (STC-1), GLP-1 production induced by free fatty acids decreases, so in the intestine, GPR120 agonists stimulate GLP-1 production. It has been suggested that it exhibits an obesity-suppressing action (Non-patent Document 13).
　しかしながら、EPCにおけるGPR120の発現や、骨髄細胞からEPCへの分化におけるGPR120の関与については、全く報告がなされていない。 However, there is no report on the expression of GPR120 in EPC or the involvement of GPR120 in the differentiation from bone marrow cells to EPC.
国際公開2004/065960号パンフレットInternational Publication No. 2004/065960 Pamphlet 国際公開2005/051373号パンフレットInternational Publication 2005/051373 Pamphlet
　したがって、本発明の目的は、EPCで特異的に発現し、その分化及び機能に重要な役割を担う分子を同定し、当該分子を創薬ターゲットとしてEPCの分化・増殖あるいは末梢血への動員等を調節する物質を探索する方法を提供することである。また、本発明の別の目的は、当該分子の調節薬を用いてEPCの機能低下が関連する種々の疾患を予防・治療する手段を提供することである。本発明のさらに別の目的は、当該分子を指標としたEPCの選別・定量方法、並びにEPCが関連する疾患の診断方法を提供することである。 Therefore, the object of the present invention is to identify a molecule that is specifically expressed in EPC and plays an important role in its differentiation and function, and uses the molecule as a drug discovery target for EPC differentiation / proliferation or mobilization to peripheral blood, etc. It is to provide a method for searching for a substance that regulates the above. Another object of the present invention is to provide means for preventing / treating various diseases associated with a decrease in EPC function using a modulator of the molecule. Still another object of the present invention is to provide a method for selecting and quantifying EPC using the molecule as an index, and a method for diagnosing a disease associated with EPC.
　上記の目的を達成すべく、本発明者らはまず、マウスにおいて、分化段階の異なる2種のEPC（large EPCとsmall EPC）における種々の受容体（GPCR、チロシンキナーゼ型受容体、核内受容体）遺伝子の発現を調べ、2種のEPC間で発現量が大きく変化する遺伝子群を抽出した。本発明者らは、これらのうち、large EPCで最も発現が増加したGPR120に着目し、さらに検討を重ねた。その結果、GPR120はマウス骨髄細胞由来および末梢血中単核球由来のsmall及びlarge EPC、並びにヒト骨髄細胞由来のsmall及びlarge EPCで特異的に発現し、骨髄細胞や内皮細胞では発現していないことを見出した。また、GPR120ノックアウト（KO）マウス由来骨髄細胞のコロニーフォーミングアッセイの結果、GPR120が骨髄細胞からlarge EPCへの分化に関与していることが明らかとなった。さらに、比較的高週齢のGPR120 KOマウスでは骨髄由来EPC数が野生型マウスに比べて減少する一方、末梢血中のEPC数は野生型マウスに比べて顕著に上昇することを見出した。以上の知見から、GPR120はEPCの特異的マーカーであり、骨髄細胞からEPCへの分化、EPCの増殖、末梢血へのEPCの動員に関与していることが示された。
　本発明者らはこれらの知見に基づいてさらに鋭意研究を行った結果、本発明を完成するに至った。 In order to achieve the above object, the present inventors first made various receptors (GPCR, tyrosine kinase type receptor, nuclear receptor) in two types of EPCs having different differentiation stages (large EPC and small EPC) in mice. We examined gene expression and extracted a group of genes whose expression levels varied greatly between the two EPCs. Of these, the inventors focused on GPR120, the expression of which was most increased in large EPC, and further studied. As a result, GPR120 is specifically expressed in small and large EPCs derived from mouse bone marrow cells and peripheral blood mononuclear cells, and small and large EPCs derived from human bone marrow cells, but not in bone marrow cells or endothelial cells. I found out. In addition, as a result of the colony forming assay of bone marrow cells derived from GPR120 knockout (KO) mice, it was revealed that GPR120 is involved in the differentiation of bone marrow cells into large EPCs. Furthermore, it was found that the number of bone marrow-derived EPCs decreased in GPR120 KO mice of relatively older age compared to wild type mice, while the number of EPCs in peripheral blood was significantly increased compared to wild type mice. These findings indicate that GPR120 is a specific marker for EPC and is involved in the differentiation of bone marrow cells into EPC, proliferation of EPC, and recruitment of EPC to peripheral blood.
As a result of further intensive studies based on these findings, the present inventors have completed the present invention.
　即ち、本発明は、
［１］配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチド、あるいは該蛋白質またはその部分ペプチドを産生する細胞を用いることを特徴とする、血管内皮前駆細胞の分化・増殖調節または末梢血中の血管内皮細胞増加物質のスクリーニング方法、
［２］配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質をコードする核酸またはその部分ポリヌクレオチド、あるいは該蛋白質またはその部分ペプチドに対する抗体をさらに用いることを特徴とする、上記［１］記載の方法、
［３］脂肪酸もしくは脂肪酸と前記蛋白質との結合性を変化させる低分子化合物またはその塩をさらに用いることを特徴とする、上記［１］記載の方法、
［４］細胞が血管内皮前駆細胞またはその前駆細胞である、上記［１］記載の方法、
［５］血管形成能の調節が予防・治療効果を示し得る疾患の予防・治療物質を選択するための、上記［１］記載の方法、
［６］以下のいずれかの物質を含有してなる血管内皮前駆細胞の分化・増殖促進剤、
（ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチド
（ｂ）上記（ａ）の蛋白質またはその部分ペプチドをコードする塩基配列を含む核酸
（ｃ）上記（ａ）の蛋白質の発現を増強する化合物
（ｄ）上記（ａ）の蛋白質の活性を増強する化合物
［７］血管形成能の増強が予防・治療効果を示し得る疾患の予防・治療のための、上記［６］記載の剤、
［８］以下のいずれかの物質を含有してなる血管内皮前駆細胞の分化増殖抑制剤、
（ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する中和抗体
（ｂ）上記（ａ）の蛋白質をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸
（ｃ）上記（ａ）の蛋白質の発現を阻害する化合物
（ｄ）上記（ａ）の蛋白質の活性を阻害する化合物
［９］血管形成能の抑制が予防・治療効果を示し得る疾患の予防・治療のための、上記［７］記載の剤、
［１０］以下のいずれかの物質を含有してなる末梢血中の血管内皮前駆細胞増加剤、
（ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する中和抗体
（ｂ）上記（ａ）の蛋白質をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸
（ｃ）上記（ａ）の蛋白質の発現を阻害する化合物
（ｄ）上記（ａ）の蛋白質の活性を阻害する化合物
［１１］以下のいずれかの物質を含有してなる血管内皮前駆細胞の選別試薬、
（ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する抗体
（ｂ）上記（ａ）の蛋白質をコードする核酸またはその部分ポリヌクレオチド
［１２］細胞含有試料に上記［１１］記載の試薬を接触させることを特徴とする、該試料中の血管内皮前駆細胞の選別方法、
［１３］哺乳動物から採取した細胞含有試料における、配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその遺伝子の発現を測定することを特徴とする、該試料中の血管内皮前駆細胞量の測定方法、
［１４］血管内皮前駆細胞量の異常が関連する疾患の診断のための、上記［１３］記載の方法、［１５］哺乳動物に対して、以下のいずれかの物質を投与することを特徴とする血管内皮前駆細胞の分化・増殖促進方法、
（ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチド
（ｂ）上記（ａ）の蛋白質またはその部分ペプチドをコードする塩基配列を含む核酸
（ｃ）上記（ａ）の蛋白質の発現を増強する化合物
（ｄ）上記（ａ）の蛋白質の活性を増強する化合物
［１６］血管内皮前駆細胞の分化・増殖促進剤を製造するための、以下のいずれかの物質の使用、
（ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチド
（ｂ）上記（ａ）の蛋白質またはその部分ペプチドをコードする塩基配列を含む核酸
（ｃ）上記（ａ）の蛋白質の発現を増強する化合物
（ｄ）上記（ａ）の蛋白質の活性を増強する化合物
［１７］哺乳動物に対して、以下のいずれかの物質を投与することを特徴とする血管内皮前駆細胞の分化増殖抑制方法、
（ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する中和抗体
（ｂ）上記（ａ）の蛋白質をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸
（ｃ）上記（ａ）の蛋白質の発現を阻害する化合物
（ｄ）上記（ａ）の蛋白質の活性を阻害する化合物
［１８］血管内皮前駆細胞の分化・増殖抑制剤を製造するための、以下のいずれかの物質の使用、
（ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する中和抗体
（ｂ）上記（ａ）の蛋白質をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸
（ｃ）上記（ａ）の蛋白質の発現を阻害する化合物
（ｄ）上記（ａ）の蛋白質の活性を阻害する化合物
［１９］哺乳動物に対して、以下のいずれかの物質を投与することを特徴とする末梢血中の血管内皮前駆細胞増加方法、
（ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する中和抗体
（ｂ）上記（ａ）の蛋白質をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸
（ｃ）上記（ａ）の蛋白質の発現を阻害する化合物
（ｄ）上記（ａ）の蛋白質の活性を阻害する化合物
［２０］末梢血中の血管内皮前駆細胞増加剤を製造するための、以下のいずれかの物質の使用、
（ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する中和抗体
（ｂ）上記（ａ）の蛋白質をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸
（ｃ）上記（ａ）の蛋白質の発現を阻害する化合物
（ｄ）上記（ａ）の蛋白質の活性を阻害する化合物
に関する。 That is, the present invention
[1] Use of a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof, or a cell producing the protein or the partial peptide. A method for regulating differentiation / proliferation of vascular endothelial progenitor cells or a method for screening a substance for increasing vascular endothelial cells in peripheral blood,
[2] A nucleic acid encoding a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial polynucleotide thereof, or an antibody against the protein or the partial peptide is further used. The method according to [1] above, characterized in that
[3] The method according to [1] above, further comprising using a fatty acid or a low molecular weight compound that changes the binding property between the fatty acid and the protein, or a salt thereof.
[4] The method according to [1] above, wherein the cells are vascular endothelial progenitor cells or progenitor cells thereof,
[5] The method according to [1] above, for selecting a prophylactic / therapeutic substance for a disease whose regulation of angiogenic ability can exhibit a prophylactic / therapeutic effect
[6] An agent for promoting differentiation / proliferation of vascular endothelial progenitor cells, comprising any of the following substances:
(A) a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof (b) a base sequence encoding the protein of (a) or a partial peptide thereof (C) a compound that enhances the expression of the protein of (a) (d) a compound that enhances the activity of the protein of (a) [7] a disease in which the enhancement of angiogenic ability can exhibit a preventive / therapeutic effect The agent according to [6] above, for the prevention and treatment of
[8] An agent for inhibiting differentiation and proliferation of vascular endothelial progenitor cells, comprising any of the following substances:
(A) a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a partial peptide thereof (b) a base sequence encoding the protein of (a) above (C) a compound that inhibits the expression of the protein (a) (d) a compound that inhibits the activity of the protein (a) [9] an angiogenic ability The agent according to [7] above, for the prevention / treatment of a disease whose suppression can exhibit a prevention / treatment effect,
[10] An agent for increasing vascular endothelial progenitor cells in peripheral blood, comprising any of the following substances:
(A) a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a partial peptide thereof (b) a base sequence encoding the protein of (a) above (C) a compound that inhibits expression of the protein of (a) above (d) a compound [11] that inhibits the activity of the protein of (a) above A screening reagent for vascular endothelial progenitor cells, comprising
(A) an antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof; (b) a nucleic acid encoding the protein of (a) above or a portion thereof A method for selecting vascular endothelial progenitor cells in a sample, which comprises contacting the reagent according to the above [11] with a polynucleotide [12] cell-containing sample,
[13] Measuring the expression of a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a gene thereof in a cell-containing sample collected from a mammal Measuring the amount of vascular endothelial progenitor cells in the sample,
[14] The method according to [13] above for diagnosing a disease associated with an abnormal amount of vascular endothelial progenitor cells, and [15] administering any of the following substances to a mammal: To promote differentiation / proliferation of vascular endothelial progenitor cells,
(A) a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof (b) a base sequence encoding the protein of (a) or a partial peptide thereof (C) A compound that enhances the expression of the protein of (a) (d) A compound that enhances the activity of the protein of (a) [16] To produce a differentiation / proliferation promoter for vascular endothelial progenitor cells Use of any of the following substances:
(A) a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof (b) a base sequence encoding the protein of (a) or a partial peptide thereof (C) a compound that enhances the expression of the protein of (a) (d) a compound that enhances the activity of the protein of (a) [17] Any of the following substances is administered to a mammal: A method for inhibiting differentiation and proliferation of vascular endothelial progenitor cells,
(A) a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a partial peptide thereof (b) a base sequence encoding the protein of (a) above (C) a compound that inhibits the expression of the protein of (a) (d) a compound that inhibits the activity of the protein of (a) [18] vascular endothelial progenitor cells Use of any of the following substances to produce a differentiation / proliferation inhibitor:
(A) a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a partial peptide thereof (b) a base sequence encoding the protein of (a) above (C) a compound that inhibits the expression of the protein of (a) (d) a compound that inhibits the activity of the protein of (a) [19] A method for increasing vascular endothelial progenitor cells in peripheral blood, comprising administering any of the following substances:
(A) a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a partial peptide thereof (b) a base sequence encoding the protein of (a) above (C) a compound that inhibits the expression of the protein of (a) (d) a compound that inhibits the activity of the protein of (a) [20] in peripheral blood Use of any of the following substances for producing an agent for increasing vascular endothelial progenitor cells,
(A) a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a partial peptide thereof (b) a base sequence encoding the protein of (a) above (C) a compound that inhibits expression of the protein of (a) above (d) a compound that inhibits the activity of the protein of (a) above.
　GPR120は骨髄細胞からEPCへの分化・EPCの増殖に関与するので、GPR120もしくはそれを産生する細胞に被験物質を接触させ、該受容体の発現又は活性（例、リガンド結合活性、シグナル伝達活性など）の変動を調べることにより、EPCの分化・増殖を調節する薬剤をスクリーニングすることができる。また、GPR120の抑制は末梢血へのEPCの動員を促進することから、同様の手法により末梢血中のEPCを増加させる薬剤をスクリーニングすることもできる。
　GPR120は、造血細胞・末梢血細胞ではEPC特異的に高発現することから、EPCのマーカーとして、EPCの選別・定量、EPC量の異常が関連する疾患の診断に用いることができる。 Since GPR120 is involved in differentiation from bone marrow cells to EPC and proliferation of EPC, GPR120 or cells producing it are brought into contact with a test substance, and the expression or activity of the receptor (eg, ligand binding activity, signal transduction activity, etc.) ) Can be screened for drugs that modulate EPC differentiation / proliferation. Moreover, since suppression of GPR120 promotes the mobilization of EPC to peripheral blood, drugs that increase EPC in peripheral blood can also be screened by the same technique.
Since GPR120 is highly expressed specifically in EPC in hematopoietic cells and peripheral blood cells, it can be used as a marker for EPC in the diagnosis of diseases associated with EPC selection / quantification and abnormal EPC amount.
large EPC（左図）およびsmall EPCのコロニー（右図）の顕微鏡写真である。下図は、各EPCコロニーにおいて、細胞をLDL/Lectin/DAPIで３重染色して得られた染色像を示す。It is a microphotograph of colonies of large EPC (left figure) and small EPC (right figure). The lower figure shows a stained image obtained by triple staining of cells with LDL / Lectin / DAPI in each EPC colony. マウス（Ａ）およびヒト（Ｂ）GPR120遺伝子のTaqMan PCR発現解析における典型的な検量線パターンを示す図である。It is a figure which shows the typical calibration curve pattern in TaqMan (TM) PCR expression analysis of a mouse | mouth (A) and a human (B) GPR120 gene. マウス骨髄細胞由来および末梢血中単核球由来EPCにおけるGPR120遺伝子の発現を示す図である。It is a figure which shows the expression of GPR120 gene in mouse bone marrow cell origin and peripheral blood mononuclear cell origin EPC. ヒト骨髄細胞、EPCおよび内皮細胞におけるGPR120遺伝子の発現を示す図である。It is a figure which shows the expression of GPR120 gene in a human bone marrow cell, EPC, and endothelial cell. C57BL/6JマウスおよびGPR120 KOマウスの骨髄細胞由来EPCにおけるGPR120遺伝子の発現を示す図である。It is a figure which shows the expression of the GPR120 gene in the bone marrow cell origin EPC of a C57BL / 6J mouse | mouth and a GPR120 KO mouse | mouth. C57BL/6JマウスおよびGPR120 KOマウスにおける骨髄細胞のEPCへの分化能を示す図である。It is a figure which shows the differentiation ability to the EPC of the bone marrow cell in C57BL / 6J mouse | mouth and GPR120RKO mouse | mouth. C57BL/6JマウスおよびGPR120 KOマウスにおける末梢血中のEPC数を示す図である。It is a figure which shows the EPC number in the peripheral blood in a C57BL / 6J mouse | mouth and a GPR120 KO mouse | mouth. C57BL/6JマウスおよびGPR120 KOマウスにおける(A) 体重(BW)、(B) 血漿中グルコース濃度 (PG)、(C) 糖化ヘモグロビン値 (GHB)の変化を示す図である。図の各カラム中、左バーがC57BL/6Jマウス、右バーがGPR120 KOマウスをそれぞれ示す。FIG. 4 shows changes in (A) body weight (BW), (B) plasma glucose concentration (PG), and (C) glycated hemoglobin value (GHB) in C57BL / 6J mice and GPR120 KO mice. In each column of the figure, the left bar shows C57BL / 6J mice, and the right bar shows GPR120 KO mice.
（発明の詳細な説明）
（１）EPC分化・増殖調節または末梢血中のEPC増加物質のスクリーニング方法
　本発明は、GPR120蛋白質またはその部分ペプチド、あるいは該蛋白質またはその部分ペプチドを産生する細胞を用いることを特徴とする、EPCの分化・増殖調節または末梢血中のEPC増加物質のスクリーニング方法（以下、「本発明のスクリーニング方法」ともいう）を提供する。 (Detailed description of the invention)
(1) EPC differentiation / proliferation regulation or screening method for EPC increasing substance in peripheral blood The present invention uses GPR120 protein or a partial peptide thereof, or a cell that produces the protein or a partial peptide thereof. Of differentiation / proliferation of EPC or a method for screening an EPC-increasing substance in peripheral blood (hereinafter also referred to as “screening method of the present invention”).
　本発明のスクリーニング方法で用いられるGPR120は、配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むレセプター蛋白質である。
　GPR120蛋白質は、ヒトもしくは他の温血動物（例えば、サル，ウシ，ウマ，ブタ，ヒツジ，ヤギ，ウサギ，マウス，ラット，モルモット，ハムスター，ニワトリなど）の細胞［例えば、肝細胞、脾細胞、神経細胞、グリア細胞、膵β細胞、骨髄細胞、メサンギウム細胞、ランゲルハンス細胞、表皮細胞、上皮細胞、内皮細胞（例：血管内皮細胞）、杯細胞、内皮細胞、平滑筋細胞、線維芽細胞、線維細胞、筋細胞、脂肪細胞、免疫細胞（例：マクロファージ，Ｔ細胞、Ｂ細胞、ナチュラルキラー細胞、肥満細胞、好中球、好塩基球、好酸球、単球）、巨核球、滑膜細胞、軟骨細胞、骨細胞、骨芽細胞、破骨細胞、乳腺細胞、腸内分泌細胞もしくは間質細胞、またはこれら細胞の前駆細胞（例、脂肪前駆細胞、血管内皮前駆細胞）、幹細胞もしくは癌細胞など］、またはそれらの細胞が存在するあらゆる組織もしくは器官［例えば、脳、脳の各部位（例：嗅球、扁桃核、大脳基底球、海馬、視床、視床下部、大脳皮質、延髄、小脳）、脊髄、下垂体、胃、膵臓、腎臓、肝臓、生殖腺、甲状腺、胆嚢、骨髄、副腎、皮膚、筋肉（例：骨格筋）、肺、消化管（例：大腸、小腸）、血管、心臓、胸腺、脾臓、顎下腺、末梢血、前立腺、睾丸、卵巣、胎盤、子宮、骨、関節、脂肪組織（例：褐色脂肪組織、白色脂肪組織）など］、好ましくは骨髄細胞から分化した血管内皮前駆細胞もしくはその前駆細胞、脂肪細胞もしくはその前駆細胞、腸内分泌細胞などに由来する天然のGPR120蛋白質であってもよい。
　また後述のように、化学的に合成された蛋白質もしくは無細胞蛋白質合成系を利用して生化学的に合成された蛋白質であってもよい。あるいは、上記アミノ酸配列をコードする塩基配列を含む核酸を導入された、後述の形質転換体から産生される組換え蛋白質であってもよい。 GPR120 used in the screening method of the present invention is a receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.
GPR120 protein is a cell of humans or other warm-blooded animals (eg, monkeys, cows, horses, pigs, sheep, goats, rabbits, mice, rats, guinea pigs, hamsters, chickens, etc.) [eg, hepatocytes, spleen cells, Nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells (eg, vascular endothelial cells), goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibers Cells, muscle cells, adipocytes, immune cells (eg macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes , Chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, enteroendocrine cells or stromal cells, or precursor cells of these cells (eg, adipose precursor cells, vascular endothelial precursor cells), stem cells or Cancer cells, etc.], or any tissue or organ in which those cells are present [eg, brain, brain regions (eg, olfactory bulb, amygdaloid nucleus, basal sphere, hippocampus, hypothalamus, cerebral cortex, medulla, cerebellum) ), Spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle (eg, skeletal muscle), lung, digestive tract (eg, large intestine, small intestine), blood vessel, heart Thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue (eg, brown adipose tissue, white adipose tissue)], preferably blood vessels differentiated from bone marrow cells Natural GPR120 protein derived from endothelial progenitor cells or progenitor cells thereof, adipocytes or progenitor cells thereof, enteroendocrine cells, or the like may be used.
Further, as described later, it may be a chemically synthesized protein or a biochemically synthesized protein using a cell-free protein synthesis system. Or the recombinant protein produced from the below-mentioned transformant into which the nucleic acid containing the base sequence which codes the said amino acid sequence was introduce | transduced may be sufficient.
　「配列番号２、４または６に示されるアミノ酸配列と実質的に同一のアミノ酸配列」としては、配列番号２、４または６に示されるアミノ酸配列と約60％以上、好ましくは約70％以上、より好ましくは約80％以上、さらに好ましくは約90％以上、特に好ましくは約95％以上、最も好ましくは約97％以上の相同性を有するアミノ酸配列などが挙げられる。ここで「相同性」とは、当該技術分野において公知の数学的アルゴリズムを用いて２つのアミノ酸配列をアラインさせた場合の、最適なアラインメント（好ましくは、該アルゴリズムは最適なアラインメントのために配列の一方もしくは両方へのギャップの導入を考慮し得るものである）における、オーバーラップする全アミノ酸残基に対する同一アミノ酸および類似アミノ酸残基の割合（％）を意味する。「類似アミノ酸」とは物理化学的性質において類似したアミノ酸を意味し、例えば、芳香族アミノ酸（Phe、Trp、Tyr）、脂肪族アミノ酸（Ala、Leu、Ile、Val）、極性アミノ酸（Gln、Asn）、塩基性アミノ酸（Lys、Arg、His）、酸性アミノ酸（Glu、Asp）、水酸基を有するアミノ酸（Ser、Thr）、側鎖の小さいアミノ酸（Gly、Ala、Ser、Thr、Met）などの同じグループに分類されるアミノ酸が挙げられる。このような類似アミノ酸による置換は蛋白質の表現型に変化をもたらさない（即ち、保存的アミノ酸置換である）ことが予測される。保存的アミノ酸置換の具体例は当該技術分野で周知であり、種々の文献に記載されている（例えば、Bowieら，Science, 247： 1306-1310 (1990)を参照）。
　本明細書におけるアミノ酸配列の相同性は、相同性計算アルゴリズムNCBI BLAST（National Center for Biotechnology Information Basic Local Alignment Search Tool）を用い、以下の条件（期待値=10；ギャップを許す；マトリクス=BLOSUM62；フィルタリング=OFF）にて計算することができる。アミノ酸配列の相同性を決定するための他のアルゴリズムとしては、例えば、Karlinら, Proc. Natl. Acad. Sci. USA, 90： 5873-5877 (1993)に記載のアルゴリズム［該アルゴリズムはNBLASTおよびXBLASTプログラム（version 2.0）に組み込まれている（Altschulら, Nucleic Acids Res., 25： 3389-3402 (1997)）］、Needlemanら, J. Mol. Biol., 48： 444-453 (1970)に記載のアルゴリズム［該アルゴリズムはGCGソフトウェアパッケージ中のGAPプログラムに組み込まれている］、MyersおよびMiller, CABIOS, 4: 11-17 (1988)に記載のアルゴリズム［該アルゴリズムはCGC配列アラインメントソフトウェアパッケージの一部であるALIGNプログラム（version 2.0）に組み込まれている］、Pearsonら, Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988)に記載のアルゴリズム［該アルゴリズムはGCGソフトウェアパッケージ中のFASTAプログラムに組み込まれている］等が挙げられ、それらも同様に好ましく用いられ得る。
　より好ましくは、配列番号２、４または６に示されるアミノ酸配列と実質的に同一のアミノ酸配列とは、配列番号２、４または６に示されるアミノ酸配列と約60％以上、好ましくは約70％以上、より好ましくは約80％以上、さらに好ましくは約90％以上、特に好ましくは約95％以上、最も好ましくは約97％以上の同一性を有するアミノ酸配列である。 As the “amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6,” the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 is about 60% or more, preferably about 70% or more, More preferred is an amino acid sequence having a homology of about 80% or more, more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97% or more. As used herein, “homology” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm uses a sequence of sequences for optimal alignment). The ratio of the same amino acid residue and similar amino acid residues to all overlapping amino acid residues in the case of introducing a gap into one or both). “Similar amino acids” means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn). ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and have been described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).
The homology of the amino acid sequences in this specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; filtering) = OFF). Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithms include NBLAST and XBLAST. (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [The algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [the algorithm is part of the CGC sequence alignment software package Embedded in the ALIGN program (version 2.0), Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm is FASTA in the GCG software package. Program It includes built-in 'or the like, may they likewise preferably used.
More preferably, the amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 is about 60% or more, preferably about 70%, with the amino acid sequence shown in SEQ ID NO: 2, 4 or 6. More preferably, it is an amino acid sequence having an identity of about 80% or more, more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97% or more.
　「配列番号２、４または６に示されるアミノ酸配列と実質的に同一のアミノ酸配列を含む蛋白質」とは、前記の「配列番号２、４または６に示されるアミノ酸配列と実質的に同一のアミノ酸配列」を含み、かつ配列番号２、４または６に示されるアミノ酸配列を含む蛋白質と実質的に同質の活性を有する蛋白質を意味する。
　実質的に同質の活性としては、例えば、リガンド（例、脂肪酸等の生理的リガンド、アゴニスト／アンタゴニスト (例えば、特許文献２参照)）結合活性、シグナル伝達活性（例、ホスホリパーゼCβ (PLCβ) 活性化、細胞内Ca2+濃度上昇、蛋白質キナーゼC (PKC) 活性化等）、EPCの分化促進活性、EPCの増殖促進活性などが挙げられる。ここで「実質的に同質」とは、それらの活性が定性的（例えば、生理学的または薬理学的）に同じであることを示す。したがって、リガンド結合活性、シグナル伝達活性、EPC分化・増殖促進活性などの活性は同等（例えば、約0.5～約2倍）であることが好ましいが、これらの活性の程度や蛋白質の分子量などの量的要素は異なっていてもよい。
　リガンド結合活性、シグナル伝達活性、EPCの分化・増殖は、自体公知の方法に準じて測定することができる。 “Protein comprising an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6” means the above-mentioned “amino acid substantially identical to the amino acid sequence shown in SEQ ID NO: 2, 4 or 6”. It means a protein having an activity substantially equivalent to that of a protein comprising the “sequence” and comprising the amino acid sequence shown in SEQ ID NO: 2, 4 or 6.
The substantially homogeneous activity includes, for example, ligand (eg, physiological ligand such as fatty acid, agonist / antagonist (eg, see Patent Document 2)) binding activity, signal transduction activity (eg, activation of phospholipase Cβ (PLCβ)) , Intracellular Ca 2+ concentration increase, protein kinase C (PKC) activation, etc.), EPC differentiation promoting activity, EPC proliferation promoting activity, and the like. Here, “substantially the same quality” indicates that their activities are qualitatively (for example, physiologically or pharmacologically) the same. Accordingly, it is preferable that the activities such as ligand binding activity, signal transduction activity, and EPC differentiation / proliferation promoting activity are equivalent (for example, about 0.5 to about 2 times), but the amount of these activities and the amount of protein such as molecular weight The target elements may be different.
Ligand binding activity, signal transduction activity, and EPC differentiation / proliferation can be measured according to a method known per se.
　また、本発明におけるGPR120には、例えば、(1)配列番号２、４または６に示されるアミノ酸配列のうち1または2個以上（例えば、1～100個程度、好ましくは1～50個程度、より好ましくは1～30個程度、さらに好ましくは1～10個程度、特に好ましくは1～数（2、3、4もしくは5）個）のアミノ酸が欠失したアミノ酸配列、(2)配列番号２、４または６に示されるアミノ酸配列に1または2個以上（例えば、1～100個程度、好ましくは1～50個程度、より好ましくは1～30個程度、さらに好ましくは1～10個程度、特に好ましくは1～数（2、3、4もしくは5）個）のアミノ酸が付加したアミノ酸配列、(3)配列番号２、４または６に示されるアミノ酸配列に1または2個以上（例えば、1～50個程度、好ましくは1～30個程度、より好ましくは1～10個程度、さらに好ましくは1～数（2、3、4もしくは5）個）のアミノ酸が挿入されたアミノ酸配列、(4)配列番号２、４または６に示されるアミノ酸配列のうち1または2個以上（例えば、1～50個程度、好ましくは1～30個程度、より好ましくは1～10個程度、さらに好ましくは1～数（2、3、4もしくは5）個）のアミノ酸が他のアミノ酸で置換されたアミノ酸配列、または(5)それらを組み合わせたアミノ酸配列を含む蛋白質なども含まれる。
　上記のようにアミノ酸配列が挿入、欠失または置換されている場合、その挿入、欠失または置換の位置は、蛋白質の活性が保持される限り特に限定されない。 The GPR120 in the present invention includes, for example, (1) one or more of the amino acid sequences represented by SEQ ID NO: 2, 4 or 6 (for example, about 1 to 100, preferably about 1 to 50, More preferably, about 1 to 30, more preferably about 1 to 10, particularly preferably 1 to several (2, 3, 4, or 5) amino acid sequences, (2) SEQ ID NO: 2 1 or 2 or more (for example, about 1 to 100, preferably about 1 to 50, more preferably about 1 to 30, more preferably about 1 to 10) in the amino acid sequence shown in 4 or 6; Particularly preferred is an amino acid sequence to which 1 to several (2, 3, 4 or 5) amino acids are added, (3) one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 (for example, 1 About 50, preferably about 1-30, more preferably about 1-10, and even more preferably 1-number ( 2, 3, 4 or 5) amino acid sequence inserted, (4) one or more of the amino acid sequences shown in SEQ ID NO: 2, 4 or 6 (for example, about 1 to 50) Preferably about 1 to 30, more preferably about 1 to 10, more preferably 1 to several (2, 3, 4 or 5) amino acids are substituted with other amino acids, or (5 ) Proteins containing amino acid sequences combining them are also included.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the activity of the protein is maintained.
　本発明におけるGPR120は、好ましくは、配列番号２に示されるアミノ酸配列からなるヒトGPR120蛋白質（GenBankアクセッション番号：NP_859529）、配列番号４に示されるアミノ酸配列からなるマウスGPR120蛋白質（GenBankアクセッション番号：NP_861413）、配列番号６に示されるアミノ酸配列からなるラットGPR120蛋白質（GenBankアクセッション番号：NP_001040553）、あるいは他の哺乳動物におけるそのオルソログ（例えば、GenBankにアクセッション番号XP_534968として登録されているイヌオルソログ等）である（マウス、ラットおよびイヌGPR120はそれぞれヒトGPR120に対して約83％、約81％および約85％のアミノ酸同一性を有する）。 The GPR120 in the present invention is preferably a human GPR120 protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (GenBank accession number: NP_859529) and a mouse GPR120 protein consisting of the amino acid sequence shown in SEQ ID NO: 4 (GenBank accession number: NP_861413), rat GPR120 protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (GenBank accession number: NP_001040553), or an ortholog thereof in other mammals (for example, a dog ortholog registered in GenBank as accession number XP_534968) (Mouse, rat and dog GPR120 have about 83%, about 81% and about 85% amino acid identity to human GPR120, respectively).
　本明細書において、蛋白質およびペプチドは、ペプチド標記の慣例に従って左端がN末端（アミノ末端）、右端がC末端（カルボキシル末端）で記載される。配列番号２に示されるアミノ酸配列を含む蛋白質をはじめとする、本発明のGPR120は、C末端がカルボキシル基（-COOH）、カルボキシレート(-COO-）、アミド（-CONH2）またはエステル（-COOR）の何れであってもよい。
　ここでエステルにおけるＲとしては、例えば、メチル、エチル、n-プロピル、イソプロピル、n-ブチルなどのC1-6アルキル基；例えば、シクロペンチル、シクロヘキシルなどのC3-8シクロアルキル基；例えば、フェニル、α-ナフチルなどのC6-12アリール基；例えば、ベンジル、フェネチルなどのフェニル－C1-2アルキル基；α－ナフチルメチルなどのα-ナフチル－C1-2アルキル基などのC7-14アラルキル基；ピバロイルオキシメチル基などが用いられる。
　GPR120がC末端以外にカルボキシル基（またはカルボキシレート）を有している場合、カルボキシル基がアミド化またはエステル化されているものも本発明のGPR120に含まれる。この場合のエステルとしては、例えば上記したＣ末端のエステルなどが用いられる。
　さらに、本発明のGPR120には、N末端のアミノ酸残基のアミノ基が保護基（例えば、ホルミル基、アセチル基などのC1-6アルカノイルなどのC1-6アシル基など）で保護されているもの、生体内で切断されて生成し得るN末端のグルタミン残基がピログルタミン酸化したもの、分子内のアミノ酸の側鎖上の置換基（例えば-OH、-SH、アミノ基、イミダゾール基、インドール基、グアニジノ基など）が適当な保護基（例えば、ホルミル基、アセチル基などのC1-6アルカノイル基などのC1-6アシル基など）で保護されているもの、あるいは糖鎖が結合したいわゆる糖蛋白質などの複合蛋白質なども含まれる。 In the present specification, proteins and peptides are described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation. The GPR120 of the present invention, including a protein comprising the amino acid sequence shown in SEQ ID NO: 2, has a C-terminal carboxyl group (—COOH), carboxylate (—COO − ), amide (—CONH 2 ) or ester (— COOR).
Here, as R in the ester, for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; C 6-12 aryl groups such as α-naphthyl; phenyl-C 1-2 alkyl groups such as benzyl and phenethyl; C 7- such as α-naphthyl-C 1-2 alkyl groups such as α-naphthylmethyl; 14 aralkyl group; pivaloyloxymethyl group is used.
When GPR120 has a carboxyl group (or carboxylate) other than the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the GPR120 of the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used.
Furthermore, in the GPR120 of the present invention, the amino group of the N-terminal amino acid residue is protected with a protecting group (for example, a C 1-6 acyl group such as C 1-6 alkanoyl such as formyl group, acetyl group, etc.). N-terminal glutamine residue that can be cleaved in vivo is pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (eg, —OH, —SH, amino group, imidazole group, indole group, guanidino group, etc.) a suitable protecting group (e.g., formyl group, those protected by C 1-6 an acyl group) such as C 1-6 alkanoyl group such as acetyl group, or a sugar chain-binding Also included are complex proteins such as so-called glycoproteins.
　GPR120の部分ペプチド（以下、単に「本発明の部分ペプチド」と略称する場合もある）は、上記したGPR120の部分アミノ酸配列を含むペプチドであり、且つGPR120と実質的に同質の活性を有する限り、何れのものであってもよい。ここで「実質的に同質の活性」とは上記と同意義を示す。また、「実質的に同質の活性」の測定はGPR120の場合と同様に行なうことができる。
　具体的には、本発明の部分ペプチドとして、例えば、配列番号２、４または６に示されるアミノ酸配列のうち、リガンドである脂肪酸との結合に関わる領域（例えばヒトGPR120の場合、例えば配列番号２に示されるアミノ酸配列中アミノ酸番号1-45、99-112、178-204および306-311で示される細胞外領域など）および該リガンドとの相互作用を介したシグナル伝達に関わる領域（例えば、三量体Ｇ蛋白質のαサブユニットと結合してそのGDP・GTP交換反応を促進する活性を有する領域（以下、「Gα活性化ドメイン」ともいう）など）を含む部分アミノ酸配列を有するものなどが用いられる。本発明の部分ペプチドは、上記のリガンドとの結合に関与する領域とシグナル伝達に関与する領域を含む限りそのサイズに特に制限はないが、好ましくは100個以上の部分アミノ酸配列を含むもの、より好ましくは200個以上の部分アミノ酸配列を含むものが挙げられる。該部分アミノ酸配列は一個の連続した部分アミノ酸配列であってもよく、あるいは不連続な複数の部分アミノ酸配列が連結されたものであってもよい。 As long as the partial peptide of GPR120 (hereinafter sometimes simply referred to as “partial peptide of the present invention”) is a peptide containing the partial amino acid sequence of GPR120 described above and has substantially the same activity as GPR120, Any one may be used. Here, “substantially the same quality of activity” has the same meaning as described above. Further, “substantially the same quality of activity” can be measured in the same manner as in the case of GPR120.
Specifically, as a partial peptide of the present invention, for example, among the amino acid sequences shown in SEQ ID NO: 2, 4 or 6, a region involved in binding to a fatty acid that is a ligand (for example, in the case of human GPR120, for example, SEQ ID NO: 2). In the amino acid sequence represented by amino acid Nos. 1-45, 99-112, 178-204 and 306-311) and a region involved in signal transduction through interaction with the ligand (for example, three Those having a partial amino acid sequence containing a region (hereinafter also referred to as “Gα activation domain”) having the activity of binding to the α subunit of the monomeric G protein and promoting its GDP / GTP exchange reaction are used. It is done. The partial peptide of the present invention is not particularly limited in size as long as it includes a region involved in binding to the above ligand and a region involved in signal transduction, but preferably contains a partial amino acid sequence of 100 or more. Preferably, those containing 200 or more partial amino acid sequences are included. The partial amino acid sequence may be a single continuous partial amino acid sequence, or may be a concatenation of a plurality of discontinuous partial amino acid sequences.
　また、本発明の部分ペプチドはC末端がカルボキシル基（-COOH）、カルボキシレート（-COO-）、アミド（-CONH2）またはエステル（-COOR）の何れであってもよい。ここでエステルにおけるＲとしては、GPR120について前記したと同様のものが挙げられる。本発明の部分ペプチドがC末端以外にカルボキシル基（またはカルボキシレート）を有している場合、カルボキシル基がアミド化またはエステル化されているものも本発明の部分ペプチドに含まれる。この場合のエステルとしては、例えば、C末端のエステルと同様のものなどが用いられる。
　さらに、本発明の部分ペプチドには、上記したGPR120と同様に、N末端のアミノ酸残基のアミノ基が保護基で保護されているもの、N末端のグルタミン残基がピログルタミン酸化したもの、分子内のアミノ酸の側鎖上の置換基が適当な保護基で保護されているもの、あるいは糖鎖が結合したいわゆる糖ペプチドなどの複合ペプチドなども含まれる。 In the partial peptide of the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO − ), an amide (—CONH 2 ), or an ester (—COOR). Here, examples of R in the ester include the same as those described above for GPR120. When the partial peptide of the present invention has a carboxyl group (or carboxylate) in addition to the C-terminus, those in which the carboxyl group is amidated or esterified are also included in the partial peptide of the present invention. As the ester in this case, for example, the same ester as the C-terminal ester is used.
Furthermore, in the partial peptide of the present invention, as in GPR120 described above, the amino group of the N-terminal amino acid residue is protected with a protecting group, the N-terminal glutamine residue is pyroglutamine oxidized, Examples include those in which a substituent on the side chain of the amino acid is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound.
　本発明で用いられるGPR120またはその部分ペプチドは塩の形態であってもよい。例えば、生理学的に許容される酸（例：無機酸、有機酸）や塩基（例：アルカリ金属）などとの塩が用いられ、とりわけ生理学的に許容される酸付加塩が好ましい。この様な塩としては、例えば、無機酸（例えば、塩酸、リン酸、臭化水素酸、硫酸）との塩、あるいは有機酸（例えば、酢酸、ギ酸、プロピオン酸、フマル酸、マレイン酸、コハク酸、酒石酸、クエン酸、リンゴ酸、蓚酸、安息香酸、メタンスルホン酸、ベンゼンスルホン酸）との塩などが用いられる。 GPR120 or a partial peptide thereof used in the present invention may be in the form of a salt. For example, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or base (eg, alkali metal) is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
　GPR120は、前述した哺乳動物の細胞または組織から、自体公知の蛋白質の精製方法によって製造することができる。具体的には、哺乳動物の組織または細胞をホモジナイズし、低速遠心により細胞デブリスを除去した後、上清を高速遠心して細胞膜含有画分を沈澱させ（必要に応じて密度勾配遠心などにより細胞膜画分を精製し）、該画分を逆相クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィーなどのクロマトグラフィー等に付すことにより、GPR120またはその塩を調製することができる。 GPR120 can be produced from the aforementioned mammalian cells or tissues by a known protein purification method. Specifically, after homogenizing mammalian tissue or cells and removing cell debris by low-speed centrifugation, the supernatant is centrifuged at high speed to precipitate a cell membrane-containing fraction (if necessary, cell membrane fractionation by density gradient centrifugation or the like). GPR120 or a salt thereof can be prepared by subjecting the fraction to chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography, and the like.
　GPR120またはその部分ペプチド（以下、これらの化学合成の説明においては、特にことわらない限り、これらを包括して単に「GPR120」という）は、公知のペプチド合成法に従って製造することもできる。
　ペプチド合成法は、例えば、固相合成法、液相合成法のいずれであってもよい。GPR120を構成し得る部分ペプチドもしくはアミノ酸と残余部分とを縮合し、生成物が保護基を有する場合は保護基を脱離することにより目的とする蛋白質を製造することができる。
　ここで、縮合や保護基の脱離は、自体公知の方法、例えば、以下の(1)～(5)に記載された方法に従って行われる。
(1) M. BodanszkyおよびM.A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966年)
(2) SchroederおよびLuebke, The Peptide, Academic Press, New York (1965年)
(3) 泉屋信夫他、ペプチド合成の基礎と実験、丸善(株) （1975年）
(4) 矢島治明および榊原俊平、生化学実験講座 1、蛋白質の化学IV 205 (1977年)
(5) 矢島治明監修、続医薬品の開発、第14巻、ペプチド合成、広川書店 GPR120 or a partial peptide thereof (hereinafter referred to simply as “GPR120” in the description of these chemical syntheses unless otherwise specified) can also be produced according to known peptide synthesis methods.
The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. When the partial peptide or amino acid capable of constituting GPR120 is condensed with the remaining portion, and the product has a protecting group, the target protein can be produced by removing the protecting group.
Here, the condensation and the removal of the protecting group are carried out according to a method known per se, for example, the method described in the following (1) to (5).
(1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(4) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV 205 (1977)
(5) Supervised by Haruaki Yajima, Development of follow-up drugs, Volume 14, Peptide synthesis, Hirokawa Shoten
　このようにして得られたGPR120は、公知の精製法により単離・精製することができる。ここで、精製法としては、例えば、溶媒抽出、蒸留、カラムクロマトグラフィー、液体クロマトグラフィー、再結晶、これらの組み合わせなどが挙げられる。
　上記方法で得られるGPR120が遊離体である場合には、該遊離体を公知の方法あるいはそれに準じる方法によって適当な塩に変換することができるし、逆にGPR120が塩として得られた場合には、該塩を公知の方法あるいはそれに準じる方法によって遊離体または他の塩に変換することができる。 The GPR120 thus obtained can be isolated and purified by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
When GPR120 obtained by the above method is a free form, the free form can be converted to an appropriate salt by a known method or a method according thereto, and conversely, when GPR120 is obtained as a salt. The salt can be converted into a free form or other salt by a known method or a method analogous thereto.
　GPR120の合成には、通常市販の蛋白質合成用樹脂を用いることができる。そのような樹脂としては、例えば、クロロメチル樹脂、ヒドロキシメチル樹脂、ベンズヒドリルアミン樹脂、アミノメチル樹脂、4-ベンジルオキシベンジルアルコール樹脂、4-メチルベンズヒドリルアミン樹脂、PAM樹脂、4-ヒドロキシメチルメチルフェニルアセトアミドメチル樹脂、ポリアクリルアミド樹脂、4-(2’,4’-ジメトキシフェニル-ヒドロキシメチル)フェノキシ樹脂、4-(2’,4’-ジメトキシフェニル-Fmocアミノエチル)フェノキシ樹脂などを挙げることができる。このような樹脂を用い、α-アミノ基と側鎖官能基を適当に保護したアミノ酸を、目的とするGPR120の配列通りに、自体公知の各種縮合方法に従い、樹脂上で縮合させる。反応の最後に樹脂から蛋白質等を切り出すと同時に各種保護基を除去し、さらに高希釈溶液中で分子内ジスルフィド結合形成反応を実施し、目的のGPR120またはそのアミド体を取得する。 For the synthesis of GPR120, generally commercially available resin for protein synthesis can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2', 4'-dimethoxyphenyl-Fmoc aminoethyl) phenoxy resin, etc. it can. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the intended GPR120 according to various condensation methods known per se. At the end of the reaction, proteins and the like are cut out from the resin, and at the same time, various protecting groups are removed, and an intramolecular disulfide bond forming reaction is carried out in a highly diluted solution to obtain the target GPR120 or its amide.
　上記した保護アミノ酸の縮合に関しては、蛋白質合成に使用できる各種活性化試薬を用いることができるが、特に、カルボジイミド類がよい。カルボジイミド類としては、DCC、N,N’-ジイソプロピルカルボジイミド、N-エチル-N’-(3-ジメチルアミノプロリル）カルボジイミドなどが用いられる。これらによる活性化にはラセミ化抑制添加剤（例えば、HOBt、HOOBt)とともに保護アミノ酸を直接樹脂に添加するか、または、対称酸無水物またはHOBtエステルあるいはHOOBtエステルとしてあらかじめ保護アミノ酸の活性化を行なった後に樹脂に添加することができる。 For the above condensation of protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimide, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (eg, HOBt, HOOBt), or the protected amino acid is activated in advance as a symmetric anhydride, HOBt ester or HOOBt ester. Can then be added to the resin.
　保護アミノ酸の活性化や樹脂との縮合に用いられる溶媒は、蛋白質縮合反応に使用しうることが知られている溶媒から適宜選択されうる。例えば、N,N-ジメチルホルムアミド，N,N-ジメチルアセトアミド，N-メチルピロリドンなどの酸アミド類、塩化メチレン，クロロホルムなどのハロゲン化炭化水素類、トリフルオロエタノールなどのアルコール類、ジメチルスルホキシドなどのスルホキシド類、ピリジンなどのアミン類，ジオキサン，テトラヒドロフランなどのエーテル類、アセトニトリル，プロピオニトリルなどのニトリル類、酢酸メチル，酢酸エチルなどのエステル類あるいはこれらの適宜の混合物などが用いられる。反応温度は蛋白質結合形成反応に使用され得ることが知られている範囲から適宜選択され、通常約-20℃～50℃の範囲から適宜選択される。活性化されたアミノ酸誘導体は通常1.5～4倍過剰で用いられる。ニンヒドリン反応を用いたテストの結果、縮合が不十分な場合には保護基の脱離を行うことなく縮合反応を繰り返すことにより十分な縮合を行なうことができる。反応を繰り返しても十分な縮合が得られないときには、無水酢酸またはアセチルイミダゾールを用いて未反応アミノ酸をアセチル化することができる。 The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range that is known to be usable for protein bond forming reaction, and is usually selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. If sufficient condensation is not obtained even after repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.
　原料の反応に関与すべきでない官能基の保護ならびに保護基、およびその保護基の脱離、反応に関与する官能基の活性化などは公知の基または公知の手段から適宜選択しうる。
　原料のアミノ基の保護基としては、例えば、Z、Boc、ターシャリーペンチルオキシカルボニル、イソボルニルオキシカルボニル、4-メトキシベンジルオキシカルボニル、Cl-Z、Br-Z、アダマンチルオキシカルボニル、トリフルオロアセチル、フタロイル、ホルミル、2-ニトロフェニルスルフェニル、ジフェニルホスフィノチオイル、Fmocなどが用いられる。
　カルボキシル基は、例えば、アルキルエステル化（例えば、メチル、エチル、プロピル、ブチル、ターシャリーブチル、シクロペンチル、シクロヘキシル、シクロヘプチル、シクロオクチル、2-アダマンチルなどの直鎖状、分枝状もしくは環状アルキルエステル化）、アラルキルエステル化（例えば、ベンジルエステル、4-ニトロベンジルエステル、4-メトキシベンジルエステル、4-クロロベンジルエステル、ベンズヒドリルエステル化）、フェナシルエステル化、ベンジルオキシカルボニルヒドラジド化、ターシャリーブトキシカルボニルヒドラジド化、トリチルヒドラジド化などによって保護することができる。
　セリンの水酸基は、例えば、エステル化またはエーテル化によって保護することができる。このエステル化に適する基としては、例えば、アセチル基などの低級アルカノイル基、ベンゾイル基などのアロイル基、ベンジルオキシカルボニル基、エトキシカルボニル基などの炭酸から誘導される基などが用いられる。また、エーテル化に適する基としては、例えば、ベンジル基、テトラヒドロピラニル基、t-ブチル基などである。
　チロシンのフェノール性水酸基の保護基としては、例えば、Bzl、Cl2-Bzl、2-ニトロベンジル、Br-Z、ターシャリーブチルなどが用いられる。
　ヒスチジンのイミダゾールの保護基としては、例えば、Tos、4-メトキシ-2,3,6-トリメチルベンゼンスルホニル、DNP、ベンジルオキシメチル、Bum、Boc、Trt、Fmocなどが用いられる。 The protection of the functional group that should not be involved in the reaction of the raw material, the protection group, the removal of the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.
Examples of the protective group for the amino group of the raw material include Z, Boc, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc, etc. are used.
The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl). ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonyl hydrazide, tertiary butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include groups derived from carbonic acid such as lower alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, benzyloxycarbonyl groups, and ethoxycarbonyl groups. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl 2 -Bzl, 2-nitrobenzyl, Br-Z, tertiary butyl and the like.
Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like.
　保護基の除去（脱離）方法としては、例えば、Pd-黒あるいはPd-炭素などの触媒の存在下での水素気流中での接触還元や、また、無水フッ化水素、メタンスルホン酸、トリフルオロメタンスルホン酸、トリフルオロ酢酸あるいはこれらの混合液などによる酸処理や、ジイソプロピルエチルアミン、トリエチルアミン、ピペリジン、ピペラジンなどによる塩基処理、また液体アンモニア中ナトリウムによる還元なども用いられる。上記酸処理による脱離反応は、一般に約-20℃～40℃の温度で行なわれるが、酸処理においては、例えば、アニソール、フェノール、チオアニソール、メタクレゾール、パラクレゾール、ジメチルスルフィド、1,4-ブタンジチオール、1,2-エタンジチオールなどのようなカチオン捕捉剤の添加が有効である。また、ヒスチジンのイミダゾール保護基として用いられる2,4-ジニトロフェニル基はチオフェノール処理により除去され、トリプトファンのインドール保護基として用いられるホルミル基は上記の1,2-エタンジチオール、1,4-ブタンジチオールなどの存在下の酸処理による脱保護以外に、希水酸化ナトリウム溶液、希アンモニアなどによるアルカリ処理によっても除去される。 Examples of methods for removing (eliminating) protecting groups include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, and anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 -Addition of a cation scavenger such as butanedithiol or 1,2-ethanedithiol is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine was removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan was the above 1,2-ethanedithiol and 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.
　原料のカルボキシル基の活性化されたものとしては、例えば、対応する酸無水物、アジド、活性エステル〔アルコール（例えば、ペンタクロロフェノール、2,4,5-トリクロロフェノール、2,4-ジニトロフェノール、シアノメチルアルコール、パラニトロフェノール、HONB、N-ヒドロキシスクシミド、N-ヒドロキシフタルイミド、HOBt）とのエステル〕などが用いられる。原料のアミノ基の活性化されたものとしては、例えば、対応するリン酸アミドが用いられる。 Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.
　GPR120のアミド体を得る別の方法としては、例えば、まず、カルボキシ末端アミノ酸のα-カルボキシル基をアミド化して保護した後、アミノ基側にペプチド鎖を所望の鎖長まで延ばした後、該ペプチド鎖のＮ末端のα-アミノ基の保護基のみを除いた蛋白質（ペプチド）とＣ末端のカルボキシル基の保護基のみを除去した蛋白質（ペプチド）とを製造し、この両蛋白質（ペプチド）を上記したような混合溶媒中で縮合させる。縮合反応の詳細については上記と同様である。縮合により得られた保護蛋白質（保護ペプチド）を精製した後、上記方法によりすべての保護基を除去し、所望の粗蛋白質（粗ペプチド）を得ることができる。この粗蛋白質（粗ペプチド）は既知の各種精製手段を駆使して精製し、主要画分を凍結乾燥することで所望のGPR120のアミド体を得ることができる。
　GPR120のエステル体は、例えば、カルボキシ末端アミノ酸のα-カルボキシル基を所望のアルコール類と縮合しアミノ酸エステルとした後、上記GPR120のアミド体の場合と同様にして得ることができる。 As another method for obtaining an amide form of GPR120, for example, first, the α-carboxyl group of the carboxy terminal amino acid is amidated and protected, and then the peptide chain is extended to the desired chain length on the amino group side and then the peptide A protein (peptide) from which only the protecting group of the α-amino group at the N-terminus of the chain is removed and a protein (peptide) from which only the protecting group of the carboxyl group at the C-terminal has been removed are prepared. Condensation in a mixed solvent as described above. The details of the condensation reaction are the same as described above. After purifying the protected protein (protected peptide) obtained by the condensation, all the protecting groups are removed by the above method to obtain the desired crude protein (crude peptide). This crude protein (crude peptide) can be purified by using various known purification means, and the main fraction can be lyophilized to obtain the desired amide form of GPR120.
The ester form of GPR120 can be obtained in the same manner as in the case of the amide form of GPR120, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester.
　本発明の部分ペプチドは、GPR120の全長蛋白質を適当なペプチダーゼで切断することによっても製造することができる。 The partial peptide of the present invention can also be produced by cleaving the full-length protein of GPR120 with an appropriate peptidase.
　さらに、GPR120は、それをコードする核酸を含有する形質転換体を培養し、得られる培養物からGPR120を分離・精製することによって製造することもできる。GPR120またはその部分ペプチドをコードする核酸はDNAであってもRNAであってもよく、あるいはDNA/RNAキメラであってもよい。好ましくはDNAが挙げられる。また、該核酸は二本鎖であっても、一本鎖であってもよい。二本鎖の場合は、二本鎖DNA、二本鎖RNAまたはDNA:RNAのハイブリッドでもよい。一本鎖の場合は、センス鎖（即ち、コード鎖）であっても、アンチセンス鎖（即ち、非コード鎖）であってもよい。
　GPR120またはその部分ペプチドをコードするDNAとしては、ゲノムDNA、ヒトもしくは他の温血動物（例えば、サル，ウシ，ウマ，ブタ，ヒツジ，ヤギ，ウサギ，マウス，ラット，モルモット，ハムスター，ニワトリなど）の細胞［例えば、肝細胞、脾細胞、神経細胞、グリア細胞、膵β細胞、骨髄細胞、メサンギウム細胞、ランゲルハンス細胞、表皮細胞、上皮細胞、内皮細胞（例：血管内皮細胞）、杯細胞、内皮細胞、平滑筋細胞、線維芽細胞、線維細胞、筋細胞、脂肪細胞、免疫細胞（例：マクロファージ，Ｔ細胞、Ｂ細胞、ナチュラルキラー細胞、肥満細胞、好中球、好塩基球、好酸球、単球）、巨核球、滑膜細胞、軟骨細胞、骨細胞、骨芽細胞、破骨細胞、乳腺細胞、腸内分泌細胞もしくは間質細胞、またはこれら細胞の前駆細胞（例、脂肪前駆細胞、血管内皮前駆細胞）、幹細胞もしくは癌細胞など］、またはそれらの細胞が存在するあらゆる組織もしくは器官［例えば、脳、脳の各部位（例：嗅球、扁桃核、大脳基底球、海馬、視床、視床下部、大脳皮質、延髄、小脳）、脊髄、下垂体、胃、膵臓、腎臓、肝臓、生殖腺、甲状腺、胆嚢、骨髄、副腎、皮膚、筋肉（例：骨格筋）、肺、消化管（例：大腸、小腸）、血管、心臓、胸腺、脾臓、顎下腺、末梢血、前立腺、睾丸、卵巣、胎盤、子宮、骨、関節、脂肪組織（例：褐色脂肪組織、白色脂肪組織）など］、好ましくは骨髄細胞から分化した血管内皮前駆細胞もしくはその前駆細胞、脂肪細胞もしくはその前駆細胞、腸内分泌細胞などに由来するcDNA、合成DNAなどが挙げられる。GPR120またはその部分ペプチドをコードするゲノムDNAおよびcDNAは、上記した細胞・組織より調製したゲノムDNA画分および全RNAもしくはmRNA画分をそれぞれ鋳型として用い、Polymerase Chain Reaction（以下、「PCR法」と略称する）およびReverse Transcriptase-PCR（以下、「RT-PCR法」と略称する）によって直接増幅することもできる。あるいは、GPR120またはその部分ペプチドをコードするゲノムDNAおよびcDNAは、上記した細胞・組織より調製したゲノムDNAおよび全RNAもしくはmRNAの断片を適当なベクター中に挿入して調製されるゲノムDNAライブラリーおよびcDNAライブラリーから、コロニーもしくはプラークハイブリダイゼーション法またはPCR法などにより、それぞれクローニングすることもできる。ライブラリーに使用するベクターは、バクテリオファージ、プラスミド、コスミド、ファージミドなどいずれであってもよい。 Furthermore, GPR120 can also be produced by culturing a transformant containing the nucleic acid encoding it, and separating and purifying GPR120 from the resulting culture. The nucleic acid encoding GPR120 or a partial peptide thereof may be DNA or RNA, or may be a DNA / RNA chimera. Preferably, DNA is used. The nucleic acid may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
As DNA encoding GPR120 or a partial peptide thereof, genomic DNA, human or other warm-blooded animals (eg, monkeys, cows, horses, pigs, sheep, goats, rabbits, mice, rats, guinea pigs, hamsters, chickens, etc.) Cells [eg, hepatocytes, spleen cells, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells (eg, vascular endothelial cells), goblet cells, endothelium Cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils) Monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, enteroendocrine cells or stromal cells, or precursor cells of these cells (e.g., Fat precursor cells, endothelial progenitor cells), stem cells or cancer cells, etc.], or any tissue or organ in which those cells are present [eg, brain, brain regions (eg, olfactory bulb, amygdala, basal sphere, hippocampus) , Thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle (eg skeletal muscle), lung, digestion Tube (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue (eg, brown adipose tissue, white adipose tissue) Etc.], preferably vascular endothelial progenitor cells differentiated from bone marrow cells or progenitor cells thereof, adipocytes or progenitor cells thereof, cDNA derived from enteroendocrine cells, synthetic DNA, and the like. Genomic DNA and cDNA encoding GPR120 or its partial peptides are prepared using Polymerase Chain Reaction (hereinafter referred to as “PCR method”) using the genomic DNA fraction and total RNA or mRNA fraction prepared from the cells and tissues as templates. (Abbreviated) and Reverse Transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”). Alternatively, genomic DNA and cDNA encoding GPR120 or a partial peptide thereof can be prepared by inserting a genomic DNA prepared from the cells and tissues described above and a total RNA or mRNA fragment into an appropriate vector and It can also be cloned from a cDNA library by colony or plaque hybridization method or PCR method, respectively. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
　GPR120をコードするDNAとしては、例えば、配列番号１、３または５に示される塩基配列を含有するDNA、または配列番号１、３または５に示される塩基配列とハイストリンジェントな条件下でハイブリダイズする塩基配列を含有し、前記したGPR120と実質的に同質の活性（例、リガンド結合活性、シグナル伝達活性、EPC分化・増殖促進活性など）を有する蛋白質をコードするDNAなどが挙げられる。
　配列番号１、３または５に示される塩基配列とハイストリンジェントな条件下でハイブリダイズできるDNAとしては、例えば、配列番号１、３または５に示される塩基配列と約60％以上、好ましくは約70％以上、より好ましくは約80％以上、さらに好ましくは約90％以上、特に好ましくは約95％以上の相同性を有する塩基配列を含有するDNAなどが用いられる。
　本明細書における塩基配列の相同性は、相同性計算アルゴリズムNCBI BLAST（National Center for Biotechnology Information Basic Local Alignment Search Tool）を用い、以下の条件（期待値=10；ギャップを許す；フィルタリング=ON；マッチスコア=1；ミスマッチスコア=-3）にて計算することができる。塩基配列の相同性を決定するための他のアルゴリズムとしては、上記したアミノ酸配列の相同性計算アルゴリズムが同様に好ましく例示される。 As DNA encoding GPR120, for example, DNA containing the base sequence shown in SEQ ID NO: 1, 3 or 5 or hybridized with the base sequence shown in SEQ ID NO: 1, 3 or 5 under highly stringent conditions And a DNA encoding a protein having substantially the same quality as GPR120 described above (eg, ligand binding activity, signal transduction activity, EPC differentiation / proliferation promoting activity, etc.).
Examples of DNA capable of hybridizing under high stringency conditions with the base sequence shown in SEQ ID NO: 1, 3 or 5 include, for example, about 60% or more, preferably about A DNA containing a base sequence having a homology of 70% or more, more preferably about 80% or more, further preferably about 90% or more, particularly preferably about 95% or more is used.
The homology of the nucleotide sequences in the present specification uses the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; allow gap; filtering = ON; match) It can be calculated by score = 1; mismatch score = -3). As another algorithm for determining the homology of a base sequence, the above-mentioned algorithm for calculating homology of amino acid sequences is also preferably exemplified.
　ハイブリダイゼーションは、自体公知の方法あるいはそれに準じる方法、例えば、モレキュラー・クローニング（Molecular Cloning）第２版（J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989）に記載の方法などに従って行なうことができる。また、市販のライブラリーを使用する場合、ハイブリダイゼーションは、添付の使用説明書に記載の方法に従って行なうことができる。ハイブリダイゼーションは、好ましくは、ストリンジェントな条件に従って行なうことができる。
　ハイストリンジェントな条件としては、例えば、6×SSC（sodium chloride/sodium citrate）中45℃でのハイブリダイゼーション反応の後、0.2×SSC／0.1％ SDS中65℃での一回以上の洗浄などが挙げられる。当業者は、ハイブリダイゼーション溶液の塩濃度、ハイブリダゼーション反応の温度、プローブ濃度、プローブの長さ、ミスマッチの数、ハイブリダイゼーション反応の時間、洗浄液の塩濃度、洗浄の温度等を適宜変更することにより、所望のストリンジェンシーに容易に調節することができる。 Hybridization is carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can do. When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be preferably performed according to stringent conditions.
High stringent conditions include, for example, a hybridization reaction at 45 ° C. in 6 × SSC (sodium chloride / sodium citrate), followed by one or more washings at 65 ° C. in 0.2 × SSC / 0.1% SDS. Can be mentioned. A person skilled in the art may appropriately change the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc. Thus, the desired stringency can be easily adjusted.
　GPR120をコードするDNAは、好ましくは、配列番号１に示される、ヒトGPR120蛋白質をコードする塩基配列を含むDNA（GenBankアクセッション番号：NM_181745）、配列番号３に示される、マウスGPR120蛋白質をコードする塩基配列を含むDNA（GenBankアクセッション番号：NM_181748）、配列番号５に示される、ラットGPR120蛋白質をコードする塩基配列を含むDNA（GenBankアクセッション番号：NM_001047088）、あるいは他の哺乳動物におけるそのオルソログ（例えば、GenBankにアクセッション番号XM_534968として登録されているイヌオルソログ等）である。他の哺乳動物におけるオルソログは、例えば、配列番号１に示される塩基配列をクエリーにして、ヒト以外の哺乳動物のゲノムおよび/またはcDNAのデータベースに対してBLASTやFASTAを用いて検索をかけるか、あるいは、例えばJackson研究所から提供されるMouse Genome Informatics（http://www.informatics.jax.org/）でaccession番号や遺伝子記号/遺伝子名をキーワードにして検索をかけ、ヒットしたデータのMammalian Orthologyの情報にアクセスする等によって、その配列情報を取得することができる。 The DNA encoding GPR120 preferably encodes a mouse GPR120 protein shown in SEQ ID NO: 3, which is a DNA containing the base sequence encoding human GPR120 protein shown in SEQ ID NO: 1 (GenBank accession number: NM_181745) DNA containing the nucleotide sequence (GenBank accession number: NM_181748), DNA containing the nucleotide sequence encoding the rat GPR120 protein shown in SEQ ID NO: 5 (GenBank accession number: NM_001047088), or its ortholog in other mammals ( For example, a dog ortholog registered as an accession number XM_534968 in GenBank). Orthologs in other mammals can be searched using BLAST or FASTA, for example, by querying the genome and / or cDNA database of mammals other than humans by querying the base sequence shown in SEQ ID NO: 1. Or, for example, Mouse Genome Informatics provided by Jackson Laboratories (http://www.informatics.jax.org/) is searched using the accession number or gene symbol / gene name as a keyword, and Mammalian Orthology of the hit data The array information can be acquired by accessing the information.
　本発明の部分ペプチドをコードするDNAは、配列番号２、４または６に示されるアミノ酸配列の一部と同一もしくは実質的に同一のアミノ酸配列を有するペプチドをコードする塩基配列を含むものであればいかなるものであってもよい。具体的には、本発明の部分ペプチドをコードするDNAとしては、例えば、（１）配列番号１、３または５に示される塩基配列の部分塩基配列を有するDNA、または（２）配列番号１、３または５に示される塩基配列を有するDNAとハイストリンジェントな条件下でハイブリダイズする塩基配列を有し、前記したGPR120と実質的に同質の活性（例、リガンド結合活性、シグナル伝達活性、EPC分化・増殖促進活性など）を有するペプチドをコードするDNAなどが用いられる。 As long as the DNA encoding the partial peptide of the present invention includes a base sequence encoding a peptide having the same or substantially the same amino acid sequence as part of the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 It can be anything. Specifically, as the DNA encoding the partial peptide of the present invention, for example, (1) DNA having a partial base sequence of the base sequence shown in SEQ ID NO: 1, 3 or 5, or (2) SEQ ID NO: 1, It has a base sequence that hybridizes with DNA having the base sequence shown in 3 or 5 under highly stringent conditions, and has substantially the same activity as GPR120 described above (eg, ligand binding activity, signal transduction activity, EPC DNA encoding a peptide having differentiation / proliferation promoting activity) is used.
　GPR120またはその部分ペプチドをコードするDNAは、該GPR120またはその部分ペプチドをコードする塩基配列の一部分を有する合成DNAプライマーを用いてPCR法によって増幅するか、または適当な発現ベクターに組み込んだDNAを、GPR120蛋白質の一部あるいは全領域をコードするDNA断片もしくは合成DNAを標識したものとハイブリダイゼーションすることによってクローニングすることができる。ハイブリダイゼーションは、例えば、モレキュラー・クローニング（Molecular Cloning）第２版（前述）に記載の方法などに従って行なうことができる。また、市販のライブラリーを使用する場合、ハイブリダイゼーションは、該ライブラリーに添付された使用説明書に記載の方法に従って行なうことができる。 A DNA encoding GPR120 or a partial peptide thereof is amplified by PCR using a synthetic DNA primer having a part of a base sequence encoding the GPR120 or a partial peptide thereof, or DNA incorporated into an appropriate expression vector is used. Cloning can be carried out by hybridization with a DNA fragment encoding a part or the entire region of GPR120 protein or a labeled synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning Second Edition (described above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.
　DNAの塩基配列は、公知のキット、例えば、MutanTM-super Express Km（宝酒造（株））、MutanTM-K（宝酒造（株））等を用いて、ODA-LA PCR法、Gapped duplex法、Kunkel法等の自体公知の方法あるいはそれらに準じる方法に従って変換することができる。 The DNA base sequence is determined using a known kit such as Mutan ™ -super Express Km (Takara Shuzo), Mutan ™ -K (Takara Shuzo), etc., using the ODA-LA PCR method, the Gapped duplex method, Conversion can be performed according to a method known per se such as the Kunkel method or a method analogous thereto.
　クローン化されたDNAは、目的によりそのまま、または所望により制限酵素で消化するか、リンカーを付加した後に、使用することができる。該DNAはその5’末端側に翻訳開始コドンとしてのATGを有し、また3’末端側には翻訳終止コドンとしてのTAA、TGAまたはTAGを有していてもよい。これらの翻訳開始コドンや翻訳終止コドンは、適当な合成DNAアダプターを用いて付加することができる。 The cloned DNA can be used as it is depending on the purpose, or after digestion with a restriction enzyme or addition of a linker as desired. The DNA may have ATG as a translation initiation codon on the 5 'end side and TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
　GPR120またはその部分ペプチドをコードするDNAを含む発現ベクターは、例えば、GPR120をコードするDNAから目的とするDNA断片を切り出し、該DNA断片を適当な発現ベクター中のプロモーターの下流に連結することにより製造することができる。
　発現ベクターとしては、大腸菌由来のプラスミド（例、pBR322，pBR325，pUC12，pUC13）；枯草菌由来のプラスミド（例、pUB110，pTP5，pC194）；酵母由来プラスミド（例、pSH19，pSH15）；昆虫細胞発現プラスミド（例：pFast-Bac）；動物細胞発現プラスミド（例：pA1-11、pXT1、pRc/CMV、pRc/RSV、pcDNAI/Neo）；λファージなどのバクテリオファージ；バキュロウイルスなどの昆虫ウイルスベクター（例：BmNPV、AcNPV）；レトロウイルス、レンチウイルス、ワクシニアウイルス、アデノウイルス、アデノ随伴ウイルス、ヘルペスウイルスなどの動物ウイルスベクターなどが用いられる。
　プロモーターとしては、遺伝子の発現に用いる宿主に対応して適切なプロモーターであればいかなるものでもよい。
　例えば、宿主が動物細胞である場合、SRαプロモーター、SV40プロモーター、LTRプロモーター、CMV（サイトメガロウイルス）プロモーター、RSV（ラウス肉腫ウイルス）プロモーター、MoMuLV（モロニーマウス白血病ウイルス）LTR、HSV-TK（単純ヘルペスウイルスチミジンキナーゼ）プロモーターなどが用いられる。なかでも、CMVプロモーター、SRαプロモーターなどが好ましい。
　宿主がエシェリヒア属菌である場合、trpプロモーター、lacプロモーター、recAプロモーター、λPLプロモーター、lppプロモーター、T7プロモーターなどが好ましい。
　宿主がバチルス属菌である場合、SPO1プロモーター、SPO2プロモーター、penPプロモーターなどが好ましい。
　宿主が酵母である場合、PHO5プロモーター、PGKプロモーター、GAPプロモーター、ADHプロモーターなどが好ましい。
　宿主が昆虫細胞である場合、ポリヘドリンプロモーター、P10プロモーターなどが好ましい。 An expression vector containing DNA encoding GPR120 or a partial peptide thereof is produced, for example, by excising a target DNA fragment from DNA encoding GPR120 and ligating the DNA fragment downstream of a promoter in an appropriate expression vector. can do.
Expression vectors include plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194); yeast-derived plasmids (eg, pSH19, pSH15); insect cell expression Plasmid (eg, pFast-Bac); animal cell expression plasmid (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophage such as λ phage; insect virus vector such as baculovirus ( Examples: BmNPV, AcNPV); animal virus vectors such as retroviruses, lentiviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, herpes viruses, etc. are used.
The promoter may be any promoter as long as it is appropriate for the host used for gene expression.
For example, when the host is an animal cell, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex) Virus thymidine kinase) promoter or the like is used. Of these, CMV promoter, SRα promoter and the like are preferable.
When the host is Escherichia, trp promoter, lac promoter, recA promoter, λP L promoter, lpp promoter, T7 promoter and the like are preferable.
When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable.
When the host is yeast, the PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable.
When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
　発現ベクターとしては、上記の他に、所望によりエンハンサー、スプライシングシグナル、ポリＡ付加シグナル、選択マーカー、SV40複製起点（以下、SV40 oriと略称する場合がある）などを含有しているものを用いることができる。選択マーカーとしては、例えば、ジヒドロ葉酸還元酵素遺伝子（以下、dhfrと略称する場合がある、メソトレキセート（MTX）耐性）、アンピシリン耐性遺伝子（以下、amprと略称する場合がある）、ネオマイシン耐性遺伝子（以下、neorと略称する場合がある、G418耐性）等が挙げられる。特に、dhfr遺伝子欠損チャイニーズハムスター細胞を用い、dhfr遺伝子を選択マーカーとして使用する場合、チミジンを含まない培地によって目的遺伝子を選択することもできる。
　また、必要に応じて、宿主に合ったシグナル配列をコードする塩基配列（シグナルコドン）を、GPR120またはその部分ペプチドをコードするDNAの5’末端側に付加（またはネイティブなシグナルコドンと置換）してもよい。例えば、宿主がエシェリヒア属菌である場合、PhoA・シグナル配列、OmpA・シグナル配列などが；宿主がバチルス属菌である場合、α-アミラーゼ・シグナル配列、サブチリシン・シグナル配列などが；宿主が酵母である場合、MFα・シグナル配列、SUC2・シグナル配列などが；宿主が動物細胞である場合、インスリン・シグナル配列、α-インターフェロン・シグナル配列、抗体分子・シグナル配列などがそれぞれ用いられる。 In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori), etc. is used as desired. Can do. Examples of the selection marker include a dihydrofolate reductase gene (hereinafter abbreviated as dhfr, methotrexate (MTX) resistance), an ampicillin resistance gene (hereinafter abbreviated as amp r ), a neomycin resistance gene ( hereinafter sometimes abbreviated as neo r, include G418 resistance) and the like. In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selection marker, the target gene can also be selected using a medium that does not contain thymidine.
If necessary, a base sequence (signal codon) encoding a signal sequence suitable for the host is added (or replaced with a native signal codon) to the 5 ′ end of DNA encoding GPR120 or a partial peptide thereof. May be. For example, when the host is Escherichia, PhoA signal sequence, OmpA, signal sequence, etc .; when the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc .; host is yeast In some cases, MFα • signal sequence, SUC2 • signal sequence, etc .; when the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. are used respectively.
　上記したGPR120またはその部分ペプチドをコードするDNAを含む発現ベクターで宿主を形質転換し、得られる形質転換体を培養することによって、GPR120またはその部分ペプチドを製造することができる。
　宿主としては、例えば、エシェリヒア属菌、バチルス属菌、酵母、昆虫細胞、昆虫、動物細胞などが用いられる。
　エシェリヒア属菌としては、例えば、エシェリヒア・コリ（Escherichia coli）K12・DH1〔プロシージングズ・オブ・ザ・ナショナル・アカデミー・オブ・サイエンシイズ・オブ・ザ・ユーエスエー（Proc. Natl. Acad. Sci. USA），60巻，160 (1968)〕，エシェリヒア・コリJM103〔ヌクレイック・アシッズ・リサーチ（Nucleic Acids Research），9巻，309 (1981)〕，エシェリヒア・コリJA221〔ジャーナル・オブ・モレキュラー・バイオロジー（Journal of Molecular Biology），120巻，517 (1978)〕，エシェリヒア・コリHB101〔ジャーナル・オブ・モレキュラー・バイオロジー，41巻，459 (1969)〕，エシェリヒア・コリC600〔ジェネティックス（Genetics），39巻，440 (1954)〕などが用いられる。
　バチルス属菌としては、例えば、バチルス・サブチルス（Bacillus subtilis）MI114〔ジーン (Gene)，24巻，255 (1983)〕，バチルス・サブチルス207-21〔ジャーナル・オブ・バイオケミストリー（Journal of Biochemistry），95巻，87 (1984)〕などが用いられる。
　酵母としては、例えば、サッカロマイセス・セレビシエ（Saccharomyces cerevisiae）AH22，AH22R-，NA87-11A，DKD-5D，20B-12、シゾサッカロマイセス・ポンベ（Schizosaccharomyces pombe）NCYC1913，NCYC2036、ピキア・パストリス（Pichia pastoris）KM71などが用いられる。 GPR120 or a partial peptide thereof can be produced by transforming a host with an expression vector containing a DNA encoding GPR120 or a partial peptide thereof and culturing the resulting transformant.
As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
Examples of the genus Escherichia include, for example, Escherichia coli K12 • DH1 [Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA). ), 60, 160 (1968)], Escherichia coli JM103 (Nucleic Acids Research, 9, 309 (1981)), Escherichia coli JA221 [Journal of Molecular Biology ( Journal of Molecular Biology), 120, 517 (1978)], Escherichia coli HB101 [Journal of Molecular Biology, 41, 459 (1969)], Escherichia coli C600 [Genetics, 39 Vol., 440 (1954)] is used.
Examples of the genus Bacillus include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], Bacillus subtilis 207-21 [Journal of Biochemistry, 95, 87 (1984)].
Examples of the yeast include Saccharomyces cerevisiae AH22, AH22R − , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris (Pichia pastoris (Pichia pastoris) KM71 etc. are used.
　昆虫細胞としては、例えば、ウイルスがAcNPVの場合、夜盗蛾の幼虫由来株化細胞（Spodoptera frugiperda cell；Sf細胞）、Trichoplusia niの中腸由来のMG1細胞、Trichoplusia niの卵由来のHigh FiveTM細胞、Mamestra brassicae由来の細胞、Estigmena acrea由来の細胞などが用いられる。ウイルスがBmNPVの場合、昆虫細胞としては、蚕由来株化細胞（Bombyx mori N 細胞；BmN細胞）などが用いられる。該Sf細胞としては、例えば、Sf9細胞（ATCC CRL1711）、Sf21細胞（以上、Vaughn, J.L.ら、イン・ヴィボ（In Vivo）,13, 213-217,(1977)）などが用いられる。
　昆虫としては、例えば、カイコの幼虫などが用いられる〔前田ら、ネイチャー（Nature），315巻，592 (1985)〕。 Insect cells include, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five TM cells derived from eggs of Trichoplusia ni , Cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used. When the virus is BmNPV, moth-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used as insect cells. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)) and the like.
Examples of insects include silkworm larvae [Maeda et al., Nature, 315, 592 (1985)].
　動物細胞としては、例えば、サルCOS-7細胞、サルVero細胞、チャイニーズハムスター卵巣細胞（以下、CHO細胞と略記）、dhfr遺伝子欠損CHO細胞（以下、CHO(dhfr-)細胞と略記）、マウスL細胞，マウスAtT-20細胞、マウスミエローマ細胞，ラットGH3細胞、ヒトFL細胞、HeLa細胞、HepG2細胞、HEK293細胞などが用いられる。 Examples of animal cells include monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO cells (hereinafter abbreviated as CHO (dhfr − ) cells), mouse L Cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, human FL cells, HeLa cells, HepG2 cells, HEK293 cells and the like are used.
　形質転換は、宿主の種類に応じ、公知の方法に従って実施することができる。
　エシェリヒア属菌は、例えば、プロシージングズ・オブ・ザ・ナショナル・アカデミー・オブ・サイエンシイズ・オブ・ザ・ユーエスエー（Proc. Natl. Acad. Sci. USA），69巻，2110 (1972)やジーン（Gene），17巻，107 (1982)などに記載の方法に従って形質転換することができる。
　バチルス属菌は、例えば、モレキュラー・アンド・ジェネラル・ジェネティックス（Molecular ＆ General Genetics），168巻，111 (1979)などに記載の方法に従って形質転換することができる。
　酵母は、例えば、メソッズ・イン・エンザイモロジー（Methods in Enzymology），194巻，182-187 (1991)、プロシージングズ・オブ・ザ・ナショナル・アカデミー・オブ・サイエンシイズ・オブ・ザ・ユーエスエー（Proc. Natl. Acad. Sci. USA），75巻，1929 (1978)などに記載の方法に従って形質転換することができる。
　昆虫細胞および昆虫は、例えば、バイオ／テクノロジー（Bio/Technology）,6巻，47-55 (1988)などに記載の方法に従って形質転換することができる。
　動物細胞は、例えば、細胞工学別冊８　新細胞工学実験プロトコール，263-267 (1995)（秀潤社発行）、ヴィロロジー（Virology），52巻，456 (1973)に記載の方法に従って形質転換することができる。 Transformation can be performed according to a known method depending on the type of host.
Escherichia bacteria, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972) and Gene ( Gene), 17, 107 (1982), and the like.
Bacillus can be transformed according to the method described in, for example, Molecular & General Genetics, 168, 111 (1979).
Yeast is, for example, Methods in Enzymology, 194, 182-287 (1991), Proceedings of the National Academy of Sciences of USA ( Proc. Natl. Acad. Sci. USA), 75, 1929 (1978).
Insect cells and insects can be transformed according to the method described in, for example, Bio / Technology, Vol. 6, 47-55 (1988).
Animal cells should be transformed according to the method described in, for example, Cell Engineering Annex 8 New Cell Engineering Experimental Protocol, 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). Can do.
　形質転換体の培養は、宿主の種類に応じ、公知の方法に従って実施することができる。
　例えば、宿主がエシェリヒア属菌またはバチルス属菌である形質転換体を培養する場合、培養に使用される培地としては液体培地が好ましい。また、培地は、形質転換体の生育に必要な炭素源、窒素源、無機物などを含有することが好ましい。ここで、炭素源としては、例えば、グルコース、デキストリン、可溶性澱粉、ショ糖などが；窒素源としては、例えば、アンモニウム塩類、硝酸塩類、コーンスチープ・リカー、ペプトン、カゼイン、肉エキス、大豆粕、バレイショ抽出液などの無機または有機物質が；無機物としては、例えば、塩化カルシウム、リン酸二水素ナトリウム、塩化マグネシウムなどがそれぞれ挙げられる。また、培地には、酵母エキス、ビタミン類、生長促進因子などを添加してもよい。培地のpHは、好ましくは約5～約8である。
　宿主がエシェリヒア属菌である形質転換体を培養する場合の培地としては、例えば、グルコース、カザミノ酸を含むM9培地〔ミラー（Miller），ジャーナル・オブ・エクスペリメンツ・イン・モレキュラー・ジェネティックス (Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972〕が好ましい。必要により、プロモーターを効率よく働かせるために、例えば、3β-インドリルアクリル酸のような薬剤を培地に添加してもよい。
　宿主がエシェリヒア属菌である形質転換体の培養は、通常約15～約43℃で、約3～約24時間行なわれる。必要により、通気や撹拌を行ってもよい。
　宿主がバチルス属菌である形質転換体の培養は、通常約30～約40℃で、約6～約24時間行なわれる。必要により、通気や撹拌を行ってもよい。
　宿主が酵母である形質転換体を培養する場合の培地としては、例えば、バークホールダー（Burkholder）最小培地〔Bostian, K.L.ら，プロシージングズ・オブ・ザ・ナショナル・アカデミー・オブ・サイエンシイズ・オブ・ザ・ユーエスエー（Proc. Natl. Acad. Sci. USA），77巻，4505 (1980)〕や0.5％カザミノ酸を含有するSD培地〔Bitter, G.A.ら，プロシージングズ・オブ・ザ・ナショナル・アカデミー・オブ・サイエンシイズ・オブ・ザ・ユーエスエー（Proc. Natl. Acad. Sci. USA），81巻，5330 (1984)〕などが挙げられる。培地のpHは、好ましくは約5～約8である。培養は、通常約20℃～約35℃で、約24～約72時間行なわれる。必要に応じて、通気や撹拌を行ってもよい。
　宿主が昆虫細胞または昆虫である形質転換体を培養する場合の培地としては、例えばGrace's Insect Medium〔Grace, T.C.C.，ネイチャー（Nature），195巻，788 (1962)〕に非働化した10％ウシ血清等の添加物を適宜加えたものなどが用いられる。培地のｐＨは、好ましくは約6.2～約6.4である。培養は、通常約27℃で、約3～約5日間行なわれる。必要に応じて通気や撹拌を行ってもよい。
　宿主が動物細胞である形質転換体を培養する場合の培地としては、例えば、約5～約20%の胎児ウシ血清を含む最小必須培地（MEM）〔サイエンス（Science），122巻，501 (1952)〕，ダルベッコ改変イーグル培地（DMEM）〔ヴィロロジー（Virology），8巻，396 (1959)〕，RPMI 1640培地〔ジャーナル・オブ・ザ・アメリカン・メディカル・アソシエーション（The Journal of the American Medical Association），199巻，519 (1967)〕，199培地〔プロシージング・オブ・ザ・ソサイエティ・フォー・ザ・バイオロジカル・メディスン（Proceeding of the Society for the Biological Medicine），73巻，1 (1950)〕などが用いられる。培地のpHは、好ましくは約6～約8である。培養は、通常約30℃～約40℃で、約15～約60時間行なわれる。必要に応じて通気や撹拌を行ってもよい。
　以上のようにして、形質転換体の細胞内または細胞外にGPR120またはその部分ペプチドを製造せしめることができる。 The culture of the transformant can be performed according to a known method depending on the type of the host.
For example, when culturing a transformant whose host is an Escherichia or Bacillus genus, a liquid medium is preferable as a medium used for the culture. Moreover, it is preferable that a culture medium contains a carbon source, a nitrogen source, an inorganic substance, etc. which are required for the growth of a transformant. Here, as the carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc .; As the nitrogen source, for example, ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, Inorganic or organic substances such as potato extract; examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added to the medium. The pH of the medium is preferably about 5 to about 8.
As a medium for culturing a transformant whose host is an Escherichia bacterium, for example, an M9 medium containing glucose and casamino acids [Miller, Journal of Experiments in Molecular Genetics ( Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3β-indolylacrylic acid may be added to the medium in order to make the promoter work efficiently.
Culturing of a transformant whose host is an Escherichia bacterium is usually performed at about 15 to about 43 ° C. for about 3 to about 24 hours. If necessary, aeration or agitation may be performed.
Culturing of the transformant whose host is Bacillus is usually performed at about 30 to about 40 ° C. for about 6 to about 24 hours. If necessary, aeration or agitation may be performed.
As a medium for culturing a transformant whose host is yeast, for example, a Burkholder minimum medium [Bostian, KL et al., Proceedings of the National Academy of Sciences of Science The USA (Proc. Natl. Acad. Sci. USA), 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Bitter, GA, et al., Proceedings of the National Academy・ Of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. The pH of the medium is preferably about 5 to about 8. The culture is usually performed at about 20 ° C. to about 35 ° C. for about 24 to about 72 hours. Aeration and agitation may be performed as necessary.
As a medium for culturing a transformant whose host is an insect cell or an insect, for example, 10% bovine serum deactivated in Grace's Insect Medium [Grace, TCC, Nature, 195, 788 (1962)] The thing etc. which added additives, such as these, etc. suitably are used. The pH of the medium is preferably about 6.2 to about 6.4. The culture is usually performed at about 27 ° C. for about 3 to about 5 days. You may perform ventilation | gas_flowing and stirring as needed.
As a medium for culturing a transformant whose host is an animal cell, for example, a minimum essential medium (MEM) containing about 5 to about 20% fetal bovine serum [Science, 122, 501 (1952 ], Dulbecco's Modified Eagle Medium (DMEM) [Virology, Vol. 8, 396 (1959)], RPMI 1640 Medium [The Journal of the American Medical Association, 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] Used. The pH of the medium is preferably about 6 to about 8. The culture is usually performed at about 30 ° C. to about 40 ° C. for about 15 to about 60 hours. You may perform ventilation | gas_flowing and stirring as needed.
As described above, GPR120 or a partial peptide thereof can be produced inside or outside the transformant.
　前記形質転換体を培養して得られる培養物からGPR120またはその部分ペプチドを自体公知の方法に従って分離精製することができる。
　例えば、GPR120またはその部分ペプチドを培養菌体あるいは細胞の細胞質から抽出する場合、培養物から公知の方法で集めた菌体あるいは細胞を適当な緩衝液に懸濁し、超音波、リゾチームおよび／または凍結融解などによって菌体あるいは細胞を破壊した後、遠心分離やろ過により可溶性蛋白質の粗抽出液を得る方法などが適宜用いられる。該緩衝液は、尿素や塩酸グアニジンなどの蛋白質変性剤や、トリトンX-100TMなどの界面活性剤を含んでいてもよい。一方、膜画分からGPR120またはその部分ペプチドを抽出する場合は、上記と同様に菌体あるいは細胞を破壊した後、低速遠心で細胞デブリスを沈澱除去し、上清を高速遠心して細胞膜含有画分を沈澱させる（必要に応じて密度勾配遠心などにより細胞膜画分を精製する）などの方法が用いられる。また、GPR120またはその部分ペプチドが菌体（細胞）外に分泌される場合には、培養物から遠心分離またはろ過等により培養上清を分取するなどの方法が用いられる。
　このようにして得られた可溶性画分、膜画分あるいは培養上清中に含まれるGPR120またはその部分ペプチドの単離・精製は、自体公知の方法に従って行うことができる。このような方法としては、塩析や溶媒沈澱法などの溶解度を利用する方法；透析法、限外ろ過法、ゲルろ過法、およびＳＤＳ－ポリアクリルアミドゲル電気泳動法などの主として分子量の差を利用する方法；イオン交換クロマトグラフィーなどの荷電の差を利用する方法；アフィニティークロマトグラフィーなどの特異的親和性を利用する方法；逆相高速液体クロマトグラフィーなどの疎水性の差を利用する方法；等電点電気泳動法などの等電点の差を利用する方法；などが用いられる。これらの方法は、適宜組み合わせることもできる。 GPR120 or a partial peptide thereof can be separated and purified from a culture obtained by culturing the transformant according to a method known per se.
For example, when GPR120 or a partial peptide thereof is extracted from cultured cells or cytoplasm of cells, the cells or cells collected from the culture by a known method are suspended in an appropriate buffer, and are subjected to ultrasound, lysozyme and / or freezing. For example, a method of obtaining a crude extract of soluble protein by centrifugation or filtration after disrupting cells or cells by thawing or the like is appropriately used. The buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride and a surfactant such as Triton X-100 ™ . On the other hand, when extracting GPR120 or its partial peptide from the membrane fraction, after disrupting the cells or cells in the same manner as described above, the cell debris is precipitated and removed by low-speed centrifugation, and the supernatant is centrifuged at high speed to obtain the cell membrane-containing fraction. A method such as precipitation (purifying a cell membrane fraction by density gradient centrifugation or the like as required) is used. When GPR120 or a partial peptide thereof is secreted outside the cells (cells), a method of separating the culture supernatant from the culture by centrifugation or filtration is used.
Isolation and purification of GPR120 or a partial peptide thereof contained in the thus obtained soluble fraction, membrane fraction or culture supernatant can be performed according to a method known per se. Examples of such methods include the use of solubilities such as salting out and solvent precipitation; mainly the differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. A method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography; A method using a difference in isoelectric point, such as point electrophoresis, is used. These methods can be combined as appropriate.
　かくして得られる蛋白質またはペプチドが遊離体である場合には、自体公知の方法あるいはそれに準じる方法によって、該遊離体を塩に変換することができ、蛋白質またはペプチドが塩として得られた場合には、自体公知の方法あるいはそれに準じる方法により、該塩を遊離体または他の塩に変換することができる。
　なお、形質転換体が産生するGPR120またはその部分ペプチドを、精製前または精製後に適当な蛋白修飾酵素を作用させることにより、任意に修飾を加えたり、ポリペプチドを部分的に除去することもできる。該蛋白修飾酵素としては、例えば、トリプシン、キモトリプシン、アルギニルエンドペプチダーゼ、プロテインキナーゼ、グリコシダーゼなどが用いられる。
　かくして得られるGPR120またはその部分ペプチドの存在は、GPR120に対する抗体を用いたエンザイムイムノアッセイやウエスタンブロッティングなどにより確認することができる。 When the protein or peptide thus obtained is a free form, the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein or peptide is obtained as a salt, The salt can be converted into a free form or other salt by a method known per se or a method analogous thereto.
The GPR120 produced by the transformant or a partial peptide thereof can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The presence of GPR120 or a partial peptide thereof thus obtained can be confirmed by enzyme immunoassay or Western blotting using an antibody against GPR120.
　さらに、GPR120またはその部分ペプチドは、上記のGPR120またはその部分ペプチドをコードするDNAに対応するRNAを鋳型として、ウサギ網状赤血球ライセート、コムギ胚芽ライセート、大腸菌ライセートなどからなる無細胞蛋白質翻訳系を用いてインビトロ合成することができる。あるいは、さらにRNAポリメラーゼを含む無細胞転写／翻訳系を用いて、GPR120またはその部分ペプチドをコードするDNAを鋳型としても合成することができる。無細胞蛋白質転写・翻訳系は市販のものを用いることもできるし、それ自体既知の方法、具体的には大腸菌抽出液はPratt J.M.ら, “Transcription and Tranlation”, Hames B.D.およびHiggins S.J.編, IRL Press, Oxford 179-209 (1984)に記載の方法等に準じて調製することもできる。市販の細胞ライセートとしては、大腸菌由来のものはE.coli S30 extract system (Promega社製)やRTS 500 Rapid Tranlation System (Roche社製)等が挙げられ、ウサギ網状赤血球由来のものはRabbit Reticulocyte Lysate System (Promega社製)等、さらにコムギ胚芽由来のものはPROTEIOSTM (TOYOBO社製)等が挙げられる。このうちコムギ胚芽ライセートを用いたものが好適である。コムギ胚芽ライセートの作製法としては、例えばJohnston F.B.ら, Nature, 179, 160-161 (1957)あるいはErickson A.H.ら, Meth. Enzymol., 96, 38-50 (1996)等に記載の方法を用いることができる。
　蛋白質合成のためのシステムまたは装置としては、バッチ法(Pratt,J.M.ら (1984) 前述)や、アミノ酸、エネルギー源等を連続的に反応系に供給する連続式無細胞蛋白質合成システム（Spirin A.S.ら, Science, 242, 1162-1164 (1988)）、透析法（木川ら、第21回日本分子生物学会、WID6）、あるいは重層法（PROTEIOSTM Wheat germ cell-free protein synthesis core kit取扱説明書：TOYOBO社製）等が挙げられる。さらには、合成反応系に、鋳型のRNA、アミノ酸、エネルギー源等を必要時に供給し、合成物や分解物を必要時に排出する方法（特開2000-333673）等を用いることができる。 Furthermore, GPR120 or a partial peptide thereof uses a cell-free protein translation system comprising a rabbit reticulocyte lysate, a wheat germ lysate, an Escherichia coli lysate, etc., using an RNA corresponding to the DNA encoding the GPR120 or the partial peptide as a template. It can be synthesized in vitro. Alternatively, a cell-free transcription / translation system further containing RNA polymerase can be used to synthesize a DNA encoding GPR120 or a partial peptide thereof as a template. The cell-free protein transcription / translation system can be a commercially available one, or a known method, specifically, Escherichia coli extract is Pratt JM et al., “Transcription and Tranlation”, Hames BD and Higgins SJ, IRL It can also be prepared according to the method described in Press, Oxford 179-209 (1984). Examples of commercially available cell lysates include E. coli S30 extract system (Promega) and RTS 500 Rapid Tranlation System (Roche) derived from E. coli, and Rabbit Reticulocyte Lysate System derived from rabbit reticulocytes. Examples of those derived from wheat germ include Proteios ™ (manufactured by TOYOBO). Of these, those using wheat germ lysate are preferred. As a method for producing wheat germ lysate, for example, the method described in Johnston FB et al., Nature, 179, 160-161 (1957) or Erickson AH et al., Meth. Enzymol., 96, 38-50 (1996), etc. should be used. Can do.
As a system or apparatus for protein synthesis, a batch method (Pratt, JM et al. (1984) mentioned above) or a continuous cell-free protein synthesis system (Spirin AS et al. , Science, 242, 1162-1164 (1988)), dialysis method (Kikawa et al., 21st Japan Molecular Biology Society, WID6) or multi-layer method (PROTEIOS TM Wheat germ cell-free protein synthesis core kit instruction manual: TOYOBO Etc.). Furthermore, a method of supplying template RNA, amino acid, energy source and the like to the synthesis reaction system when necessary, and discharging the synthesized product and degradation product when necessary (Japanese Patent Laid-Open No. 2000-333673) can be used.
　本発明で用いられる「GPR120またはその部分ペプチドを産生する細胞」としては、生来GPR120を発現する細胞（例えば、EPC（large EPC、small EPC）、脂肪細胞、腸内分泌細胞等）およびその前駆細胞（例えば、骨髄細胞、脂肪前駆細胞等）、並びに前記GPR120またはその部分ペプチドをコードする核酸を導入された形質転換細胞などが挙げられる。形質転換細胞を用いてGPR120またはその部分ペプチドの発現を指標にしてスクリーニングを行う場合、GPR120またはその部分ペプチドをコードする核酸の発現を制御するプロモーターおよび／またはエンハンサー／サプレッサーとして、GPR120遺伝子の内在のプロモーターおよび／またはエンハンサー／サプレッサー配列を用いることが好ましい。このようなGPR120遺伝子の調節配列は、自体公知の方法により、哺乳動物の細胞・組織から抽出したゲノムDNAから単離・クローン化することができる。
　GPR120またはその部分ペプチドを産生する細胞として、好ましくはEPC（large EPC、small EPC）およびその前駆細胞（骨髄細胞）が挙げられる。これらの細胞を用いると、内在のGPR120の活性もしくは発現を指標としてEPCの分化・増殖調節または末梢血中のEPC増加物質をスクリーニングできるだけでなく、実際に骨髄細胞からEPCへの分化や、EPCの増殖速度を調節し得るか否かの検証を行うこともできる。尚、本明細書において「骨髄細胞」とは、特にことわらない限り、骨髄に存在するEPCの前駆細胞全般を含むものとする。例えば、代表的なEPCの前駆細胞は、各種細胞表面抗原マーカーの発現に関し、c-kit+，Sca-1+，lineage marker-で特徴づけられる画分から取得することができる。 As used herein, “cells producing GPR120 or a partial peptide thereof” include cells that naturally express GPR120 (eg, EPC (large EPC, small EPC), adipocytes, enteroendocrine cells, etc.) and progenitor cells thereof ( For example, bone marrow cells, preadipocytes, etc.), and transformed cells into which a nucleic acid encoding GPR120 or a partial peptide thereof has been introduced. When screening is performed using the transformed cells using the expression of GPR120 or a partial peptide thereof as an index, the promoter and / or enhancer / suppressor that regulates the expression of the nucleic acid encoding GPR120 or the partial peptide is used as an endogenous GPR120 gene. It is preferred to use promoters and / or enhancer / suppressor sequences. Such a regulatory sequence of GPR120 gene can be isolated and cloned from genomic DNA extracted from mammalian cells and tissues by a method known per se.
The cells that produce GPR120 or a partial peptide thereof preferably include EPC (large EPC, small EPC) and its precursor cells (bone marrow cells). Using these cells, not only can EPC differentiation / proliferation regulation or EPC increase in peripheral blood be screened using the activity or expression of endogenous GPR120 as an index, but differentiation from bone marrow cells to EPC, It is also possible to verify whether the growth rate can be adjusted. In the present specification, “bone marrow cells” include all EPC progenitor cells present in bone marrow unless otherwise specified. For example, typical EPC progenitor cells can be obtained from fractions characterized by c-kit + , Sca-1 + , and lineage marker − for the expression of various cell surface antigen markers.
（１ａ）GPR120とリガンドとの結合性を変化させる化合物のスクリーニング
　上記のようにして得られるGPR120またはその部分ペプチド、または天然もしくは組換えGPR120発現系を用いたアフィニティーアッセイ系を利用することによって、GPR120とリガンドとの結合性を変化させる化合物またはその塩を効率よくスクリーニングすることができる。
　このような化合物には、（a）GPR120に結合して細胞刺激活性（例えば、共役GαにおけるGTP-GDP交換反応促進、PLCβ活性化、イノシトールリン酸産生調節、細胞内Ca2+取り込み、PKC活性化、EPCの分化促進、EPCの増殖促進、インスリン分泌促進、血糖低下、ACTH分泌抑制、成長ホルモン分泌調節など）を発揮する化合物（アゴニスト）、（b）GPR120に結合するが該細胞刺激活性を有しない化合物（アンタゴニスト）、（c）GPR120とリガンドとの結合力を増強する化合物、あるいは（d）GPR120とリガンドとの結合力を減少させる化合物などが含まれる。
　GPR120は、骨髄細胞からEPCへの分化（本明細書において単に「EPCの分化」という場合は、骨髄細胞からEPCへの分化を意味する）およびEPCの増殖を促進する作用を有するので、GPR120のアゴニストまたはGPR120とリガンドとの結合力を増強する化合物は、EPCの分化・増殖を促進することができる。一方、GPR120のアンタゴニストまたはGPR120とリガンドとの結合力を減少させる化合物は、EPCの分化・増殖を抑制することができる。また、GPR120の抑制は末梢血へのEPCの動員を促進することから、GPR120のアンタゴニストまたはGPR120とリガンドとの結合力を減少させる化合物は、末梢血中EPCを増加させることにも有用である。 (1a) Screening of a compound that changes the binding property between GPR120 and a ligand By utilizing an affinity assay system using GPR120 or a partial peptide thereof obtained as described above, or a natural or recombinant GPR120 expression system, GPR120 Can be efficiently screened for a compound or its salt that alters the binding property between a ligand and a ligand.
Such compounds include (a) GPR120 binding to cell stimulating activity (eg, GTP-GDP exchange reaction acceleration in conjugated Gα, PLCβ activation, regulation of inositol phosphate production, intracellular Ca 2+ uptake, PKC activity) , EPC differentiation promotion, EPC proliferation promotion, insulin secretion promotion, hypoglycemia, ACTH secretion suppression, growth hormone secretion regulation, etc.) (b) A compound that binds to GPR120 but exhibits cell-stimulating activity A compound that does not have (antagonist), (c) a compound that enhances the binding force between GPR120 and the ligand, or (d) a compound that decreases the binding force between GPR120 and the ligand.
Since GPR120 has an action of promoting differentiation of bone marrow cells to EPC (in the present specification, “differentiation of EPC” simply means differentiation of bone marrow cells to EPC) and proliferation of EPC, A compound that enhances the binding force between an agonist or GPR120 and a ligand can promote differentiation and proliferation of EPC. On the other hand, an antagonist of GPR120 or a compound that decreases the binding force between GPR120 and a ligand can suppress the differentiation / proliferation of EPC. In addition, since inhibition of GPR120 promotes the recruitment of EPC to peripheral blood, an antagonist of GPR120 or a compound that decreases the binding force between GPR120 and a ligand is also useful for increasing EPC in peripheral blood.
　したがって、本発明は、GPR120またはその部分ペプチドと、生理的リガンドである脂肪酸または脂肪酸とGPR120との結合性を変化させる公知の低分子化合物（以下、「サロゲートリガンド」という場合がある）とを、試験化合物の存在下および非存在下で接触させ、両条件下におけるGPR120またはその部分ペプチドの活性を比較することを特徴とする、EPCの分化・増殖調節（促進もしくは抑制）または末梢血中のEPC増加物質のスクリーニング方法を提供する。
　上記スクリーニング方法において、GPR120またはその部分ペプチドの活性は、該蛋白質またはペプチドに対するリガンド（脂肪酸またはサロゲートリガンド）の結合量、あるいは上記細胞刺激活性などを指標として測定される。 Therefore, the present invention includes GPR120 or a partial peptide thereof and a known low-molecular compound (hereinafter sometimes referred to as “surrogate ligand”) that changes the binding property of fatty acid that is a physiological ligand or fatty acid and GPR120. EPC differentiation / proliferation regulation (promotion or suppression) or EPC in peripheral blood, characterized by contacting in the presence and absence of a test compound and comparing the activity of GPR120 or its partial peptide under both conditions A method for screening increased substances is provided.
In the screening method described above, the activity of GPR120 or a partial peptide thereof is measured using the binding amount of a ligand (fatty acid or surrogate ligand) to the protein or peptide, or the cell stimulating activity as an index.
　より具体的には、本発明は、
(1)標識した脂肪酸またはサロゲートリガンドと、GPR120またはその部分ペプチドとを、試験化合物の存在下および非存在下で接触させた場合における、標識した脂肪酸またはサロゲートリガンドのGPR120またはその部分ペプチドに対する結合量を測定し、比較することを特徴とする、EPCの分化・増殖調節または末梢血中のEPC増加物質のスクリーニング方法、
(2)標識した脂肪酸またはサロゲートリガンドと、GPR120またはその部分ペプチドを産生する細胞またはその膜画分とを、試験化合物の存在下および非存在下で接触させた場合における、標識した脂肪酸またはサロゲートリガンドの該細胞または膜画分に対する結合量を測定し、比較することを特徴とする、EPCの分化・増殖調節または末梢血中のEPC増加物質のスクリーニング方法、
(3)標識した脂肪酸またはサロゲートリガンドと、GPR120またはその部分ペプチドをコードするDNAを含有する形質転換体を培養することによりその細胞膜上に発現させた該蛋白質またはペプチドとを、試験化合物の存在下および非存在下で接触させた場合における、標識した脂肪酸またはサロゲートリガンドのGPR120またはその部分ペプチドに対する結合量を測定し、比較することを特徴とする、EPCの分化・増殖調節または末梢血中のEPC増加物質のスクリーニング方法、
(4)脂肪酸またはサロゲートリガンドと、GPR120またはその部分ペプチドを細胞膜上に発現する細胞とを、試験化合物の存在下および非存在下で接触させた場合における、GPR120またはその部分ペプチドを介した細胞刺激活性（例えば、共役GαにおけるGTP-GDP交換反応促進、PLCβ活性化、イノシトールリン酸産生調節、細胞内Ca2+取り込み、PKC活性化など）を測定し、比較することを特徴とする、EPCの分化・増殖調節または末梢血中のEPC増加物質のスクリーニング方法、および
(5)脂肪酸またはサロゲートリガンドと、GPR120またはその部分ペプチドをコードするDNAを含有する形質転換体を培養することによりその細胞膜上に発現させた該蛋白質またはペプチドとを、試験化合物の存在下および非存在下で接触させた場合における、GPR120またはその部分ペプチドを介した細胞刺激活性（例えば、共役GαにおけるGTP-GDP交換反応促進、PLCβ活性化、イノシトールリン酸産生調節、細胞内Ca2+取り込み、PKC活性化など）を測定し、比較することを特徴とする、EPCの分化・増殖調節または末梢血中のEPC増加物質のスクリーニング方法を提供する。 More specifically, the present invention provides:
(1) Binding amount of labeled fatty acid or surrogate ligand to GPR120 or its partial peptide when the labeled fatty acid or surrogate ligand is contacted with GPR120 or its partial peptide in the presence or absence of the test compound EPC differentiation / proliferation regulation or screening method for EPC increasing substance in peripheral blood, characterized by measuring and comparing
(2) A labeled fatty acid or surrogate ligand when the labeled fatty acid or surrogate ligand is contacted with a cell producing GPR120 or a partial peptide thereof or a membrane fraction thereof in the presence and absence of a test compound A method of screening for a substance that regulates differentiation / proliferation of EPC or an EPC-increasing substance in peripheral blood, which comprises measuring and comparing the binding amount of the cells to the cell or membrane fraction,
(3) A labeled fatty acid or surrogate ligand and the protein or peptide expressed on the cell membrane by culturing a transformant containing DNA encoding GPR120 or a partial peptide thereof in the presence of a test compound EPC in the differentiation or proliferation of EPC or EPC in peripheral blood, characterized by measuring and comparing the amount of labeled fatty acid or surrogate ligand bound to GPR120 or its partial peptide when contacted in the absence of Increasing substance screening method,
(4) Cell stimulation via GPR120 or its partial peptide when fatty acid or surrogate ligand is contacted with cells expressing GPR120 or its partial peptide on the cell membrane in the presence or absence of the test compound EPCs characterized by measuring and comparing activities (eg, GTP-GDP exchange reaction acceleration in conjugated Gα, PLCβ activation, regulation of inositol phosphate production, intracellular Ca 2+ uptake, PKC activation, etc.) Methods for screening for differentiation / proliferation regulation or EPC increasing substances in peripheral blood, and
(5) Fatty acid or surrogate ligand and the protein or peptide expressed on the cell membrane by culturing a transformant containing DNA encoding GPR120 or a partial peptide thereof in the presence or absence of the test compound Cell stimulation activity via GPR120 or a partial peptide thereof when contacted in the presence (for example, promotion of GTP-GDP exchange reaction in conjugated Gα, activation of PLCβ, regulation of inositol phosphate production, intracellular Ca 2+ uptake, The present invention provides a screening method for EPC differentiation / proliferation regulation or EPC increasing substance in peripheral blood, characterized by measuring and comparing PKC activation and the like.
　以下に本発明のスクリーニング方法をさらに具体的に説明する。
　まず、本発明のスクリーニング方法に用いるGPR120またはその部分ペプチドとしては、上記したGPR120またはその部分ペプチドを含有するものであれば何れのものであってもよいが、細胞外領域の生理的なコンフォーメーションを再現し得るという点から、GPR120またはその部分ペプチドを産生する哺乳動物の臓器の細胞膜画分が好適である。しかし、特にヒト由来の臓器は入手が困難なことから、スクリーニングに用いられるものとしては、組換え体を用いて大量発現させたヒト由来のGPR120またはその部分ペプチドなどが適している。 Hereinafter, the screening method of the present invention will be described more specifically.
First, GPR120 or a partial peptide thereof used in the screening method of the present invention may be any as long as it contains GPR120 or the partial peptide described above, but physiological conformation of the extracellular region. Is preferably a cell membrane fraction of a mammalian organ that produces GPR120 or a partial peptide thereof. However, since human-derived organs are particularly difficult to obtain, human-derived GPR120 or a partial peptide thereof expressed in large quantities using a recombinant is suitable for use in screening.
　GPR120またはその部分ペプチドを製造するには、前述の方法が用いられるが、GPR120またはその部分ペプチドをコードするDNAを哺乳動物細胞や昆虫細胞で発現させることにより行なうことが好ましい。目的とする蛋白質部分をコードするDNA断片にはcDNAが用いられるが、必ずしもこれに制約されるものではない。例えば、遺伝子断片や合成DNAを用いてもよい。GPR120またはその部分ペプチドをコードするDNA断片を宿主動物（昆虫）細胞に導入し、それらを効率よく発現させるためには、該DNA断片を、SV40由来のプロモーター、レトロウイルスのプロモーター、メタロチオネインプロモーター、ヒトヒートショックプロモーター、サイトメガロウイルスプロモーター、SRαプロモーター、昆虫を宿主とするバキュロウイルスに属する核多角体病ウイルス（nuclear polyhedrosis virus；NPV）のポリヘドリンプロモーターなどの下流に組み込むのが好ましい。 In order to produce GPR120 or a partial peptide thereof, the above-mentioned method is used, but it is preferable to carry out expression in mammalian cells or insect cells of DNA encoding GPR120 or a partial peptide thereof. CDNA is used as a DNA fragment encoding a target protein portion, but is not necessarily limited thereto. For example, gene fragments or synthetic DNA may be used. In order to introduce a DNA fragment encoding GPR120 or a partial peptide thereof into a host animal (insect) cell and to express them efficiently, the DNA fragment can be expressed using an SV40-derived promoter, a retroviral promoter, a metallothionein promoter, human It is preferably incorporated downstream of a heat shock promoter, a cytomegalovirus promoter, an SRα promoter, a polyhedrin promoter of a nuclear polyhedrosis virus (NPV) belonging to baculoviruses with insect hosts.
　上記スクリーニング方法において、GPR120またはその部分ペプチドを産生する細胞を用いる場合、該細胞をグルタルアルデヒド、ホルマリンなどで固定化してもよい。固定化方法はそれ自体公知の方法に従って行なうことができる。
　GPR120またはその部分ペプチドを産生する細胞とは、GPR120またはその部分ペプチドを発現した宿主細胞をいうが、該宿主細胞としては、動物細胞、昆虫細胞などが用いられる。
　前記細胞膜画分とは、細胞を破砕した後、それ自体公知の方法で得られる細胞膜が多く含まれる画分のことをいう。細胞の破砕方法としては、Potter－Elvehjem型ホモジナイザーで細胞を押し潰す方法、ワーリングブレンダーやポリトロン（Kinematica社製）による破砕、超音波による破砕、フレンチプレスなどで加圧しながら細胞を細いノズルから噴出させることによる破砕などが挙げられる。細胞膜の分画には、分画遠心分離法や密度勾配遠心分離法などの遠心力による分画法が主として用いられる。例えば、細胞破砕液を低速（500rpm～3000rpm）で短時間（通常、約1～10分）遠心し、上清をさらに高速（15000rpm～30000rpm）で通常30分～2時間遠心し、得られる沈澱を膜画分とする。該膜画分中には、発現したGPR120またはその部分ペプチドと細胞由来のリン脂質や膜蛋白質などの膜成分が多く含まれる。
　あるいは、各種の脂質を適当な比率、好ましくは真核生物由来細胞の細胞膜におけるそれに近い比率で混合した溶液から常法により調製される人工脂質二重層中で、前記のようにして取得したGPR120またはその部分ペプチドを再構成させたものを、前記細胞膜画分として使用することができる。人工脂質二重層を構成する脂質としては、ホスファチジルコリン（PC）、ホスファチジルセリン（PS）、コレステロール（Ch）、ホスファチジルイノシトール（PI）、ホスファチジルエタノールアミン（PE）等が挙げられ、これら１種または２種以上を適当な比率で混合したものが好ましく使用される。例えば、GPR120またはその部分ペプチドを組み込んだ人工脂質二重層（プロテオリポソーム）は、以下の方法により調製することができる。即ち、まず、PC:PI:Ch=12:12:1の混合脂質クロロホルム溶液を適当量ガラスチューブに分取し、窒素ガス蒸気でクロロホルムを蒸発させて脂質をフィルム状に乾燥させた後、適当な緩衝液を加えて懸濁、次いで超音波処理により均一に分散させ、コール酸ナトリウム等の界面活性剤を含む緩衝液をさらに加えて脂質を完全に懸濁する。ここに、精製した融合蛋白質を適量添加し、氷中で時々攪拌しながら20～30分間程度インキュベートした後、適当な緩衝液に対して透析する。約100,000×gで30～60分間遠心して沈渣を回収することにより、所望のプロテオリポソームを得ることができる。 In the above screening method, when cells that produce GPR120 or a partial peptide thereof are used, the cells may be fixed with glutaraldehyde, formalin or the like. The immobilization method can be performed according to a method known per se.
A cell that produces GPR120 or a partial peptide thereof refers to a host cell that expresses GPR120 or a partial peptide thereof. Examples of the host cell include animal cells and insect cells.
The cell membrane fraction refers to a fraction containing a lot of cell membranes obtained by a method known per se after disrupting cells. Cell disruption methods include crushing cells with a Potter-Elvehjem homogenizer, disrupting with a Waring blender or polytron (manufactured by Kinematica), disrupting with ultrasonic waves, and pressurizing with a French press while ejecting cells from a thin nozzle. Crushing by things. For fractionation of the cell membrane, a fractionation method using centrifugal force such as a fractional centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate is centrifuged at low speed (500 rpm to 3000 rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further centrifuged at high speed (15000 rpm to 30000 rpm) for usually 30 minutes to 2 hours. Is the membrane fraction. The membrane fraction is rich in the expressed GPR120 or a partial peptide thereof and membrane components such as cell-derived phospholipids and membrane proteins.
Alternatively, GPR120 obtained as described above in an artificial lipid bilayer prepared by a conventional method from a solution in which various lipids are mixed at an appropriate ratio, preferably a ratio close to that in the cell membrane of a eukaryotic cell. The reconstituted partial peptide can be used as the cell membrane fraction. Examples of the lipid constituting the artificial lipid bilayer include phosphatidylcholine (PC), phosphatidylserine (PS), cholesterol (Ch), phosphatidylinositol (PI), phosphatidylethanolamine (PE), etc., one or two of these What mixed the above by the appropriate ratio is used preferably. For example, an artificial lipid bilayer (proteoliposome) incorporating GPR120 or a partial peptide thereof can be prepared by the following method. That is, first, dispense a suitable amount of mixed lipid chloroform solution of PC: PI: Ch = 12: 12: 1 into a glass tube, evaporate chloroform with nitrogen gas vapor and dry the lipid into a film, A buffer solution containing a surfactant such as sodium cholate is further added to completely suspend the lipid. An appropriate amount of the purified fusion protein is added thereto, incubated for about 20 to 30 minutes with occasional stirring in ice, and then dialyzed against an appropriate buffer. The desired proteoliposome can be obtained by collecting the sediment by centrifugation at about 100,000 × g for 30 to 60 minutes.
　GPR120またはその部分ペプチドを産生する細胞やその膜画分中のGPR120またはその部分ペプチドの量は、１細胞当たり103～108分子であるのが好ましく、105～107分子であるのがより好適である。なお、発現量が多いほど膜画分当たりのリガンド結合活性（比活性）が高くなり、高感度なスクリーニング系の構築が可能になるばかりでなく、同一ロットで大量の試料を測定できるようになる。 The amount of GPR120 or its partial peptide in a cell producing GPR120 or its partial peptide or its membrane fraction is preferably 10 3 to 10 8 molecules, more preferably 10 5 to 10 7 molecules per cell. More preferred. In addition, the higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, making it possible not only to construct a highly sensitive screening system, but also to measure a large amount of samples in the same lot. .
　GPR120またはその部分ペプチドと脂肪酸またはサロゲートリガンドとの結合性を変化させる化合物をスクリーニングする上記の(1)～(3)を実施するためには、例えば、適当なGPR120またはその部分ペプチド含有膜画分と、標識した脂肪酸またはサロゲートリガンドとが必要である。
　GPR120またはその部分ペプチド含有膜画分としては、天然型のGPR120またはその部分ペプチド含有膜画分か、またはそれと同等の活性を有する組換えGPR120またはその部分ペプチド含有膜画分などが望ましい。ここで、同等の活性とは、同等のリガンド結合活性などを示す。 In order to carry out the above (1) to (3) for screening a compound that changes the binding property between GPR120 or a partial peptide thereof and a fatty acid or a surrogate ligand, for example, an appropriate GPR120 or a partial peptide-containing membrane fraction thereof is used. And a labeled fatty acid or surrogate ligand.
As GPR120 or a partial peptide-containing membrane fraction thereof, a natural GPR120 or a partial peptide-containing membrane fraction thereof, or a recombinant GPR120 having a similar activity or a partial peptide-containing membrane fraction thereof is desirable. Here, the equivalent activity indicates an equivalent ligand binding activity and the like.
　脂肪酸としては、GPR120またはその部分ペプチドに対するリガンドとなり得る限りいかなるであってもよいが、炭素数14～22の脂肪酸（例えば、オレイン酸、パルミトレイン酸、リノール酸、γ－リノレン酸、アラキドン酸、ドコサヘキサエン酸など）が用いられ、中でもパルミトレイン酸、リノール酸、γ－リノレン酸が好ましく用いられる。脂肪酸は遊離体であっても塩の形態であってもよい。脂肪酸の塩としては、例えば、塩基との塩（例、ナトリウム塩、カリウム塩等のアルカリ金属塩、カルシウム塩、マグネシウム塩等のアルカリ土類金属塩、アンモニウム塩、アルカノールアミン塩など）が好ましく挙げられるが、それらに限定されない。
　「脂肪酸とGPR120との結合性を変化させる低分子化合物」、即ちサロゲートリガンドとしては、例えば、上記特許文献２に記載される、式： The fatty acid may be any fatty acid as long as it can serve as a ligand for GPR120 or a partial peptide thereof. Acid) and the like. Among them, palmitoleic acid, linoleic acid, and γ-linolenic acid are preferably used. The fatty acid may be a free form or a salt form. Preferred examples of the fatty acid salts include salts with bases (eg, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, ammonium salts and alkanolamine salts). However, it is not limited to them.
Examples of the “low molecular weight compound that changes the binding property between fatty acid and GPR120”, that is, the surrogate ligand, include, for example, the formula:
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
〔式中、環Ａは置換基を有していてもよい芳香環を、環Ｂは－Ｙ－ＣＯＯＨ以外にさらに置換基を有していてもよい芳香環を、ＸおよびＹはそれぞれスペーサーを表し、－Ｙ－ＣＯＯＨは環Ｂ上の任意の位置に置換している。〕で表わされる化合物もしくはその塩などが挙げられるが、それらに限定されない。 [In the formula, ring A represents an aromatic ring which may have a substituent, ring B represents an aromatic ring which may further have a substituent other than —Y—COOH, and X and Y each represent a spacer. -Y-COOH is substituted at any position on ring B. ] Or a salt thereof and the like, but are not limited thereto.
　脂肪酸またはサロゲートリガンドは、常法に従って、例えば放射性同位元素、酵素、蛍光物質、発光物質などで標識することができる。放射性同位元素としては、例えば、〔125I〕、〔131I〕、〔3H〕、〔14C〕などが用いられる。酵素としては、安定で比活性の大きなものが好ましく、例えば、β－ガラクトシダーゼ、β－グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素などが用いられる。蛍光物質としては、例えば、フルオレスカミン、フルオレッセンイソチオシアネートなどが用いられる。発光物質としては、例えば、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなどが用いられる。 The fatty acid or surrogate ligand can be labeled with, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like according to a conventional method. As the radioisotope, for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used. As the enzyme, those which are stable and have high specific activity are preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
　具体的には、GPR120またはその部分ペプチドと脂肪酸またはサロゲートリガンドとの結合性を変化させる化合物のスクリーニングを行なうには、まずGPR120またはその部分ペプチドを産生する細胞またはその膜画分を、スクリーニングに適したバッファーに懸濁することによりGPR120またはその部分ペプチド標品を調製する。バッファーは、pH4～10（望ましくはpH6～8）のリン酸バッファー、トリス－塩酸バッファーなどのGPR120またはその部分ペプチドと脂肪酸（もしくはサロゲートリガンド）との結合を阻害しないバッファーであればいずれでもよい。また、非特異的結合を低減させる目的で、CHAPS、Tween-80TM（花王－アトラス社）、ジギトニン、デオキシコレートなどの界面活性剤をバッファーに加えることもできる。さらに、プロテアーゼによるレセプターの分解を抑える目的でPMSF、ロイペプチン、E-64（ペプチド研究所製）、ペプスタチンなどのプロテアーゼ阻害剤を添加することもできる。0.01～10mlのGPR120またはその部分ペプチド懸濁液に、一定量（放射性同位元素の場合、5000cpm～500000cpm）の標識した脂肪酸またはサロゲートリガンドを添加し、同時に10-4M～10-10Mの試験化合物を共存させる。非特異的結合量（NSB）を知るために大過剰の未標識のリガンドを加えた反応チューブも用意する。反応は約0℃から50℃、望ましくは約4℃から37℃で、約20分から24時間、望ましくは約30分から3時間行う。反応後、ガラス繊維濾紙等で濾過し、適量の同バッファーで洗浄した後、ガラス繊維濾紙に残存する放射活性を液体シンチレーションカウンターまたはγ-カウンターで計測する。競合する物質がない場合のカウント(B0）から非特異的結合量（NSB）を引いたカウント（B0-NSB）を100％とした時、特異的結合量（B-NSB）が、例えば、50％以下になる試験化合物をEPCの分化・増殖調節または末梢血中のEPC増加物質の候補化合物として選択することができる。 Specifically, in order to screen for a compound that changes the binding property between GPR120 or a partial peptide thereof and a fatty acid or a surrogate ligand, first, cells that produce GPR120 or a partial peptide thereof or a membrane fraction thereof are suitable for screening. GPR120 or a partial peptide preparation thereof is prepared by suspending in a buffer. The buffer may be any buffer that does not inhibit the binding of GPR120 or a partial peptide thereof and a fatty acid (or surrogate ligand), such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a Tris-HCl buffer. For the purpose of reducing non-specific binding, a surfactant such as CHAPS, Tween-80 ™ (Kao-Atlas), digitonin, deoxycholate and the like can be added to the buffer. Furthermore, protease inhibitors such as PMSF, leupeptin, E-64 (manufactured by Peptide Institute) and pepstatin can be added for the purpose of suppressing the degradation of the receptor by the protease. Add a fixed amount (5000 cpm to 500000 cpm for radioisotope) of labeled fatty acid or surrogate ligand to 0.01 to 10 ml of GPR120 or its partial peptide suspension, and test 10 -4 M to 10 -10 M at the same time Make the compound coexist. Prepare a reaction tube with a large excess of unlabeled ligand to determine the amount of non-specific binding (NSB). The reaction is carried out at about 0 ° C. to 50 ° C., preferably about 4 ° C. to 37 ° C., for about 20 minutes to 24 hours, preferably about 30 minutes to 3 hours. After the reaction, the solution is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and then the radioactivity remaining on the glass fiber filter is measured with a liquid scintillation counter or γ-counter. When nonspecific binding amount from the count (B 0) when there is no conflicting material count obtained by subtracting the (NSB) (B 0 -NSB) was 100%, the specific binding amount (B-NSB) is, for example, Therefore, a test compound that is 50% or less can be selected as a candidate compound for EPC differentiation / proliferation regulation or an EPC increasing substance in peripheral blood.
　GPR120またはその部分ペプチドと脂肪酸またはサロゲートリガンドとの結合性を変化させる化合物をスクリーニングする上記の(4)～(5)の方法を実施するためには、例えば、GPR120またはその部分ペプチドを介する細胞刺激活性（例えば、共役GαにおけるGTP-GDP交換反応促進、PLCβ活性化、イノシトールリン酸産生調節、細胞内Ca2+取り込み、PKC活性化など）を公知の方法または市販の測定用キットなどを用いて測定する。
　具体的には、まず、GPR120またはその部分ペプチドを産生する細胞をマルチウェルプレート等に培養する。スクリーニングを行うにあたっては、前もって新鮮な培地あるいは細胞に毒性を示さない適当なバッファー（例えば、HEPES緩衝液、リン酸緩衝液、リン酸緩衝生理食塩水、トリス塩酸緩衝液、ホウ酸緩衝液、酢酸緩衝液など）に交換し、リガンド、試験化合物などを添加して一定時間インキュベートした後、細胞を抽出あるいは上清を回収して、生成した産物をそれぞれの方法に従って定量する。細胞刺激活性の指標とする物質の生成が、細胞が含有する分解酵素によって検定困難な場合は、該分解酵素に対する阻害剤を添加してアッセイを行なってもよい。
　細胞刺激活性を測定してスクリーニングを行なうには、GPR120またはその部分ペプチドを膜上に発現した適当な細胞が必要である。GPR120またはその部分ペプチドを発現した細胞としては、天然型のGPR120を産生する細胞、前述の組換えGPR120またはその部分ペプチドを発現した細胞などが望ましい。
　試験化合物としては、例えば、ペプチド、蛋白質、非ペプチド性化合物、合成化合物、発酵生産物、細胞抽出液、植物抽出液、動物組織抽出液などが用いられ、これら化合物は新規な化合物であってもよいし、公知の化合物であってもよい。 In order to carry out the above methods (4) to (5) for screening a compound that changes the binding property between GPR120 or a partial peptide thereof and a fatty acid or a surrogate ligand, for example, cell stimulation via GPR120 or a partial peptide thereof Activity (for example, promotion of GTP-GDP exchange reaction in conjugated Gα, activation of PLCβ, regulation of inositol phosphate production, intracellular Ca 2+ uptake, activation of PKC, etc.) using known methods or commercially available measurement kits, etc. taking measurement.
Specifically, first, cells that produce GPR120 or a partial peptide thereof are cultured in a multiwell plate or the like. In conducting screening, an appropriate buffer that is not toxic to fresh medium or cells in advance (eg, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetic acid) The solution is exchanged with a buffer solution, etc., and a ligand, a test compound, etc. are added and incubated for a certain period of time. Then, the cells are extracted or the supernatant is collected, and the produced product is quantified according to each method. When the production of a substance used as an index of cell stimulating activity is difficult to assay with a degrading enzyme contained in cells, an assay may be performed with an inhibitor for the degrading enzyme added.
In order to perform screening by measuring cell stimulating activity, appropriate cells expressing GPR120 or a partial peptide thereof on the membrane are required. As a cell expressing GPR120 or a partial peptide thereof, a cell producing natural GPR120, a cell expressing the aforementioned recombinant GPR120 or a partial peptide thereof, or the like is desirable.
Examples of test compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like, and these compounds may be novel compounds. It may be a known compound.
　GPR120またはその部分ペプチドと脂肪酸またはサロゲートリガンドの結合性を変化させる化合物またはその塩のスクリーニング用キットは、GPR120またはその部分ペプチド、GPR120またはその部分ペプチドを産生する細胞またはその膜画分などを含有するものである。
　本発明のスクリーニング用キットの例としては、次のものが挙げられる。
１．スクリーニング用試薬
(a)測定用緩衝液および洗浄用緩衝液
　Hanks' Balanced Salt Solution（ギブコ社製）に、0.05％のウシ血清アルブミン（シグマ社製）を加えたもの。
　孔径0.45μmのフィルターで濾過滅菌し、4℃で保存するか、あるいは用時調製しても良い。
(b)GPR120またはその部分ペプチド標品
　GPR120またはその部分ペプチドを発現させたCHO細胞を、12穴プレートに5×105個／穴で継代し、37℃、5％CO2、95％airで2日間培養したもの。
(c)標識した脂肪酸またはサロゲートリガンド
　市販の〔3H〕、〔125I〕、〔14C〕などで標識した脂肪酸またはサロゲートリガンド水溶液の状態のものを4℃あるいは-20℃にて保存し、用時に測定用緩衝液にて1μMに希釈する。
(d)脂肪酸またはサロゲートリガンドの標準液
　脂肪酸またはサロゲートリガンドを0.1％ウシ血清アルブミン（シグマ社製）を含むPBSで1mMとなるように溶解し、-20℃で保存する。 A screening kit for a compound or a salt thereof that alters the binding property between GPR120 or a partial peptide thereof and a fatty acid or a surrogate ligand contains GPR120 or a partial peptide thereof, a cell that produces GPR120 or a partial peptide thereof, a membrane fraction thereof, or the like Is.
Examples of the screening kit of the present invention include the following.
1. Screening reagents
(a) Buffer for measurement and buffer for washing Hanks' Balanced Salt Solution (Gibco) plus 0.05% bovine serum albumin (Sigma).
The solution is sterilized by filtration through a 0.45 μm filter and stored at 4 ° C. or may be prepared at the time of use.
(b) GPR120 or its partial peptide preparation CHO cells expressing GPR120 or its partial peptide are passaged to a 12-well plate at 5 × 10 5 cells / well, 37 ° C., 5% CO 2 , 95% air Cultured for 2 days.
(c) Labeled fatty acid or surrogate ligand A commercially available fatty acid or surrogate ligand aqueous solution labeled with [ 3 H], [ 125 I], [ 14 C] or the like is stored at 4 ° C. or −20 ° C., Dilute to 1 μM with buffer for measurement.
(d) Fatty acid or surrogate ligand standard solution Fatty acid or surrogate ligand is dissolved to 1 mM in PBS containing 0.1% bovine serum albumin (manufactured by Sigma) and stored at -20 ° C.
２．測定法
(i)12穴組織培養用プレートにて培養したGPR120またはその部分ペプチド発現CHO細胞を、測定用緩衝液1mlで2回洗浄した後、490μlの測定用緩衝液を各穴に加える。
(ii)10-3～10-10Mの試験化合物溶液を5μl加えた後、標識した脂肪酸またはサロゲートリガンドを5μl加え、室温にて1時間反応させる。非特異的結合量を知るためには、試験化合物の代わりに10-3Mの脂肪酸またはサロゲートリガンドの標準液を5μl加えておく。
(iii)反応液を除去し、1mlの洗浄用緩衝液で3回洗浄する。細胞（またはプレート）に結合した標識脂肪酸またはサロゲートリガンドを0.2N NaOH-1％ SDSで溶解し、4mlの液体シンチレーターA（和光純薬製）と混合する。
(iv)液体シンチレーションカウンター（ベックマン社製）を用いて放射活性を測定し、Percent Maximum Binding（PMB）を次の式〔数１〕で求める。 2. Measurement method
(i) GPR120 or its partial peptide-expressing CHO cells cultured in a 12-well tissue culture plate are washed twice with 1 ml of the measurement buffer, and 490 μl of the measurement buffer is added to each well.
(ii) After 5 μl of a test compound solution of 10 −3 to 10 −10 M is added, 5 μl of a labeled fatty acid or surrogate ligand is added and reacted at room temperature for 1 hour. To know the amount of non-specific binding, 5 μl of 10 −3 M fatty acid or surrogate ligand standard solution is added instead of the test compound.
(iii) Remove the reaction solution and wash 3 times with 1 ml of washing buffer. Labeled fatty acid or surrogate ligand bound to cells (or plates) is dissolved in 0.2N NaOH-1% SDS and mixed with 4 ml of liquid scintillator A (Wako Pure Chemical Industries, Ltd.).
(iv) The radioactivity is measured using a liquid scintillation counter (manufactured by Beckman), and the Percent Maximum Binding (PMB) is determined by the following equation [Equation 1].
〔数１〕
　　　PMB=[(B-NSB)/(B0-NSB)]×100
　PMB：Percent Maximum Binding
　B　：検体を加えた時の値
　NSB：Non-specific Binding（非特異的結合量）
　B0：最大結合量 [Equation 1]
PMB = [(B-NSB) / (B 0 -NSB)] × 100
PMB: Percent Maximum Binding
B: Value when sample is added NSB: Non-specific binding
B 0 : Maximum binding amount
　別の好ましい一実施態様においては、GPR120またはその部分ペプチドを細胞膜上に発現する細胞はさらに共役するGαを発現する。GPR120またはその部分ペプチドと共役するGαのファミリーは、Gαq/11である。好ましくは、GPR120またはその部分ペプチドおよびその共役Gαを発現する細胞としては、GPR120またはその部分ペプチドをコードするDNAおよびGαq/11をコードするDNAをコトランスフェクトされた形質転換細胞が挙げられる。
　脂肪酸またはGPR120のアゴニストがGPR120またはその部分ペプチドに結合すると、該受容体のGα活性化ドメインと共役Gαの受容体結合領域とが相互作用してGαのコンフォメーション変化を生じ、GTP/GDP結合領域からGDPが解離して速やかにGTPを結合する。Gα-GTPは効果器であるPLCβに作用してその活性を促進する。一方、アンタゴニストが結合すると、受容体のコンフォメーション変化が起こらずGα活性化ドメインは不活性化状態にあるので、活性化型のGα-GTPレベルが減少し、PLCβへの作用が阻害される。ここで、GTPの代わりに、例えば、35S標識したGTPγS等のGαのGTPase活性によって加水分解を受けないGTPアナログを系に添加しておけば、試験化合物の存在下と非存在下での膜に結合した放射活性を測定・比較することにより、GαにおけるGDP-GTP交換反応に及ぼす試験化合物の効果を評価することができ、GPR120またはその部分ペプチドのアゴニスト／アンタゴニスト活性を有する物質をスクリーニングすることができる。即ち、試験化合物の存在下で放射活性が増加すれば、該試験化合物はGPR120またはその部分ペプチドに対するアゴニスト活性を有し、放射活性が減少すれば、該試験化合物はGPR120またはその部分ペプチドに対するアンタゴニスト活性を有する。 In another preferred embodiment, cells that express GPR120 or a partial peptide thereof on the cell membrane further express conjugated Gα. The family of Gα conjugated to GPR120 or its partial peptide is Gα q / 11 . Preferably, the cells expressing GPR120 or a partial peptide thereof and its conjugated Gα include transformed cells co-transfected with DNA encoding GPR120 or its partial peptide and DNA encoding Gα q / 11 .
When an agonist of fatty acid or GPR120 binds to GPR120 or a partial peptide thereof, the Gα activation domain of the receptor interacts with the receptor binding region of conjugated Gα, resulting in a conformational change of Gα, and a GTP / GDP binding region GDP dissociates from GTP and quickly binds GTP. Gα-GTP acts on the effector PLCβ to promote its activity. On the other hand, when the antagonist binds, the conformation of the receptor does not occur and the Gα activation domain is in an inactivated state, so that the activated Gα-GTP level is decreased and the action on PLCβ is inhibited. Here, instead of GTP, for example, if a GTP analog that does not undergo hydrolysis due to GTPase activity of Gα such as 35 S-labeled GTPγS is added to the system, the membrane in the presence and absence of the test compound can be used. By measuring and comparing the radioactivity bound to Gα, the effect of the test compound on the GDP-GTP exchange reaction in Gα can be evaluated, and screening for substances having agonist / antagonist activity of GPR120 or its partial peptides Can do. That is, if the radioactivity increases in the presence of the test compound, the test compound has an agonist activity against GPR120 or a partial peptide thereof, and if the radioactivity decreases, the test compound has an antagonist activity against GPR120 or a partial peptide thereof. Have
　別の好ましい一実施態様においては、GPR120またはその部分ペプチドと共役Gαとの共発現系において、該Gαと相互作用するPLCβの活性を、試験化合物の存在下及び非存在下で比較することを特徴とするスクリーニング方法が提供される。したがって、GPR120またはその部分ペプチドおよびその共役Gαを発現する細胞はまた、該Gαからのシグナルを受容するPLCβをさらに発現する。
　PLCβ活性は、例えば、［3H］標識したホスファチジルイノシトール-4,5-二リン酸をPLCβ含有膜画分に添加し、生成するイノシトール-1,4,5-三リン酸量を、公知の手法を用いて測定することにより評価することができる。試験化合物の存在下及び非存在下でPLCβ活性を測定・比較し、試験化合物存在下で該酵素活性が増加すれば、該試験化合物はGPR120またはその部分ペプチドに対するアゴニスト活性を有し、反対にPLCβ活性が減少すれば、該試験化合物はGPR120またはその部分ペプチドに対するアンタゴニスト活性を有すると評価される。 In another preferred embodiment, in the co-expression system of GPR120 or a partial peptide thereof and conjugated Gα, the activity of PLCβ that interacts with Gα is compared in the presence and absence of a test compound. A screening method is provided. Therefore, cells expressing GPR120 or its partial peptide and its conjugated Gα also express PLCβ that receives signals from the Gα.
The PLCβ activity is determined, for example, by adding [ 3 H] -labeled phosphatidylinositol-4,5-diphosphate to the PLCβ-containing membrane fraction, and determining the amount of inositol-1,4,5-triphosphate produced by a known amount. It can evaluate by measuring using a method. PLCβ activity is measured and compared in the presence and absence of the test compound, and if the enzyme activity increases in the presence of the test compound, the test compound has agonist activity against GPR120 or its partial peptide, and conversely PLCβ If the activity decreases, the test compound is evaluated as having antagonist activity against GPR120 or a partial peptide thereof.
　スクリーニング系として無傷真核細胞を用いる場合は、GαのPLCβへの作用は、細胞に［3H］標識イノシトールを添加し、生成した［3H］標識イノシトールリン酸の放射活性を測定したり、細胞内のCa2+量を測定することによっても評価することができる。細胞内Ca2+量は、試験化合物の存在下及び非存在下で細胞を適当な時間インキュベートした後、蛍光プローブ（fura-2、indo-1、fluor-3、Calcium-Green I等）を用いて分光学的に測定するか、カルシウム感受性発光蛋白質であるエクオリン等を用いて測定することができるが、公知の他のいかなる方法を使用してもよい。蛍光プローブを用いた分光学的測定に適した装置として、FLIPR（Molecular Devices社）システムが挙げられる。 When using intact eukaryotic cells as a screening system, the action of Gα on PLCβ is measured by adding [ 3 H] -labeled inositol to the cells and measuring the radioactivity of the generated [ 3 H] -labeled inositol phosphate, It can also be evaluated by measuring the amount of intracellular Ca 2+ . For intracellular Ca 2+ levels, incubate the cells for an appropriate time in the presence and absence of the test compound, and then use a fluorescent probe (fura-2, indo-1, fluor-3, Calcium-Green I, etc.) It can be measured spectroscopically or using aequorin, which is a calcium-sensitive photoprotein, but any other known method may be used. An apparatus suitable for spectroscopic measurement using a fluorescent probe is a FLIPR (Molecular Devices) system.
　別の態様として、Ca2+によりアップレギュレートされるTPA（12-O-テトラデカノイルホルボール-13-アセテート）応答エレメント（TRE）の制御下にあるリポーター遺伝子の発現量を測定することによりCa2+量を評価する方法もある。即ち、TREを含むプロモーターの下流にリポーター蛋白質（例えば、ルシフェラーゼ、GFP、ペルオキシダーゼ、アルカリホスファターゼ等）をコードするDNAを連結した発現カセットを含むベクターを導入された真核細胞を、試験化合物の存在下及び非存在下で適当な時間培養し、細胞を破砕して得られた抽出液におけるリポーター遺伝子の発現を公知の手法を用いて測定・比較することにより、細胞内Ca2+量を評価するというものである。
　従って、試験化合物の存在下で細胞内Ca2+量（もしくはTRE制御下にあるリポーター遺伝子の発現量）が増加すれば、該試験化合物はGPR120またはその部分ペプチドに対するアゴニスト活性を有し、反対に細胞内Ca2+量（もしくはリポーター遺伝子の発現量）が減少すれば、該試験化合物はGPR120またはその部分ペプチドに対するアンタゴニスト活性を有する。 In another embodiment, by measuring the expression level of a reporter gene under the control of a TPA (12-O-tetradecanoylphorbol-13-acetate) response element (TRE) that is up-regulated by Ca 2+ There is also a method for evaluating the amount of Ca 2+ . That is, eukaryotic cells into which a vector containing an expression cassette linked with a DNA encoding a reporter protein (eg, luciferase, GFP, peroxidase, alkaline phosphatase, etc.) is introduced downstream of a promoter containing TRE in the presence of a test compound. In addition, the amount of Ca 2+ in the cell is evaluated by measuring and comparing the expression of the reporter gene in the extract obtained by culturing for an appropriate time in the absence and disrupting the cells using a known method. Is.
Therefore, if the intracellular Ca 2+ level (or the expression level of the reporter gene under TRE control) increases in the presence of the test compound, the test compound has agonist activity for GPR120 or its partial peptide, and conversely If the intracellular Ca 2+ level (or reporter gene expression level) decreases, the test compound has antagonist activity against GPR120 or a partial peptide thereof.
　上記(4)～(5)のスクリーニング方法において、脂肪酸またはサロゲートリガンドを接触させることなく、GPR120またはその部分ペプチドを産生する細胞を試験化合物の存在下および非存在下で培養した場合における、GPR120またはその部分ペプチドを介した細胞刺激活性を測定し、比較することによって、リガンドとの結合性を変化させるか否かにかかわらず、GPR120の活性を促進もしくは抑制する化合物を選択することができる。
　また、別の好ましい一実施態様においては、WO 03/055507に記載されるようにして、GPR120またはその部分ペプチドのC末端側に共役Gαを連結した融合蛋白質を、哺乳動物細胞や昆虫細胞などの宿主真核細胞で発現させることにより、GPR120またはその部分ペプチドを恒常的に活性化することができる。この場合、脂肪酸またはサロゲートリガンドを用いることなく、アゴニストやインバースアゴニストをスクリーニングすることが可能である。 In the screening method according to the above (4) to (5), GPR120 or GPR120 when cells producing GPR120 or a partial peptide thereof are cultured in the presence and absence of a test compound without contacting with a fatty acid or a surrogate ligand. By measuring and comparing the cell stimulating activity via the partial peptide, a compound that promotes or suppresses the activity of GPR120 can be selected regardless of whether or not the binding property to the ligand is changed.
In another preferred embodiment, as described in WO 03/055507, a fusion protein in which conjugated Gα is linked to the C-terminal side of GPR120 or a partial peptide thereof is used in mammalian cells, insect cells, and the like. By expressing it in a host eukaryotic cell, GPR120 or a partial peptide thereof can be constitutively activated. In this case, it is possible to screen for agonists and inverse agonists without using fatty acids or surrogate ligands.
　さらに別の好ましい一実施態様においては、宿主細胞として酵母（例えば、パン酵母など）が用いられる。酵母は唯一のGPCRとして接合フェロモンα因子の受容体であるSTE2を有する。リガンドの刺激を受けると、STE2は共役GαであるGPA1を活性化してGβ/Gγと解離する。解離したGβ/GγからのキナーゼカスケードシグナルによりFUS1の発現が誘導され、接合が起こる。ここでSTE2を破壊して、代わりにGPR120またはその部分ペプチドを酵母で発現させ、さらにGPA1プロモーターの制御下にGPR120またはその部分ペプチドの共役Gαを共発現させれば、脂肪酸（もしくはGPR120のアゴニスト）の刺激によりGPR120またはその部分ペプチドがあたかもSTE2と同様に細胞内シグナル伝達を媒介する。ここでGPA1プロモーターの制御下にGPR120またはその部分ペプチドと共役するGαを共発現させ、さらにFUS1に栄養要求性変異を相補する遺伝子やレポーター遺伝子（例えば、LacZ）などを融合させれば、容易にシグナルの活性化を検出することができる。この方法の詳細については、例えば、Yeast, 16: 11-22 (2000)等に記載されている。 In yet another preferred embodiment, yeast (eg, baker's yeast) is used as the host cell. Yeast has STE2, which is the receptor for mating pheromone alpha factor, as the only GPCR. When stimulated by a ligand, STE2 activates GPA1, which is a conjugated Gα, and dissociates from Gβ / Gγ. Kinase cascade signals from dissociated Gβ / Gγ induce FUS1 expression and conjugation occurs. Here, if STE2 is destroyed and GPR120 or a partial peptide thereof is expressed in yeast instead, and GPR120 or a conjugated Gα of the partial peptide is co-expressed under the control of the GPA1 promoter, a fatty acid (or an agonist of GPR120) GPR120 or a partial peptide thereof mediates intracellular signal transduction as if by STE2. Here, Gα coupled to GPR120 or its partial peptide is co-expressed under the control of the GPA1 promoter, and a gene supplementing an auxotrophic mutation or a reporter gene (for example, LacZ) is fused to FUS1. Signal activation can be detected. Details of this method are described in, for example, Yeast, 16: 11-22 (2000).
　さらに別の一実施態様においては、GPR120またはその部分ペプチドを産生する細胞は、非ヒト哺乳動物個体の形態で提供される。該動物個体の状態は特に制限されないが、例えば、db/dbマウス、ob/obマウス、KKAyマウス、Zucker fattyラット、WHHLウサギ等の疾患（肥満、糖尿病、高血圧、動脈硬化など）モデル動物であってもよい。使用される動物の飼育条件に特に制限はないが、SPFグレード以上の環境下で飼育されたものであることが好ましい。試験化合物の該細胞との接触は、該動物個体への試験化合物の投与によって行われる。投与経路は特に制限されないが、例えば、静脈内投与、動脈内投与、皮下投与、皮内投与、腹腔内投与、経口投与、気道内投与、直腸投与等が挙げられる。投与量も特に制限はないが、例えば、１回量として約0.5～20 mg／kgを、1日1～5回、好ましくは1日1～3回、1～14日間投与することができる。尚、リガンドの該細胞との接触は、該動物個体に内在する生理的リガンドである脂肪酸の作用により代替され得る。 In yet another embodiment, the cell producing GPR120 or a partial peptide thereof is provided in the form of a non-human mammal individual. The state of the individual animal is not particularly limited. For example, it is a model animal of a disease (obesity, diabetes, hypertension, arteriosclerosis, etc.) such as db / db mouse, ob / ob mouse, KKAy mouse, Zuckerfatty rat, and WHHL rabbit. May be. There are no particular restrictions on the breeding conditions of the animals to be used, but it is preferable that the animals are raised in an environment of SPF grade or higher. Contact of the test compound with the cells is effected by administration of the test compound to the animal individual. The administration route is not particularly limited, and examples thereof include intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, intraperitoneal administration, oral administration, intratracheal administration, and rectal administration. The dose is not particularly limited. For example, a dose of about 0.5 to 20 mg / kg can be administered 1 to 5 times a day, preferably 1 to 3 times a day for 1 to 14 days. The contact of the ligand with the cell can be replaced by the action of a fatty acid that is a physiological ligand inherent in the animal individual.
　上記のスクリーニング方法を用いて得られるGPR120またはその部分ペプチドに対するアゴニストは、GPR120またはその部分ペプチドに対する脂肪酸が有する生理活性と同様の作用を有しているので、GPR120を介したシグナル伝達作用、EPCの分化・増殖促進作用を増強する安全で低毒性な医薬として有用である。GPR120またはその部分ペプチドに対するアンタゴニストは、GPR120またはその部分ペプチドに対する脂肪酸が有する生理活性を抑制することができるので、GPR120を介したシグナル伝達作用、EPCの分化・増殖促進作用を抑制し、また末梢血中のEPC増加作用を示す安全で低毒性な医薬として有用である。GPR120またはその部分ペプチドと脂肪酸との結合力を増強する化合物は、GPR120またはその部分ペプチドに対する脂肪酸が有する生理活性を増強するための安全で低毒性な医薬として有用である。GPR120またはその部分ペプチドと脂肪酸との結合力を減少させる化合物は、GPR120またはその部分ペプチドに対する脂肪酸が有する生理活性を減少させるための安全で低毒性な医薬として有用である。 The agonist for GPR120 or its partial peptide obtained by using the above screening method has the same action as the physiological activity of fatty acid for GPR120 or its partial peptide. It is useful as a safe and low-toxic drug that enhances differentiation / proliferation promoting action. Since antagonists to GPR120 or its partial peptide can suppress the physiological activity of fatty acids for GPR120 or its partial peptide, it suppresses GPR120-mediated signal transduction and EPC differentiation / proliferation-promoting effects, as well as peripheral blood. It is useful as a safe and low-toxic drug that exhibits an increase in EPC. A compound that enhances the binding force between GPR120 or a partial peptide thereof and a fatty acid is useful as a safe and low toxic pharmaceutical for enhancing the physiological activity of the fatty acid for GPR120 or a partial peptide thereof. A compound that decreases the binding force between GPR120 or a partial peptide thereof and a fatty acid is useful as a safe and low-toxic pharmaceutical for reducing the physiological activity of fatty acids for GPR120 or a partial peptide thereof.
（１ｂ）GPR120の発現量を変化させる化合物のスクリーニング
　前述の通り、GPR120はEPCの分化・増殖を促進する作用を有し、また、GPR120の抑制は末梢血へのEPCの動員を促進することから、GPR120の発現量を変化させる化合物は、EPCの分化・増殖を調節し、また末梢血中のEPCを増加させることができる。
　従って、本発明はまた、GPR120またはその部分ペプチドを産生する細胞における該蛋白質またはペプチド（あるいはそれをコードする遺伝子）の発現を、試験化合物の存在下と非存在下で比較することを特徴とする、EPCの分化・増殖調節または末梢血中のEPC増加物質のスクリーニング方法を提供する。本方法において用いられる細胞、試験化合物の種類、試験化合物と細胞との接触の態様などは、上記したGPR120またはその部分ペプチドの活性を指標とする方法と同様である。 (1b) Screening of compounds that change the expression level of GPR120 As described above, GPR120 has an action of promoting differentiation and proliferation of EPC, and suppression of GPR120 promotes mobilization of EPC to peripheral blood. A compound that changes the expression level of GPR120 can regulate differentiation and proliferation of EPC and increase EPC in peripheral blood.
Therefore, the present invention is also characterized in that the expression of the protein or peptide (or gene encoding it) in cells producing GPR120 or a partial peptide thereof is compared in the presence and absence of the test compound. The present invention provides a screening method for EPC differentiation / proliferation regulation or EPC increasing substance in peripheral blood. The cells used in this method, the type of test compound, the mode of contact between the test compound and the cells, and the like are the same as those described above using the activity of GPR120 or a partial peptide thereof as an index.
　GPR120またはその部分ペプチドの発現量は、前記したGPR120またはその部分ペプチドをコードするDNAとハイストリンジェントな条件下でハイブリダイズし得る核酸、即ち、配列番号１、３または５に示される塩基配列もしくはその部分塩基配列またはその相補鎖配列からなるDNAとハイストリンジェントな条件下でハイブリダイズし得る核酸（以下、「本発明の検出用核酸」という場合がある）を用いて、GPR120またはその部分ペプチドのmRNAを検出することにより、RNAレベルで測定することができる。あるいは、該発現量は、GPR120またはその部分ペプチドに対する抗体（以下、「本発明の検出用抗体」という場合がある）を用いて、該蛋白質またはペプチドを検出することにより、蛋白質レベルで測定することもできる。
　従って、より具体的には、本発明は、
（a）GPR120またはその部分ペプチドを産生する細胞を試験化合物の存在下および非存在下で培養し、両条件下における該蛋白質またはペプチドをコードするmRNAの量を、本発明の検出用核酸を用いて測定、比較することを特徴とする、EPCの分化・増殖調節または末梢血中のEPC増加物質のスクリーニング方法、および
（b）GPR120またはその部分ペプチドを産生する細胞を試験化合物の存在下および非存在下で培養し、両条件下における該蛋白質またはペプチドの量を、本発明の検出用抗体を用いて測定、比較することを特徴とする、EPCの分化・増殖調節または末梢血中のEPC増加物質のスクリーニング方法を提供する。 The expression level of GPR120 or a partial peptide thereof is a nucleic acid that can hybridize with the above-described DNA encoding GPR120 or the partial peptide under high stringency conditions, that is, the nucleotide sequence represented by SEQ ID NO: 1, 3, or 5 or GPR120 or a partial peptide thereof using a nucleic acid capable of hybridizing under high stringency conditions with a DNA comprising the partial base sequence or the complementary strand sequence (hereinafter sometimes referred to as “the nucleic acid for detection of the present invention”) Can be measured at the RNA level. Alternatively, the expression level is measured at the protein level by detecting the protein or peptide using an antibody against GPR120 or a partial peptide thereof (hereinafter sometimes referred to as “the detection antibody of the present invention”). You can also.
Therefore, more specifically, the present invention
(A) Cells that produce GPR120 or a partial peptide thereof are cultured in the presence and absence of a test compound, and the amount of mRNA encoding the protein or peptide under both conditions is determined using the nucleic acid for detection of the present invention. A method of screening for EPC differentiation / proliferation or screening for an EPC-increasing substance in peripheral blood, and (b) cells that produce GPR120 or a partial peptide thereof in the presence or absence of a test compound. EPC differentiation / proliferation regulation or increase of EPC in peripheral blood, characterized by measuring and comparing the amount of the protein or peptide under both conditions using the detection antibody of the present invention. A method for screening a substance is provided.
　例えば、GPR120またはその部分ペプチドのmRNA量または蛋白質（ペプチド）量の測定は、具体的には以下のようにして行うことができる。
（i）正常あるいは疾患モデル非ヒト哺乳動物（例えば、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど、より具体的には、肥満マウス、糖尿病マウス、高血圧ラット、動脈硬化ウサギなど）に対して、薬剤（例えば、抗肥満薬、抗糖尿病薬、降圧薬、血管作用薬など）あるいは物理的ストレス（例えば、浸水ストレス、電気ショック、明暗、低温など）などを与え、一定時間経過した後に、末梢血あるいは特定の臓器（例えば、骨髄、臓器障害局所など）、組織（例えば、骨髄組織、病変組織など）あるいは細胞を得る。
　得られた細胞等に含まれるGPR120のmRNAは、例えば、通常の方法により細胞等からmRNAを抽出し、例えば、RT-PCRなどの手法を用いることにより定量することができ、あるいは自体公知のノーザンブロット解析により定量することもできる。一方、GPR120蛋白質量は、ウェスタンブロット解析や以下に詳述する各種イムノアッセイ法を用いて定量することができる。
（ii）GPR120またはその部分ペプチドをコードする核酸を導入した形質転換体を上記の方法に従って作製し、該形質転換体に含まれるGPR120またはその部分ペプチドあるいはそれをコードするmRNAを、上記（i）と同様にして定量、解析することができる。 For example, the measurement of the amount of mRNA or protein (peptide) of GPR120 or a partial peptide thereof can be specifically performed as follows.
(I) Normal or disease model non-human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc. More specifically, obese mouse, diabetic mouse, hypertensive rat, arteriosclerosis Rabbits, etc.) are given drugs (eg, anti-obesity drugs, anti-diabetic drugs, antihypertensive drugs, vasoactive drugs, etc.) or physical stress (eg, water stress, electric shock, light / dark, low temperature, etc.) After a lapse of time, peripheral blood or a specific organ (for example, bone marrow, organ lesion local area, etc.), tissue (for example, bone marrow tissue, diseased tissue, etc.) or cells are obtained.
The mRNA of GPR120 contained in the obtained cells and the like can be quantified by, for example, extracting mRNA from cells and the like by a normal method, for example, using a technique such as RT-PCR, or a known Northern per se It can also be quantified by blot analysis. On the other hand, the amount of GPR120 protein can be quantified using Western blot analysis or various immunoassay methods described in detail below.
(Ii) A transformant into which a nucleic acid encoding GPR120 or a partial peptide thereof has been introduced is prepared according to the method described above, and GPR120 or a partial peptide thereof or mRNA encoding the transformant contained in the transformant is prepared as described in (i) Quantitative analysis can be performed in the same manner.
　GPR120またはその部分ペプチドの発現量を変化させる物質のスクリーニングは、
（i）正常あるいは疾患モデル非ヒト哺乳動物に対して、薬剤などを与える一定時間前（30分前～24時間前、好ましくは30分前～12時間前、より好ましくは1時間前～6時間前）もしくは一定時間後（30分後～3日後、好ましくは1時間後～2日後、より好ましくは1時間後～24時間後）、または薬剤などと同時に試験化合物を投与し、投与から一定時間が経過した後（30分後～3日後、好ましくは1時間後～2日後、より好ましくは1時間後～24時間後）、該動物から単離した細胞に含まれるGPR120をコードするmRNA量、あるいはGPR120蛋白質量を定量、解析することにより、あるいは
（ii）形質転換体を常法に従って培養する際に試験化合物を培地もしくは緩衝液中に添加し、一定時間インキュベート後（1日後～7日後、好ましくは1日後～3日後、より好ましくは2日後～3日後）、該形質転換体に含まれるGPR120またはその部分ペプチドをコードするmRNA量、あるいは該蛋白質（ペプチド）量を定量、解析することにより行うことができる。 Screening for substances that alter the expression level of GPR120 or its partial peptides
(I) A certain time before giving a drug to a normal or disease model non-human mammal (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 hour to 6 hours) Before) or after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), or at the same time as the drug, etc. After 30 minutes (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the amount of mRNA encoding GPR120 contained in cells isolated from the animal, Alternatively, by quantifying and analyzing the amount of GPR120 protein, or (ii) when culturing a transformant according to a conventional method, a test compound is added to a medium or a buffer, and incubated for a certain period of time (after 1 to 7 days, Preferably after 1 to 3 days, more preferably 2 days 1-3 days after), mRNA quantity encoding GPR120 or its partial peptide contained in the transformant, or protein (peptide) amount quantitation can be carried out by analyzing.
　上記（b）のスクリーニング方法に用いられる本発明の検出用抗体は、GPR120またはその部分ペプチドを特異的に認識する抗体であれば特に制限されず、ポリクローナル抗体、モノクローナル抗体の何れであってもよい。抗体のアイソタイプは特に限定されないが、好ましくはIgG、IgMまたはIgA、特に好ましくはIgGが挙げられる。また、該抗体は、標的抗原を特異的に認識し結合するための相補性決定領域（CDR）を少なくとも有するものであれば特に制限はなく、完全抗体分子の他、例えばFab、Fab'、F(ab’)2等のフラグメント、scFv、scFv-Fc、ミニボディー、ダイアボディー等の遺伝子工学的に作製されたコンジュゲート分子、あるいはポリエチレングリコール（PEG）等の蛋白質安定化作用を有する分子等で修飾されたそれらの誘導体などであってもよい。GPR120またはその部分ペプチドに対する抗体は、GPR120またはその部分ペプチドを抗原として用い、自体公知の抗体または抗血清の製造法に従って製造することができる。
　以下に、本発明の抗体の免疫原調製法、および該抗体の製造法について説明する。 The detection antibody of the present invention used in the screening method (b) is not particularly limited as long as it is an antibody that specifically recognizes GPR120 or a partial peptide thereof, and may be either a polyclonal antibody or a monoclonal antibody. . The isotype of the antibody is not particularly limited, but preferably IgG, IgM or IgA, particularly preferably IgG. The antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding a target antigen. In addition to a complete antibody molecule, for example, Fab, Fab ′, F (ab ') 2 such as fragments, scFv, scFv-Fc, conjugation molecules prepared by genetic engineering such as minibodies and diabodies, or molecules having protein stabilizing action such as polyethylene glycol (PEG) They may be modified derivatives thereof. An antibody against GPR120 or a partial peptide thereof can be produced according to a known method for producing an antibody or antiserum using GPR120 or a partial peptide thereof as an antigen.
Hereinafter, a method for preparing an immunogen of the antibody of the present invention and a method for producing the antibody will be described.
１）抗原の調製
　抗体を調製するために使用される抗原としては、上記したGPR120またはその部分ペプチド（抗体の製造に関する以下の説明においては、特にことわらない限り、これらを包括して単に「GPR120」という）、あるいはそれと同一の抗原決定基を1種あるいは2種以上有する（合成）ペプチドなど、何れのものも使用することができる（以下、これらを単に本発明の抗原と称することもある）。
　GPR120は、上記した通り、例えば、（a）哺乳動物の組織または細胞から公知の方法あるいはそれに準ずる方法を用いて調製、（b）ペプチド・シンセサイザー等を使用する公知のペプチド合成方法で化学的に合成、（c）GPR120またはその部分ペプチドをコードするDNAを含有する形質転換体を培養、あるいは（d）GGPR120またはその部分ペプチドをコードする核酸を鋳型として無細胞転写／翻訳系を用いて生化学的に合成することによって製造される。
（a）哺乳動物の組織または細胞からGPR120を調製する場合、その組織または細胞をホモジナイズした後、粗分画物（例、膜画分、可溶性画分）をそのまま抗原として用いることもできる。あるいは酸、界面活性剤またはアルコールなどで抽出を行い、該抽出液を、塩析、透析、ゲル濾過、逆相クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィーなどのクロマトグラフィーを組み合わせることにより精製単離することもできる。得られたGPR120をそのまま免疫原とすることもできるし、ペプチダーゼ等を用いた限定分解により部分ペプチドを調製してそれを免疫原とすることもできる。
（b）化学的に本発明の抗原を調製する場合、該合成ペプチドとしては、例えば上述の（a）の方法を用いて天然材料より精製したGPR120と同一の構造を有するもの、具体的には、GPR120のアミノ酸配列において少なくとも3個以上、好ましくは6個以上のアミノ酸からなる任意の箇所のアミノ酸配列と同一のアミノ酸配列を１種あるいは２種以上含有するペプチドなどが用いられる。
（c）DNAを含有する形質転換体を用いて本発明の抗原を製造する場合、該DNAは、公知のクローニング方法〔例えば、Molecular Cloning 2nd ed.（J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989）に記載の方法など〕に従って作製することができる。該クローニング方法とは、（1）GPR120をコードする遺伝子配列に基づきデザインしたDNAプローブを用い、ヒトcDNAライブラリーからハイブリダイゼーション法により該抗原をコードするDNAを単離するか、（2）GPR120をコードする遺伝子配列に基づきデザインしたDNAプライマーを用い、哺乳動物由来のcDNAを鋳型としてPCR法により該抗原をコードするDNAを調製し、該DNAを宿主に適合する発現ベクターに挿入する方法などが挙げられる。該発現ベクターで宿主を形質転換して得られる形質転換体を適当な培地中で培養することにより、所望の抗原を得ることができる。
（d）無細胞転写／翻訳系を利用する場合、上記（c）と同様の方法により調製した抗原をコードするDNAを挿入した発現ベクター（例えば、該DNAがT7、SP6プロモーター等の制御下におかれた発現ベクターなど）を鋳型とし、該プロモーターに適合するRNAポリメラーゼおよび基質（NTPs）を含む転写反応液を用いてmRNAを合成した後、該mRNAを鋳型として公知の無細胞翻訳系（例：大腸菌、ウサギ網状赤血球、コムギ胚芽等の抽出液）を用いて翻訳反応を行わせる方法などが挙げられる。塩濃度等を適当に調整することにより、転写反応と翻訳反応を同一反応液中で一括して行うこともできる。 1) Preparation of antigen Antigen used for preparing the antibody includes GPR120 or a partial peptide thereof (in the following description relating to the production of antibody, unless otherwise specified, these are simply referred to as “GPR120”). Or a (synthetic) peptide having one or more of the same antigenic determinants (hereinafter, these may be simply referred to as the antigen of the present invention). .
As described above, GPR120 is prepared by, for example, (a) preparing from a mammalian tissue or cell using a known method or a method equivalent thereto, and (b) chemically using a known peptide synthesis method using a peptide synthesizer or the like. Synthesis, (c) culture of transformants containing DNA encoding GPR120 or its partial peptide, or (d) biochemistry using cell-free transcription / translation system with nucleic acid encoding GGPR120 or its partial peptide as template It is manufactured by synthesizing automatically.
(A) When preparing GPR120 from a mammalian tissue or cell, after homogenizing the tissue or cell, the crude fraction (eg, membrane fraction, soluble fraction) can be used as an antigen as it is. Alternatively, extraction with an acid, surfactant, alcohol, or the like is performed, and the extract is purified by a combination of chromatography such as salting out, dialysis, gel filtration, reverse phase chromatography, ion exchange chromatography, and affinity chromatography. It can also be separated. The obtained GPR120 can be used as an immunogen as it is, or a partial peptide can be prepared by limited degradation using a peptidase or the like and used as an immunogen.
(B) When the antigen of the present invention is chemically prepared, the synthetic peptide is, for example, one having the same structure as GPR120 purified from a natural material using the method (a) described above, specifically, In the GPR120 amino acid sequence, a peptide containing one or two or more amino acid sequences identical to the amino acid sequence of any position consisting of at least 3 amino acids, preferably 6 amino acids or more, is used.
(C) When the antigen of the present invention is produced using a transformant containing DNA, the DNA can be obtained by known cloning methods [for example, Molecular Cloning 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab The method described in Press, 1989)]. The cloning method includes (1) isolating DNA encoding the antigen from a human cDNA library by hybridization using a DNA probe designed based on the gene sequence encoding GPR120, or (2) Examples include a method of preparing a DNA encoding the antigen by a PCR method using a cDNA primer designed based on the gene sequence to be encoded as a template and inserting the DNA into an expression vector suitable for the host. It is done. A desired antigen can be obtained by culturing a transformant obtained by transforming a host with the expression vector in an appropriate medium.
(D) When a cell-free transcription / translation system is used, an expression vector inserted with a DNA encoding an antigen prepared by the same method as in (c) above (for example, the DNA is under the control of a T7, SP6 promoter, etc. Synthesize mRNA using a transcription reaction solution containing RNA polymerase and substrate (NTPs) compatible with the promoter using the expressed expression vector as a template, and then use a known cell-free translation system (eg : Extraction method of Escherichia coli, rabbit reticulocyte, wheat germ, etc.) and the like. By appropriately adjusting the salt concentration and the like, the transcription reaction and the translation reaction can also be carried out collectively in the same reaction solution.
　免疫原としてはGPR120またはその部分ペプチドや、より短い部分アミノ酸配列を有するペプチドを用いることができる。部分アミノ酸配列としては、例えば3個以上の連続するアミノ酸残基からなるもの、好ましくは4個以上、より好ましくは5個以上、いっそう好ましくは6個以上の連続するアミノ酸残基からなるものが挙げられる。あるいは、該アミノ酸配列としては、例えば20個以下の連続するアミノ酸残基からなるもの、好ましくは18個以下、より好ましくは15個以下、いっそう好ましくは12個以下の連続するアミノ酸残基からなるものが挙げられる。これらのアミノ酸残基の一部（例：1ないし数個）は置換可能な基（例：Cys、水酸基等）によって置換されていてもよい。免疫原として用いられるペプチドは、このような部分アミノ酸配列を1ないし数個含むアミノ酸配列を有する。 As an immunogen, GPR120 or a partial peptide thereof, or a peptide having a shorter partial amino acid sequence can be used. Examples of the partial amino acid sequence include those consisting of 3 or more consecutive amino acid residues, preferably 4 or more, more preferably 5 or more, and even more preferably 6 or more consecutive amino acid residues. It is done. Alternatively, the amino acid sequence is composed of, for example, 20 or less consecutive amino acid residues, preferably 18 or less, more preferably 15 or less, and even more preferably 12 or less consecutive amino acid residues. Is mentioned. Some of these amino acid residues (eg, 1 to several) may be substituted with a substitutable group (eg, Cys, hydroxyl group, etc.). The peptide used as an immunogen has an amino acid sequence containing 1 to several such partial amino acid sequences.
　あるいは、GPR120を発現する哺乳動物細胞自体を、本発明の抗原として直接用いることもできる。哺乳動物細胞としては、上記（a）項で述べたような天然の細胞、上記（c）項で述べたような方法で形質転換した細胞などを用いることができる。形質転換に用いる宿主としては、ヒト、サル、ラット、マウス、ハムスター、ニワトリなどから採取した細胞であれば何れのものでも良く、HEK293、COS7、CHO-K1、NIH3T3、Balb3T3、FM3A、L929、SP2/0、P3U1、B16、またはP388などが好ましく用いられる。GPR120を発現する天然の哺乳動物細胞または形質転換した真核細胞は、組織培養に用いられる培地（例、RPMI1640等）または緩衝液（例、Hanks’ Balanced Salt Solution等）に懸濁された状態で、免疫動物に注射することができる。免疫方法としては、抗体産生を促すことのできる方法であれば何れの方法でも良く、静脈内注射、腹腔内注射、筋肉内注射または皮下注射などが好ましく用いられる。 Alternatively, mammalian cells that express GPR120 can also be used directly as the antigen of the present invention. As mammalian cells, natural cells as described in the above section (a), cells transformed by the method as described in the above section (c), and the like can be used. The host used for transformation may be any cell collected from humans, monkeys, rats, mice, hamsters, chickens, etc., HEK293, COS7, CHO-K1, NIH3T3, Balb3T3, FM3A, L929, SP2 / 0, P3U1, B16, or P388 is preferably used. Natural mammalian cells expressing GPR120 or transformed eukaryotic cells are suspended in a medium used for tissue culture (eg, RPMI1640) or buffer (eg, Hanks' Balanced Salt Solution). Can be injected into immunized animals. As an immunization method, any method can be used as long as it can promote antibody production, and intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, and the like are preferably used.
　本発明の抗原は、免疫原性を有していれば不溶化したものを直接免疫することもできるが、分子内に１ないし数個の抗原決定基しか有しない低分子量（例えば、分子量約3,000以下）の抗原（即ち、GPR120の部分ペプチド）を用いる場合には、これらの抗原は通常免疫原性の低いハプテン分子なので、適当な担体（キャリアー）に結合または吸着させた複合体として免疫することができる。担体としては天然もしくは合成の高分子を用いることができる。天然高分子としては、例えばウシ、ウサギ、ヒトなどの哺乳動物の血清アルブミンや例えばウシ、ウサギなどの哺乳動物のサイログロブリン、例えばニワトリのオボアルブミン、例えばウシ、ウサギ、ヒト、ヒツジなどの哺乳動物のヘモグロビン、キーホールリンペットヘモシアニン（KLH）などが用いられる。合成高分子としては、例えばポリアミノ酸類、ポリスチレン類、ポリアクリル類、ポリビニル類、ポリプロピレン類などの重合物または共重合物などの各種ラテックスなどが挙げられる。 The antigen of the present invention can be directly immunized as long as it has immunogenicity, but it has a low molecular weight (for example, a molecular weight of about 3,000 or less) having only one or several antigenic determinants in the molecule. ) Antigens (ie, partial peptides of GPR120), since these antigens are usually hapten molecules with low immunogenicity, they can be immunized as a complex bound or adsorbed to a suitable carrier (carrier). it can. A natural or synthetic polymer can be used as the carrier. Examples of natural polymers include serum albumin of mammals such as cows, rabbits and humans, and thyroglobulin of mammals such as cows and rabbits, such as chicken ovalbumin, such as mammals such as cows, rabbits, humans and sheep. Hemoglobin, keyhole limpet hemocyanin (KLH), etc. are used. Examples of the synthetic polymer include various latexes such as polymers or copolymers such as polyamino acids, polystyrenes, polyacryls, polyvinyls, and polypropylenes.
　該キャリアーとハプテンとの混合比は、担体に結合あるいは吸着させた抗原に対する抗体が効率よく産生されれば、どのようなものをどのような比率で結合あるいは吸着させてもよく、通常ハプテンに対する抗体の作製にあたり常用されている上記の天然もしくは合成の高分子キャリアーを、重量比でハプテン1に対し0.1～100の割合で結合あるいは吸着させたものを使用することができる。 The mixing ratio of the carrier and the hapten is such that any antibody can be bound or adsorbed at any ratio as long as an antibody against the antigen bound or adsorbed to the carrier is efficiently produced. The above-mentioned natural or synthetic polymer carrier, which is commonly used in the production of the above, can be bound or adsorbed at a ratio of 0.1 to 100 with respect to hapten 1 by weight.
　また、ハプテンとキャリアーのカプリングには、種々の縮合剤を用いることができる。例えば、チロシン、ヒスチジン、トリプトファンを架橋するビスジアゾ化ベンジジンなどのジアゾニウム化合物、アミノ基同士を架橋するグルタルアルデビトなどのジアルデヒド化合物、トルエン-2,4-ジイソシアネートなどのジイソシアネート化合物、チオール基同士を架橋するN,N’-o-フェニレンジマレイミドなどのジマレイミド化合物、アミノ基とチオール基を架橋するマレイミド活性エステル化合物、アミノ基とカルボキシル基とを架橋するカルボジイミド化合物などが好都合に用いられる。また、アミノ基同士を架橋する際にも、一方のアミノ基にジチオピリジル基を有する活性エステル試薬（例えば、3-(2-ピリジルジチオ)プロピオン酸N-スクシンイミジル（SPDP）など）を反応させた後還元することによりチオール基を導入し、他方のアミノ基にマレイミド活性エステル試薬によりマレイミド基を導入後、両者を反応させることもできる。 Also, various condensing agents can be used for coupling of the hapten and the carrier. For example, diazonium compounds such as bisdiazotized benzidine that crosslinks tyrosine, histidine, and tryptophan, dialdehyde compounds such as glutaraldehyde that crosslink amino groups, diisocyanate compounds such as toluene-2,4-diisocyanate, and thiol groups A dimaleimide compound such as N, N′-o-phenylene dimaleimide, a maleimide active ester compound that crosslinks an amino group and a thiol group, a carbodiimide compound that crosslinks an amino group and a carboxyl group, and the like are advantageously used. In addition, when cross-linking amino groups, an active ester reagent having a dithiopyridyl group on one amino group (for example, N-succinimidyl (SPDP) 3- (2-pyridyldithio) propionate) was reacted. It is also possible to introduce a thiol group by post-reduction, introduce a maleimide group into the other amino group with a maleimide active ester reagent, and then react both.
２）モノクローナル抗体の作製
　本発明の抗原は、温血動物に対して、例えば腹腔内注入、静脈注入、皮下注射、皮内注射などの投与方法によって、抗体産生が可能な部位にそれ自体単独で、あるいは担体、希釈剤とともに投与される。投与に際して抗体産生能を高めるため、完全フロイントアジュバントや不完全フロイントアジュバントを投与してもよい。投与は通常1～6週毎に1回ずつ、計2～10回程度行われる。用いられる温血動物としては、例えば、サル、ウサギ、イヌ、モルモット、マウス、ラット、ハムスター、ヒツジ、ヤギ、ロバ、ニワトリが挙げられる。抗Ig抗体産生の問題を回避するためには投与対象と同一種の哺乳動物を用いることが好ましいが、モノクローナル抗体作製には一般にマウスおよびラットが好ましく用いられる。 2) Preparation of monoclonal antibody The antigen of the present invention is administered to a warm-blooded animal by itself at a site where antibody production is possible by administration methods such as intraperitoneal injection, intravenous injection, subcutaneous injection, and intradermal injection. Or, it is administered together with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually once every 1 to 6 weeks, for a total of 2 to 10 times. Examples of warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, hamsters, sheep, goats, donkeys, and chickens. In order to avoid the problem of anti-Ig antibody production, it is preferable to use a mammal of the same species as that to be administered, but in general, mice and rats are preferably used for production of monoclonal antibodies.
　モノクローナル抗体の作製に際しては、抗原を免疫された温血動物（例：マウス、ラット）から抗体価の上昇が認められた個体を選択し、最終免疫の2～5日後に脾臓またはリンパ節を採取して抗体産生細胞を単離し、これと骨髄腫細胞とを融合させることにより抗体産生ハイブリドーマを調製することができる。血清中の抗体価の測定は、例えば標識化抗原と抗血清とを反応させた後、抗体に結合した標識剤の活性を測定することにより行うことができる。 When producing monoclonal antibodies, select individuals with elevated antibody titers from warm-blooded animals immunized with antigen (eg, mice, rats), and collect spleen or lymph nodes 2-5 days after the final immunization Thus, antibody-producing hybridomas can be prepared by isolating antibody-producing cells and fusing them with myeloma cells. The antibody titer in serum can be measured, for example, by reacting a labeled antigen with antiserum and then measuring the activity of the labeling agent bound to the antibody.
　骨髄腫細胞は多量の抗体を分泌するハイブリドーマを産生し得るものであれば特に制限はないが、自身は抗体を産生もしくは分泌しないものが好ましく、また、細胞融合効率が高いものがより好ましい。また、ハイブリドーマの選択を容易にするために、HAT（ヒポキサンチン、アミノプテリン、チミジン）感受性の株を用いることが好ましい。例えばマウス骨髄腫細胞としてはNS-1、P3U1、SP2/0、AP-1等が、ラット骨髄腫細胞としてはR210.RCY3、Y3-Ag 1.2.3等が、ヒト骨髄腫細胞としてはSKO-007、GM 1500-6TG-2、LICR-LON-HMy2、UC729-6等が挙げられる。 The myeloma cell is not particularly limited as long as it can produce a hybridoma that secretes a large amount of antibody, but it does not itself produce or secrete an antibody, and more preferably has high cell fusion efficiency. In order to facilitate selection of hybridomas, it is preferable to use a HAT (hypoxanthine, aminopterin, thymidine) sensitive strain. For example, mouse myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, etc., rat myeloma cells include R210.RCY3, Y3-Ag 1.2.3, and human myeloma cells include SKO- 007, GM 1500-6TG-2, LICR-LON-HMy2, UC729-6 and the like.
　融合操作は既知の方法、例えばケーラーとミルスタインの方法［ネイチャー（Nature）、256巻、495頁(1975年)］に従って実施することができる。融合促進剤としては、ポリエチレングリコール（PEG）やセンダイウィルスなどが挙げられるが、好ましくはPEGなどが用いられる。PEGの分子量は特に制限はないが、低毒性で且つ粘性が比較的低いPEG1000～PEG6000が好ましい。PEG濃度としては例えば10～80%程度、好ましくは30～50%程度が例示される。PEGの希釈用溶液としては無血清培地（例：RPMI1640）、5～20%程度の血清を含む完全培地、リン酸緩衝生理食塩水（PBS）、トリス緩衝液等の各種緩衝液を用いることができる。所望によりDMSO（例：10～20％程度）を添加することもできる。融合液のpHとしては、例えば4～10程度、好ましくは6～8程度が挙げられる。
　抗体産生細胞（脾細胞）数と骨髄細胞数との好ましい比率は、通常1:1～20:1程度であり、通常20～40℃、好ましくは30～37℃で通常1～10分間インキュベートすることにより効率よく細胞融合を実施できる。 The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used. The molecular weight of PEG is not particularly limited, but PEG1000 to PEG6000 having low toxicity and relatively low viscosity are preferable. Examples of the PEG concentration include about 10 to 80%, preferably about 30 to 50%. As the PEG dilution solution, various buffer solutions such as serum-free medium (eg RPMI1640), complete medium containing about 5 to 20% serum, phosphate buffered saline (PBS), Tris buffer, etc. may be used. it can. If desired, DMSO (eg, about 10 to 20%) can be added. The pH of the fusion solution is, for example, about 4 to 10, preferably about 6 to 8.
The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of bone marrow cells is usually about 1: 1 to 20: 1, and is usually incubated at 20 to 40 ° C., preferably 30 to 37 ° C. for usually 1 to 10 minutes. Cell fusion can be carried out efficiently.
　モノクローナル抗体産生ハイブリドーマは、例えば、抗原を直接あるいは担体とともに吸着させた固相（例、マイクロプレート）にハイブリドーマ培養上清を添加し、次に放射性物質や酵素などで標識した抗免疫グロブリン抗体（細胞融合に用いられる細胞がマウスの場合、抗マウス免疫グロブリン抗体が用いられる）またはプロテインAを加え、固相に結合したモノクローナル抗体を検出する方法；抗免疫グロブリン抗体またはプロテインAを吸着させた固相にハイブリドーマ培養上清を添加し、放射性物質や酵素などで標識した蛋白質を加え、固相に結合したモノクローナル抗体を検出する方法；などによりスクリーニングすることができる。 Monoclonal antibody-producing hybridomas are prepared, for example, by adding the hybridoma culture supernatant to a solid phase (eg, microplate) on which an antigen is adsorbed directly or with a carrier, and then anti-immunoglobulin antibodies (cells) labeled with radioactive substances or enzymes. When mouse used for fusion is mouse, anti-mouse immunoglobulin antibody is used) or protein A is added to detect monoclonal antibody bound to the solid phase; solid phase adsorbed with anti-immunoglobulin antibody or protein A The method can be screened by adding a hybridoma culture supernatant, adding a protein labeled with a radioactive substance or an enzyme, and detecting a monoclonal antibody bound to the solid phase.
　モノクローナル抗体の選別は、自体公知あるいはそれに準じる方法に従って行なうことができる。モノクローナル抗体の選別は、通常HAT（ヒポキサンチン、アミノプテリン、チミジン）を添加した動物細胞用培地で行うことができる。モノクローナル抗体の選別および育種用培地は、ハイブリドーマが生育できるものならばどのような培地を用いても良い。このような培地としては、例えば、1～20％、好ましくは10～20％の牛胎児血清を含むRPMI 1640培地、1～10％の牛胎児血清を含むＧＩＴ培地（和光純薬工業（株））あるいはハイブリドーマ培養用無血清培地（SFM-101、日水製薬（株））などを用いることができる。培養温度は、通常20～40℃、好ましくは約37℃である。培養時間は、通常5日～3週間、好ましくは1週間～2週間である。培養は、通常5％炭酸ガス下で行うことができる。ハイブリドーマ培養上清の抗体価は、上記の抗血清中の抗体価の測定と同様にして測定できる。 The selection of the monoclonal antibody can be performed according to a method known per se or a method analogous thereto. The selection of the monoclonal antibody can be usually performed in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. Any medium may be used as the monoclonal antibody selection and breeding medium as long as the hybridoma can grow. As such a medium, for example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal calf serum, GIT medium containing 1 to 10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) Or a serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like. The culture temperature is usually 20-40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.
　このようにして得られたモノクローナル抗体は、自体公知の方法、例えば、免疫グロブリンの分離精製法〔例、塩析法、アルコール沈殿法、等電点沈殿法、電気泳動法、イオン交換体（例、DEAE）による吸脱着法、超遠心法、ゲルろ過法、抗原結合固相あるいはプロテインAあるいはプロテインGなどの活性吸着剤により抗体のみを採取し、結合を解離させて抗体を得る特異的精製法〕に従って分離精製することができる。 The monoclonal antibody thus obtained can be obtained by a method known per se, for example, an immunoglobulin separation and purification method [eg, salting out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, , DEAE) adsorption / desorption method, ultracentrifugation, gel filtration, antigen-binding solid phase or specific purification method to obtain antibody by dissociating the binding using active adsorbent such as protein A or protein G ] Can be separated and purified according to the above.
３）ポリクローナル抗体の作製
　ポリクローナル抗体は、例えば、免疫抗原（蛋白質もしくはペプチド抗原）自体、あるいはそれとキャリアー蛋白質との複合体をつくり、上記のモノクローナル抗体の製造法と同様に温血動物に免疫を行い、該免疫動物からGPR120またはその部分ペプチドに対する抗体含有物、例えば血液、腹水、乳、卵など、好ましくは血液から採取することができる。ポリクローナル抗体の分離精製は、上記のモノクローナル抗体の分離精製と同様の免疫グロブリンの分離精製法に従って行うことができる。 3) Preparation of polyclonal antibody Polyclonal antibodies, for example, immunize warm-blooded animals in the same manner as the above-mentioned monoclonal antibody production method, by creating an immune antigen (protein or peptide antigen) itself or a complex thereof with a carrier protein. From the immunized animal, an antibody-containing product against GPR120 or a partial peptide thereof, such as blood, ascites, milk, egg, etc., can be preferably collected from blood. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as that of the monoclonal antibody.
　上記（b）のスクリーニング方法におけるGPR120またはその部分ペプチドの量の測定は、具体的には、例えば、
（i）本発明の検出用抗体と、試料液および標識化されたGPR120またはその部分ペプチドとを競合的に反応させ、該抗体に結合した標識化された蛋白質またはペプチドを検出することにより試料液中のGPR120またはその部分ペプチドを定量する方法、
（ii）試料液と、担体上に不溶化した本発明の検出用抗体および標識化された別の本発明の検出用抗体とを、同時あるいは連続的に反応させた後、不溶化担体上の標識剤の量（活性）を測定することにより、試料液中のGPR120またはその部分ペプチドを定量する方法等が挙げられる。
　上記（ii）の定量法においては、2種の抗体はGPR120の異なる部分を認識するものであることが望ましい。例えば、一方の抗体が該蛋白質のＮ端部を認識する抗体であれば、他方の抗体として該蛋白質のＣ端部と反応するものを用いることができる。
　標識物質を用いる測定法に用いられる標識剤としては、例えば、放射性同位元素、酵素、蛍光物質、発光物質などが用いられる。放射性同位元素としては、例えば、〔125I〕、〔131I〕、〔3H〕、〔14C〕、〔32P〕、〔33P〕、〔35S〕などが用いられる。上記酵素としては、安定で比活性の大きなものが好ましく、例えば、β－ガラクトシダーゼ、β－グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素などが用いられる。蛍光物質としては、例えば、フルオレスカミン、フルオレッセンイソチオシアネート、シアニン蛍光色素などが用いられる。発光物質としては、例えば、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなどが用いられる。さらに、抗体あるいは抗原と標識剤との結合にビオチン－（ストレプト）アビジン系を用いることもできる。 Specifically, the measurement of the amount of GPR120 or a partial peptide thereof in the screening method of (b) above is, for example,
(I) A sample solution by competitively reacting the detection antibody of the present invention with the sample solution and labeled GPR120 or a partial peptide thereof, and detecting the labeled protein or peptide bound to the antibody A method for quantifying GPR120 or a partial peptide thereof,
(Ii) The sample solution, the detection antibody of the present invention insolubilized on the carrier, and another labeled detection antibody of the present invention are reacted simultaneously or successively, and then the labeling agent on the insolubilized carrier A method for quantifying GPR120 or a partial peptide thereof in a sample solution by measuring the amount (activity) of the above is mentioned.
In the above quantification method (ii), it is desirable that the two types of antibodies recognize different portions of GPR120. For example, if one antibody recognizes the N-terminal part of the protein, one that reacts with the C-terminal part of the protein can be used as the other antibody.
As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. Examples of the radioisotope include [ 125 I], [ 131 I], [ 3 H], [ 14 C], [ 32 P], [ 33 P], [ 35 S] and the like. The enzyme is preferably stable and has a large specific activity. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate, cyanine fluorescent dye and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin- (strept) avidin system can be used for binding of an antibody or antigen to a labeling agent.
　本発明の検出用抗体を用いるGPR120またはその部分ペプチドの定量法は、特に制限されるべきものではなく、試料液中の抗原量に対応した、抗体、抗原もしくは抗体－抗原複合体の量を化学的または物理的手段により検出し、これを既知量の抗原を含む標準液を用いて作製した標準曲線より算出する測定法であれば、いずれの測定法を用いてもよい。例えば、ネフロメトリー、競合法、イムノメトリック法およびサンドイッチ法が好適に用いられる。感度、特異性の点で、例えば、後述するサンドイッチ法を用いるのが好ましい。 The method for quantifying GPR120 or its partial peptide using the detection antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the sample solution is chemically determined. Any measurement method may be used as long as it is a measurement method that is detected by a standard or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used. In view of sensitivity and specificity, for example, the sandwich method described later is preferably used.
　抗原あるいは抗体の不溶化にあたっては、物理吸着を用いてもよく、また通常蛋白質あるいは酵素等を不溶化・固定化するのに用いられる化学結合を用いてもよい。担体としては、アガロース、デキストラン、セルロースなどの不溶性多糖類、ポリスチレン、ポリアクリルアミド、シリコン等の合成樹脂、あるいはガラス等があげられる。 In the insolubilization of the antigen or antibody, physical adsorption may be used, or a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
　サンドイッチ法においては不溶化した本発明の検出用抗体に試料液を反応させ（１次反応）、さらに標識化した別の本発明の検出用抗体を反応させ（２次反応）た後、不溶化担体上の標識剤の量もしくは活性を測定することにより、試料液中のGPR120またはその部分ペプチドを定量することができる。１次反応と２次反応は逆の順序で行っても、また、同時に行ってもよいし、時間をずらして行ってもよい。標識化剤および不溶化の方法は前記のそれらに準じることができる。また、サンドイッチ法による免疫測定法において、固相化抗体あるいは標識化抗体に用いられる抗体は必ずしも１種類である必要はなく、測定感度を向上させる等の目的で２種類以上の抗体の混合物を用いてもよい。 In the sandwich method, a sample solution is reacted with the insolubilized detection antibody of the present invention (primary reaction), and another labeled detection antibody of the present invention is reacted (secondary reaction), and then on the insolubilized carrier. By measuring the amount or activity of the labeling agent, GPR120 or its partial peptide in the sample solution can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times. The labeling agent and the insolubilization method can be the same as those described above. Further, in the immunoassay by the sandwich method, the antibody used for the immobilized antibody or the labeled antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
　本発明の検出用抗体は、サンドイッチ法以外の測定システム、例えば、競合法、イムノメトリック法あるいはネフロメトリーなどにも用いることができる。
　競合法では、試料液中のGPR120またはその部分ペプチドと標識したGPR120またはその部分ペプチドとを抗体に対して競合的に反応させた後、未反応の標識抗原（Ｆ）と、抗体と結合した標識抗原（Ｂ）とを分離し（Ｂ／Ｆ分離）、Ｂ、Ｆいずれかの標識量を測定することにより、試料液中のGPR120またはその部分ペプチドを定量する。本反応法には、抗体として可溶性抗体を用い、ポリエチレングリコールや前記抗体（１次抗体）に対する２次抗体などを用いてＢ／Ｆ分離を行う液相法、および、１次抗体として固相化抗体を用いるか（直接法）、あるいは１次抗体は可溶性のものを用い、２次抗体として固相化抗体を用いる（間接法）固相化法とが用いられる。
　イムノメトリック法では、試料液中のGPR120またはその部分ペプチドと固相化したGPR120またはその部分ペプチドとを一定量の標識化抗体に対して競合反応させた後、固相と液相を分離するか、あるいは試料液中のGPR120またはその部分ペプチドと過剰量の標識化抗体とを反応させ、次に固相化したGPR120またはその部分ペプチドを加えて未反応の標識化抗体を固相に結合させた後、固相と液相を分離する。次に、いずれかの相の標識量を測定し試料液中の抗原量を定量する。
　また、ネフロメトリーでは、ゲル内あるいは溶液中で抗原抗体反応の結果生じた不溶性の沈降物の量を測定する。試料液中のGPR120またはその部分ペプチドの量がわずかであり、少量の沈降物しか得られない場合にもレーザーの散乱を利用するレーザーネフロメトリーなどが好適に用いられる。 The detection antibody of the present invention can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
In the competitive method, GPR120 or a partial peptide thereof in a sample solution and labeled GPR120 or a partial peptide thereof are reacted competitively with an antibody, and then an unreacted labeled antigen (F) and a label bound to the antibody. By separating the antigen (B) (B / F separation) and measuring the labeling amount of either B or F, GPR120 or its partial peptide in the sample solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol or a secondary antibody against the antibody (primary antibody), and a solid phase is used as the primary antibody. Either an antibody is used (direct method), or a primary antibody is soluble, and a solid phase antibody is used as a secondary antibody (indirect method).
In the immunometric method, GPR120 or its partial peptide in the sample solution and the immobilized GPR120 or its partial peptide are allowed to compete with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. Alternatively, GPR120 or its partial peptide in the sample solution is reacted with an excess amount of labeled antibody, and then immobilized GPR120 or its partial peptide is added to bind the unreacted labeled antibody to the solid phase. Thereafter, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the sample solution.
In nephrometry, the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of GPR120 or its partial peptide in the sample solution is small and only a small amount of precipitate is obtained.
　これら個々の免疫学的測定法を本発明の定量方法に適用するにあたっては、特別の条件、操作等の設定は必要とされない。それぞれの方法における通常の条件、操作法に当業者の通常の技術的配慮を加えて、GPR120またはその部分ペプチドの測定系を構築すればよい。これらの一般的な技術手段の詳細については、総説、成書などを参照することができる。
　例えば、入江　寛編「ラジオイムノアッセイ」（講談社、昭和４９年発行）、入江　寛編「続ラジオイムノアッセイ」（講談社、昭和５４年発行）、石川栄治ら編「酵素免疫測定法」（医学書院、昭和５３年発行）、石川栄治ら編「酵素免疫測定法」（第２版）（医学書院、昭和５７年発行）、石川栄治ら編「酵素免疫測定法」（第３版）（医学書院、昭和６２年発行）、「Methods in ENZYMOLOGY」 Vol. 70 (Immunochemical Techniques (Part A))、同書 Vol. 73 (Immunochemical Techniques (Part B))、同書 Vol. 74 (Immunochemical Techniques (Part C))、同書 Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays))、同書 Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods))、同書 Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies))（以上、アカデミックプレス社発行）などを参照することができる。
　以上のようにして、本発明の検出用抗体を用いることによって、細胞におけるGPR120またはその部分ペプチドの量を感度よく定量することができる。 In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. A measurement system for GPR120 or a partial peptide thereof may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and procedures in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Sequel Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (2nd edition) (Medical School, published in 1982), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (3rd edition) (Medical School, Showa) 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), Id. Vol. 73 (Immunochemical Techniques (Part B)), Id. Vol. 74 (Immunochemical Techniques (Part C)), Id. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid. Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid. )) (End , Inc. issued) and the like can be referred to.
As described above, by using the detection antibody of the present invention, the amount of GPR120 or a partial peptide thereof in a cell can be quantified with high sensitivity.
　例えば、上記（a）および（b）のスクリーニング法において、試験化合物の存在下におけるGPR120またはその部分ペプチドの発現量（mRNA量または蛋白質（ペプチド）量）が、試験化合物の非存在下における場合に比べて、約20%以上、好ましくは約30％以上、より好ましくは約50％以上増加した場合、該試験化合物を、GPR120の発現促進物質、従って、EPCの分化・増殖促進物質の候補として選択することができる。一方、試験化合物の存在下における該蛋白質またはペプチドの発現量が、試験化合物の非存在下における場合に比べて、約20%以上、好ましくは約30％以上、より好ましくは約50％以上減少した場合、該試験化合物を、GPR120の発現抑制物質、従って、EPCの分化・増殖抑制および／または末梢血中のEPC増加物質の候補として選択することができる。 For example, in the above screening methods (a) and (b), when the expression level of GPR120 or a partial peptide thereof (mRNA level or protein (peptide level)) in the presence of the test compound is in the absence of the test compound If the test compound is increased by about 20% or more, preferably about 30% or more, more preferably about 50% or more, the test compound is selected as a candidate for GPR120 expression promoting substance, and therefore, for EPC differentiation / proliferation promoting substance. can do. On the other hand, the expression level of the protein or peptide in the presence of the test compound was reduced by about 20% or more, preferably about 30% or more, more preferably about 50% or more, compared to the case in the absence of the test compound. In this case, the test compound can be selected as a GPR120 expression-suppressing substance, and thus a candidate for EPC differentiation / proliferation inhibition and / or an EPC increasing substance in peripheral blood.
　上記（１ａ）のスクリーニング方法により得られるGPR120の活性を促進する物質（GPR120のアゴニストまたはGPR120とリガンドとの結合性を増強する化合物）、並びに上記（１ｂ）のスクリーニング方法により得られるGPR120の発現を促進する物質は、骨髄細胞からEPCへの分化およびEPCの増殖を促進するので、EPCの作用を介する血管形成の能力を増強することにより予防および／または治療効果が得られうる疾患の予防・治療薬として有用である。そのような疾患としては、例えば、糖尿病およびその合併症（例、神経障害、腎症、足病変、血流障害）、肥満症、動脈硬化、閉塞性動脈硬化症、バージャー病、肺高血圧、心筋梗塞、脳梗塞、肝炎、肝硬変、慢性閉塞性肺疾患、創傷、骨折、骨粗鬆症、歯周病、認知症などが挙げられる。さらに、これらの物質は、自家末梢血中単核球移植において採取できる単核球数を増大させたり、骨髄移植患者における移植骨髄細胞の分化を促進させる目的で使用することもできる。
　一方、上記（１ａ）のスクリーニング方法により得られるGPR120の活性を抑制する物質（GPR120のアゴニストまたはGPR120とリガンドとの結合性を減少させる化合物）、並びに上記（１ｂ）のスクリーニング方法により得られるGPR120の発現を抑制する物質は、末梢血中のEPC数を増加させることができるので、EPCの作用を介する血管形成の能力を増強することにより予防および／または治療効果が得られうる、上記疾患の予防・治療薬として同様に有用である。さらに、これらの物質は、自家末梢血中単核球移植において採取できる単核球数を増大させる目的で使用することもできる。
　また、GPR120の活性または発現を抑制する物質は、骨髄細胞からEPCへの分化およびEPCの増殖を抑制するので、EPCの作用を介する血管形成の能力を抑制することにより予防および／または治療効果が得られうる疾患、例えば、癌、糖尿病性網膜症、リウマチ、骨関節炎などの慢性炎症などの予防・治療薬として有用である。 A substance that promotes the activity of GPR120 obtained by the screening method of (1a) (a compound that enhances the binding property between an agonist of GPR120 or GPR120 and a ligand), and the expression of GPR120 obtained by the screening method of (1b) above. The substance that promotes promotes differentiation from bone marrow cells to EPC and proliferation of EPC. Therefore, prevention and / or treatment of diseases that can have preventive and / or therapeutic effects by enhancing the ability of angiogenesis through the action of EPC Useful as a medicine. Such diseases include, for example, diabetes and its complications (eg, neuropathy, nephropathy, foot lesions, blood flow disorders), obesity, arteriosclerosis, obstructive arteriosclerosis, Buerger's disease, pulmonary hypertension, myocardium Examples include infarction, cerebral infarction, hepatitis, cirrhosis, chronic obstructive pulmonary disease, wound, bone fracture, osteoporosis, periodontal disease, dementia and the like. Furthermore, these substances can also be used for the purpose of increasing the number of mononuclear cells that can be collected in autologous peripheral blood mononuclear cell transplantation or promoting the differentiation of transplanted bone marrow cells in bone marrow transplant patients.
On the other hand, a substance that suppresses the activity of GPR120 obtained by the screening method of (1a) (a compound that decreases the binding property between an agonist of GPR120 or GPR120 and a ligand), and GPR120 obtained by the screening method of (1b) above. Since the substance that suppresses the expression can increase the number of EPCs in peripheral blood, prevention and / or therapeutic effect can be obtained by enhancing the ability of angiogenesis through the action of EPC. • It is equally useful as a therapeutic agent. Furthermore, these substances can also be used for the purpose of increasing the number of mononuclear cells that can be collected in autologous peripheral blood mononuclear cell transplantation.
In addition, since a substance that suppresses the activity or expression of GPR120 suppresses differentiation from bone marrow cells to EPC and proliferation of EPC, it has a preventive and / or therapeutic effect by suppressing the ability of angiogenesis through the action of EPC. It is useful as a preventive / therapeutic agent for diseases that can be obtained, for example, chronic inflammation such as cancer, diabetic retinopathy, rheumatism, osteoarthritis and the like.
　本発明のスクリーニング方法により得られるGPR120の発現または活性を調節（増強もしくは抑制）する物質（以下、「GPR120調節薬」という場合がある）を含有する医薬は低毒性であり、そのまま液剤として、または適当な剤型の医薬組成物として、ヒトまたは哺乳動物（例、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど）に対して経口的または非経口的（例、血管内投与、皮下投与など）に投与することができる。 A drug containing a substance that regulates (enhances or suppresses) the expression or activity of GPR120 obtained by the screening method of the present invention (hereinafter sometimes referred to as “GPR120 modulator”) has low toxicity and is used as a solution as it is or Suitable pharmaceutical forms of pharmaceutical compositions include oral or parenteral (eg, intravascular) for humans or mammals (eg, mice, rats, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.) Administration, subcutaneous administration, etc.).
　GPR120調節薬は、それ自体を投与してもよいし、または適当な医薬組成物として投与してもよい。投与に用いられる医薬組成物としては、GPR120調節薬と薬理学的に許容され得る担体、希釈剤もしくは賦形剤とを含むものであってもよい。このような医薬組成物は、経口または非経口投与に適する剤形として提供される。 The GPR120 modulator may be administered per se, or may be administered as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain a GPR120 modulator and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.
　非経口投与のための組成物としては、例えば、注射剤、坐剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含しても良い。このような注射剤は、公知の方法に従って調製できる。注射剤の調製方法としては、例えば、GPR120調節薬を通常注射剤に用いられる無菌の水性液、または油性液に溶解、懸濁または乳化することによって調製できる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液等が用いられ、適当な溶解補助剤、例えば、アルコール（例、エタノール）、ポリアルコール（例、プロピレングリコール、ポリエチレングリコール）、非イオン界面活性剤〔例、ポリソルベート８０、ＨＣＯ－５０（polyoxyethylene（50mol）adduct of hydrogenated castor oil）〕等と併用してもよい。油性液としては、例えば、ゴマ油、大豆油等が用いられ、溶解補助剤として安息香酸ベンジル、ベンジルアルコール等を併用してもよい。調製された注射液は、適当なアンプルに充填されることが好ましい。直腸投与に用いられる坐剤は、GPR120調節薬を通常の坐薬用基剤に混合することによって調製されてもよい。 As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included. Such an injection can be prepared according to a known method. As a method for preparing an injection, for example, the GPR120 modulator can be prepared by dissolving, suspending or emulsifying in a sterile aqueous liquid or oily liquid usually used for injection. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)), etc. may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. Suppositories used for rectal administration may be prepared by mixing a GPR120 modulator with a conventional suppository base.
　経口投与のための組成物としては、固体または液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤等が挙げられる。このような組成物は公知の方法によって製造され、製剤分野において通常用いられる担体、希釈剤もしくは賦形剤を含有していてもよい。錠剤用の担体、賦形剤としては、例えば、乳糖、でんぷん、蔗糖、ステアリン酸マグネシウムが用いられる。 Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.
　上記の非経口用または経口用医薬組成物は、GPR120調節薬の投与量に適合するような投薬単位の剤形に調製されることが好都合である。このような投薬単位の剤形としては、例えば、錠剤、丸剤、カプセル剤、注射剤（アンプル）、坐剤が挙げられる。GPR120調節薬は、投薬単位剤形当たり通常5～500mg、とりわけ注射剤では5～100mg、その他の剤形では10～250mg含有されていることが好ましい。 The above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dosage of the GPR120 modulator. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories. The GPR120 modulator is usually contained in an amount of 5 to 500 mg per dosage unit form, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.
　GPR120調節薬を含有する上記医薬の投与量は、投与対象、対象疾患、症状、投与ルートなどによっても異なるが、例えば、成人の糖尿病の治療・予防のために使用する場合には、GPR120調節薬を1回量として、通常0.01～20mg/kg体重程度、好ましくは0.1～10mg/kg体重程度、さらに好ましくは0.1～5mg/kg体重程度を、1日1～5回程度、好ましくは1日1～3回程度、静脈注射により投与するのが好都合である。他の非経口投与および経口投与の場合もこれに準ずる量を投与することができる。症状が特に重い場合には、その症状に応じて増量してもよい。 The dosage of the above-mentioned pharmaceutical containing a GPR120 modulator varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for the treatment and prevention of diabetes in adults, the GPR120 modulator Is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably 1 day a day. It is convenient to administer about 3 times by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
（２）EPCの分化・増殖促進剤
　上述の通り、GPR120はEPCの分化・増殖を促進するので、(a)GPR120またはその部分ペプチド、（b）GPR120またはその部分ペプチドをコードする塩基配列を含む核酸は、上記（c）GPR120の発現を増強する化合物や（d）GPR120の活性を増強する化合物と同様に、1) EPCの分化・増殖促進剤、2) EPCの作用を介する血管形成能の促進により予防・治療効果が得られうる疾患の予防・治療剤などとして使用することができる。そのような疾患としては、例えば、糖尿病およびその合併症（例、神経障害、腎症、足病変、血流障害）、肥満症、動脈硬化、閉塞性動脈硬化症、バージャー病、肺高血圧、心筋梗塞、脳梗塞、肝炎、肝硬変、慢性閉塞性肺疾患、創傷、骨折、骨粗鬆症、歯周病、認知症などが挙げられる。さらに、上記（a）または（b）の物質は、自家末梢血中単核球移植において採取できる単核球数を増大させたり、骨髄移植患者における移植骨髄細胞の分化を促進させる目的で使用することもできる。 (2) EPC differentiation / proliferation promoter As described above, GPR120 promotes EPC differentiation / proliferation, and thus includes (a) GPR120 or a partial peptide thereof, and (b) a base sequence encoding GPR120 or a partial peptide thereof. Nucleic acids, like (c) compounds that enhance the expression of GPR120 and (d) compounds that enhance the activity of GPR120, have 1) an EPC differentiation / proliferation promoter, 2) an angiogenic ability through the action of EPC. It can be used as a prophylactic / therapeutic agent for diseases that can have a prophylactic / therapeutic effect by promotion. Such diseases include, for example, diabetes and its complications (eg, neuropathy, nephropathy, foot lesions, blood flow disorders), obesity, arteriosclerosis, obstructive arteriosclerosis, Buerger's disease, pulmonary hypertension, myocardium Examples include infarction, cerebral infarction, hepatitis, cirrhosis, chronic obstructive pulmonary disease, wound, bone fracture, osteoporosis, periodontal disease, dementia and the like. Furthermore, the substance (a) or (b) is used for the purpose of increasing the number of mononuclear cells that can be collected in autologous peripheral blood mononuclear cell transplantation or promoting the differentiation of transplanted bone marrow cells in bone marrow transplant patients. You can also
　GPR120またはその部分ペプチドを上述の予防・治療剤として使用する場合、上記GPR120調節薬と同様に製剤化することができ、同様の投与経路および投与量で、ヒトまたは哺乳動物（例えば、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ウマ、ネコ、イヌ、サル、チンパンジーなど）に対して、経口的にまたは非経口的に投与することができる。 When GPR120 or a partial peptide thereof is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the above-mentioned GPR120 modulator, and can be administered to humans or mammals (for example, mice, rats, etc.) with the same administration route and dosage , Rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) orally or parenterally.
　GPR120またはその部分ペプチドをコードする塩基配列を含む核酸を上述の予防・治療剤として使用する場合、自体公知の方法に従って製剤化し、投与することができる。即ち、前記核酸を、単独あるいはレトロウイルスベクター、レンチウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター、ヘルペスウイルスベクターなどの適当な哺乳動物細胞用の発現ベクターに機能可能な態様で挿入した後、常套手段に従って製剤化することができる。該核酸は、そのままで、あるいは摂取促進のための補助剤とともに、遺伝子銃やハイドロゲルカテーテルのようなカテーテルによって投与することができる。あるいは、エアロゾル化して吸入剤として気管内に局所投与することもできる。
　さらに、体内動態の改良、半減期の長期化、細胞内取り込み効率の改善を目的に、前記核酸を単独またはリポソームなどの担体とともに製剤（注射剤）化し、静脈、皮下等に投与してもよい。 When a nucleic acid containing a base sequence encoding GPR120 or a partial peptide thereof is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated and administered according to a method known per se. That is, the nucleic acid is inserted in a functional manner into an appropriate expression vector for mammalian cells such as a retrovirus vector, lentivirus vector, adenovirus vector, adeno-associated virus vector, herpes virus vector, etc. It can be formulated according to the means. The nucleic acid can be administered as it is or together with an auxiliary agent for promoting intake by a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
Furthermore, for the purpose of improving the pharmacokinetics, extending the half-life, and improving the cellular uptake efficiency, the nucleic acid may be formulated (injection) alone or together with a carrier such as a liposome and administered intravenously, subcutaneously, etc. .
　GPR120またはその部分ペプチドをコードする塩基配列を含む核酸は、それ自体を投与してもよいし、または適当な医薬組成物として投与してもよい。投与に用いられる医薬組成物としては、前記核酸と薬理学的に許容され得る担体、希釈剤もしくは賦形剤とを含むものであってよい。このような医薬組成物は、経口または非経口投与に適する剤形として提供される。 The nucleic acid containing the base sequence encoding GPR120 or a partial peptide thereof may be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for administration may contain the nucleic acid and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.
　非経口投与のための組成物としては、例えば、注射剤、坐剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含しても良い。このような注射剤は、公知の方法に従って調製できる。注射剤の調製方法としては、例えば、前記核酸を通常注射剤に用いられる無菌の水性液、または油性液に溶解、懸濁または乳化することによって調製できる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液等が用いられ、適当な溶解補助剤、例えば、アルコール（例、エタノール）、ポリアルコール（例、プロピレングリコール、ポリエチレングリコール）、非イオン界面活性剤〔例、ポリソルベート８０、ＨＣＯ－５０（polyoxyethylene（50mol）adduct of hydrogenated castor oil）〕等と併用してもよい。油性液としては、例えば、ゴマ油、大豆油等が用いられ、溶解補助剤として安息香酸ベンジル、ベンジルアルコール等を併用してもよい。調製された注射液は、適当なアンプルに充填されることが好ましい。直腸投与に用いられる坐剤は、前記核酸を通常の坐薬用基剤に混合することによって調製されてもよい。 As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included. Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared, for example, by dissolving, suspending or emulsifying the nucleic acid in a sterile aqueous liquid or oily liquid usually used for injection. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)), etc. may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. A suppository used for rectal administration may be prepared by mixing the nucleic acid with an ordinary suppository base.
　経口投与のための組成物としては、固体または液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤等が挙げられる。このような組成物は公知の方法によって製造され、製剤分野において通常用いられる担体、希釈剤もしくは賦形剤を含有していても良い。錠剤用の担体、賦形剤としては、例えば、乳糖、でんぷん、蔗糖、ステアリン酸マグネシウムが用いられる。 Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.
　上記の非経口用または経口用医薬組成物は、活性成分の投与量に適合するような投薬単位の剤形に調製されることが好都合である。このような投薬単位の剤形としては、例えば、錠剤、丸剤、カプセル剤、注射剤（アンプル）、坐剤が挙げられる。GPR120またはその部分ペプチドをコードする塩基配列を含む核酸は、例えば、投薬単位剤形当たり通常5～500mg、とりわけ注射剤では5～100mg、その他の剤形では10～250mg含有されていることが好ましい。 The above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories. The nucleic acid containing a base sequence encoding GPR120 or a partial peptide thereof is preferably contained, for example, usually 5 to 500 mg per dosage unit dosage form, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms. .
　GPR120またはその部分ペプチドをコードする塩基配列を含む核酸を含有する上記医薬の投与量は、投与対象、対象疾患、症状、投与ルートなどによっても異なるが、例えば、成人の糖尿病の治療・予防のために使用する場合には、前記核酸を１回量として、通常0.01～20mg/kg体重程度、好ましくは0.1～10mg/kg体重程度、さらに好ましくは0.1～5mg/kg体重程度を、1日1～5回程度、好ましくは1日1～3回程度、静脈注射により投与するのが好都合である。他の非経口投与および経口投与の場合もこれに準ずる量を投与することができる。症状が特に重い場合には、その症状に応じて増量してもよい。 The dosage of the above-mentioned pharmaceutical containing a nucleic acid comprising a base sequence encoding GPR120 or a partial peptide thereof varies depending on the administration subject, target disease, symptom, administration route, etc., but for example for the treatment and prevention of diabetes in adults When used in the above, the above nucleic acid is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight per day. It is convenient to administer by intravenous injection about 5 times, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
（３）EPCの分化・増殖抑制／末梢血中のEPC増加剤
　上述の通り、GPR120はEPCの分化・増殖を促進し、また、GPR120の抑制は末梢血中のEPC数を増加させるので、(a)GPR120またはその部分ペプチドに対する中和抗体、（b）GPR120をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸は、上記（c）GPR120の発現を抑制する化合物や（d）GPR120の活性を抑制する化合物と同様に、1) EPCの分化・増殖抑制剤、2) EPCの作用を介する血管形成能の抑制により予防・治療効果が得られうる疾患の予防・治療剤、3) 末梢血中のEPC増加剤、4) 末梢血中のEPC増加により予防・治療効果が得られうる疾患の予防・治療剤などとして使用することができる。EPCの作用を介する血管形成能の抑制により予防・治療効果が得られうる疾患としては、例えば、癌、糖尿病性網膜症、リウマチ、骨関節炎などの慢性炎症などが挙げられる。また、末梢血中のEPC増加により予防・治療効果が得られうる疾患としては、例えば、糖尿病およびその合併症（例、神経障害、腎症、足病変、血流障害）、肥満症、動脈硬化、閉塞性動脈硬化症、バージャー病、肺高血圧、心筋梗塞、脳梗塞、肝炎、肝硬変、慢性閉塞性肺疾患、創傷、骨折、骨粗鬆症、歯周病、認知症などが挙げられる。さらに、上記（a）または（b）の物質は、自家末梢血中単核球移植において採取できる単核球数を増大させたり、骨髄移植患者における移植骨髄細胞の分化を促進させる目的で使用することもできる。 (3) EPC differentiation / proliferation inhibition / EPC increasing agent in peripheral blood As described above, GPR120 promotes EPC differentiation / proliferation, and suppression of GPR120 increases the number of EPCs in peripheral blood. a) a neutralizing antibody against GPR120 or a partial peptide thereof, (b) a nucleic acid comprising a base sequence complementary to the base sequence encoding GPR120 or a part thereof, (c) a compound that suppresses the expression of GPR120 or (d ) Similar to compounds that suppress GPR120 activity, 1) EPC differentiation / proliferation inhibitor, 2) Prophylactic / therapeutic agent for diseases that can have preventive / therapeutic effects by inhibiting angiogenic ability through the action of EPC, 3) EPC increasing agent in peripheral blood 4) It can be used as a prophylactic / therapeutic agent for diseases that can be prevented or treated by increasing EPC in peripheral blood. Examples of diseases that can be prevented or treated by suppressing angiogenic ability through the action of EPC include chronic inflammation such as cancer, diabetic retinopathy, rheumatism, and osteoarthritis. Examples of diseases that can be prevented or treated by increasing EPC in peripheral blood include diabetes and its complications (eg, neuropathy, nephropathy, foot lesions, blood flow disorders), obesity, arteriosclerosis , Obstructive arteriosclerosis, Buerger's disease, pulmonary hypertension, myocardial infarction, cerebral infarction, hepatitis, cirrhosis, chronic obstructive pulmonary disease, wound, fracture, osteoporosis, periodontal disease, dementia and the like. Furthermore, the substance (a) or (b) is used for the purpose of increasing the number of mononuclear cells that can be collected in autologous peripheral blood mononuclear cell transplantation or promoting the differentiation of transplanted bone marrow cells in bone marrow transplant patients. You can also
　GPR120またはその部分ペプチドに対する中和抗体は、前記した本発明の検出用抗体と同様にして製造することができる。好ましい一実施態様において、GPR120またはその部分ペプチドに対する中和抗体は、ヒトを投与対象とする医薬品として使用されることから、該抗体（好ましくはモノクローナル抗体）はヒトに投与した場合に抗原性を示す危険性が低減された抗体、具体的には、完全ヒト抗体、ヒト化抗体、マウス－ヒトキメラ抗体などであり、特に好ましくは完全ヒト抗体またはヒト化抗体である。ヒト化抗体およびキメラ抗体は、常法に従って遺伝子工学的に作製することができる。また、完全ヒト抗体は、ヒト－ヒト（もしくはマウス）ハイブリドーマより製造することも可能ではあるが、大量の抗体を安定に且つ低コストで提供するためには、ヒト抗体産生マウスやファージディスプレイ法を用いて製造することが望ましい。
　得られたGPR120またはその部分ペプチドに対する抗体がGPR120の生理作用、即ちEPCの分化・増殖促進活性を中和するか否かは、例えば、公知のコロニーフォーミングアッセイを利用して、該抗体の存在下でEPCもしくはその前駆細胞を培養し、EPCの増殖もしくはEPCへの分化能を測定することにより行うことができる。 A neutralizing antibody against GPR120 or a partial peptide thereof can be produced in the same manner as the detection antibody of the present invention described above. In a preferred embodiment, since the neutralizing antibody against GPR120 or a partial peptide thereof is used as a pharmaceutical for human administration, the antibody (preferably a monoclonal antibody) exhibits antigenicity when administered to a human. Antibodies with reduced risk, specifically, fully human antibodies, humanized antibodies, mouse-human chimeric antibodies and the like, particularly preferably fully human antibodies or humanized antibodies. Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to conventional methods. In addition, fully human antibodies can be produced from human-human (or mouse) hybridomas, but in order to provide a large amount of antibodies stably and at low cost, human antibody-producing mice and phage display methods are used. It is desirable to manufacture using.
Whether or not the antibody against the obtained GPR120 or a partial peptide thereof neutralizes the physiological action of GPR120, that is, the differentiation / proliferation promoting activity of EPC, is determined in the presence of the antibody using, for example, a known colony forming assay. Can be performed by culturing EPC or its progenitor cells and measuring EPC proliferation or EPC differentiation ability.
　GPR120またはその部分ペプチドに対する中和抗体を上述の予防・治療剤として使用する場合、上記GPR120調節薬と同様に製剤化することができ、同様の投与経路および投与量で、ヒトまたは哺乳動物（例えば、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ウマ、ネコ、イヌ、サル、チンパンジーなど）に対して、経口的にまたは非経口的に投与することができる。 When a neutralizing antibody against GPR120 or a partial peptide thereof is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as the above-mentioned GPR120 modulator, and can be administered to humans or mammals (for example, Mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) orally or parenterally.
　「GPR120をコードする塩基配列に相補的な塩基配列またはその一部」とは、GPR120のmRNAに特異的に結合することができ、且つ該mRNAからの蛋白質の翻訳を阻害し得るものであれば、その長さや位置に特に制限はないが、配列特異性の面から、標的配列に相補的もしくは実質的に相補的な部分を少なくとも10塩基以上、好ましくは約15塩基以上、より好ましくは約20塩基以上含むものである。 The “base sequence complementary to the base sequence encoding GPR120 or a part thereof” is a sequence that can specifically bind to the mRNA of GPR120 and can inhibit the translation of the protein from the mRNA. The length and position are not particularly limited, but from the viewpoint of sequence specificity, a portion complementary or substantially complementary to the target sequence is at least 10 bases or more, preferably about 15 bases or more, more preferably about 20 It contains more than a base.
　具体的には、GPR120のmRNAの塩基配列と相補的（もしくは実質的に相補的）な塩基配列またはその一部を含む核酸として、以下の(i)～(iii)のいずれかのものが好ましく例示される。
(i) GPR120のmRNAに対するアンチセンス核酸
(ii) GPR120のmRNAに対するsiRNA
(iii) GPR120のmRNAに対するsiRNAを生成し得る核酸 Specifically, the nucleic acid comprising a base sequence complementary to (or substantially complementary to) the base sequence of mRNA of GPR120 or a part thereof is preferably any of the following (i) to (iii): Illustrated.
(i) Antisense nucleic acid against GPR120 mRNA
(ii) siRNA against GPR120 mRNA
(iii) Nucleic acid capable of generating siRNA for GPR120 mRNA
(i) GPR120のmRNAに対するアンチセンス核酸
　本発明における「GPR120のmRNAに対するアンチセンス核酸」とは、該mRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸であって、標的mRNAと特異的かつ安定した二重鎖を形成して結合することにより、蛋白質合成を抑制する機能を有するものである。
　アンチセンス核酸は、2-デオキシ-D-リボースを含有しているポリデオキシリボヌクレオチド、D-リボースを含有しているポリリボヌクレオチド、プリンまたはピリミジン塩基のN-グリコシドであるその他のタイプのポリヌクレオチド、非ヌクレオチド骨格を有するその他のポリマー（例えば、市販の蛋白質核酸および合成配列特異的な核酸ポリマー）または特殊な結合を含有するその他のポリマー（但し、該ポリマーはDNAやRNA中に見出されるような塩基のペアリングや塩基の付着を許容する配置をもつヌクレオチドを含有する）などが挙げられる。それらは、二本鎖DNA、一本鎖DNA、二本鎖RNA、一本鎖RNA、DNA:RNAハイブリッドであってもよく、さらに非修飾ポリヌクレオチド（または非修飾オリゴヌクレオチド）、公知の修飾の付加されたもの、例えば当該分野で知られた標識のあるもの、キャップの付いたもの、メチル化されたもの、１個以上の天然のヌクレオチドを類縁物で置換したもの、分子内ヌクレオチド修飾のされたもの、例えば非荷電結合（例えば、メチルホスホネート、ホスホトリエステル、ホスホルアミデート、カルバメートなど）を持つもの、電荷を有する結合または硫黄含有結合（例、ホスホロチオエート、ホスホロジチオエートなど）を持つもの、例えば蛋白質（例、ヌクレアーゼ、ヌクレアーゼ・インヒビター、トキシン、抗体、シグナルペプチド、ポリ-L-リジンなど）や糖（例、モノサッカライドなど）などの側鎖基を有しているもの、インターカレント化合物（例、アクリジン、ソラレンなど）を持つもの、キレート化合物（例えば、金属、放射活性をもつ金属、ホウ素、酸化性の金属など）を含有するもの、アルキル化剤を含有するもの、修飾された結合を持つもの（例えば、αアノマー型の核酸など）であってもよい。ここで「ヌクレオシド」、「ヌクレオチド」および「核酸」とは、プリンおよびピリミジン塩基を含有するのみでなく、修飾されたその他の複素環型塩基をもつようなものを含んでいて良い。このような修飾物は、メチル化されたプリンおよびピリミジン、アシル化されたプリンおよびピリミジン、あるいはその他の複素環を含むものであってよい。修飾されたヌクレオシドおよび修飾されたヌクレオチドはまた糖部分が修飾されていてよく、例えば、１個以上の水酸基がハロゲンとか、脂肪族基などで置換されていたり、またはエーテル、アミンなどの官能基に変換されていてよい。 (i) Antisense nucleic acid against GPR120 mRNA The term “antisense nucleic acid against GPR120 mRNA” in the present invention refers to a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of the mRNA or a part thereof. Thus, it has a function of suppressing protein synthesis by forming a specific and stable double strand with the target mRNA and binding.
Antisense nucleic acids are polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages (provided that the polymer is a base such as found in DNA or RNA) And a nucleotide having a configuration that allows attachment of a base). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc. ), Sugars (eg, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, metals, radioactive metals) , Boron, an oxidizable metal, etc.), an alkylating agent, and a modified bond (for example, an α anomeric nucleic acid). Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.
　上記の通り、アンチセンス核酸はDNAであってもRNAであってもよく、あるいはDNA/RNAキメラであってもよい。アンチセンス核酸がDNAの場合、標的RNAとアンチセンスDNAとによって形成されるRNA:DNAハイブリッドは、内在性RNase Hに認識されて標的RNAの選択的な分解を引き起こすことができる。したがって、RNase Hによる分解を指向するアンチセンスDNAの場合、標的配列は、mRNA中の配列だけでなく、GPR120遺伝子の初期翻訳産物におけるイントロン領域の配列であってもよい。例えば、ヒトの場合、GPR120遺伝子は第10番染色体の10q23.33領域に存在するので、この領域のゲノム配列と、配列番号１に示されるヒトGPR120 cDNA塩基配列とをBLAST、FASTA等のホモロジー検索プログラムを用いて比較して、イントロン配列を決定することができる。 As described above, the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA: DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of GPR120 gene. For example, in the case of humans, the GPR120 gene is present in the 10q23.33 region of chromosome 10, so the homology search such as BLAST and FASTA for the genomic sequence of this region and the human GPR120 cDNA base sequence shown in SEQ ID NO: 1 Intron sequences can be determined by comparison using a program.
　本発明のアンチセンス核酸の標的領域は、該アンチセンス核酸がハイブリダイズすることにより、結果としてGPR120蛋白質への翻訳が阻害されるものであればその長さに特に制限はなく、該蛋白質をコードするmRNAの全配列であっても部分配列であってもよく、短いもので約10塩基程度、長いものでmRNAもしくは初期転写産物の全配列が挙げられる。合成の容易さや抗原性、細胞内移行性の問題等を考慮すれば、約10～約40塩基、特に約15～約30塩基からなるオリゴヌクレオチドが好ましいが、それに限定されない。具体的には、GPR120遺伝子の5'端ヘアピンループ、5'端６－ベースペア・リピート、5'端非翻訳領域、翻訳開始コドン、蛋白質コード領域、ORF翻訳終止コドン、3'端非翻訳領域、3'端パリンドローム領域または3'端ヘアピンループなどが、アンチセンス核酸の好ましい標的領域として選択しうるが、それらに限定されない。 The length of the target region of the antisense nucleic acid of the present invention is not particularly limited as long as the antisense nucleic acid hybridizes, and as a result, translation into the GPR120 protein is inhibited. The entire sequence or partial sequence of mRNA may be a short sequence of about 10 bases, and a long sequence of mRNA or the initial transcript. In view of easiness of synthesis, antigenicity, intracellular migration, etc., an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferred, but is not limited thereto. Specifically, 5 'end hairpin loop of GPR120 gene, 5' end 6-base pair repeat, 5 'end untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region , 3 ′ end palindromic region or 3 ′ end hairpin loop, etc. may be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.
　さらに、本発明のアンチセンス核酸は、GPR120のmRNAや初期転写産物とハイブリダイズして蛋白質への翻訳を阻害するだけでなく、二本鎖DNAであるこれらの遺伝子と結合して三重鎖（トリプレックス）を形成し、RNAへの転写を阻害し得るもの（アンチジーン）であってもよい。 Furthermore, the antisense nucleic acid of the present invention not only hybridizes with mRNA of GPR120 and the initial transcription product and inhibits translation into proteins, but also binds to these genes that are double-stranded DNAs and binds to triplex (tri-stranded). A plex) and can inhibit transcription to RNA (antigene).
　アンチセンス核酸を構成するヌクレオチド分子は、天然型のDNAもしくはRNAでもよいが、安定性（化学的および／または対酵素）や比活性（RNAとの親和性）を向上させるために、種々の化学修飾を含むことができる。例えば、ヌクレアーゼなどの加水分解酵素による分解を防ぐために、アンチセンス核酸を構成する各ヌクレオチドのリン酸残基（ホスフェート）を、例えば、ホスホロチオエート（PS)、メチルホスホネート、ホスホロジチオネートなどの化学修飾リン酸残基に置換することができる。また、各ヌクレオチドの糖（リボース）の2'位の水酸基を、-OR（Rは、例えばCH3（2'-O-Me）、CH2CH2OCH3（2'-O-MOE）、CH2CH2NHC(NH)NH2、CH2CONHCH3、CH2CH2CN等を示す）に置換してもよい。さらに、塩基部分（ピリミジン、プリン）に化学修飾を施してもよく、例えば、ピリミジン塩基の5位へのメチル基やカチオン性官能基の導入、あるいは2位のカルボニル基のチオカルボニルへの置換などが挙げられる。 The nucleotide molecule constituting the antisense nucleic acid may be natural DNA or RNA, but various chemicals may be used to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA). Modifications can be included. For example, in order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue. In addition, the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R is, for example, CH 3 (2′-O-Me), CH 2 CH 2 OCH 3 (2′-O-MOE), CH 2 CH 2 NHC (NH) NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN and the like may be substituted). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned.
　RNAの糖部のコンフォーメーションはC2'-endo（S型）とC3'-endo（N型）の２つが支配的であり、一本鎖RNAではこの両者の平衡として存在するが、二本鎖を形成するとN型に固定される。したがって、標的RNAに対して強い結合能を付与するために、2'酸素と4’炭素を架橋することにより、糖部のコンフォーメーションをN型に固定したRNA誘導体であるBNA(LNA)（Imanishi, T. et al., Chem. Commun., 1653-9, 2002; Jepsen, J.S. et al., Oligonucleotides, 14, 130-46, 2004）やENA（Morita, K. et al., Nucleosides Nucleotides Nucleic Acids, 22, 1619-21, 2003）もまた、好ましく用いられ得る。 The conformation of the sugar part of RNA is dominated by C2'-endo (S type) and C3'-endo (N type). In single-stranded RNA, it exists as an equilibrium between the two, but double-stranded Is fixed to the N type. Therefore, in order to give strong binding ability to the target RNA, BNA (LNA) (Imanishi) is an RNA derivative in which the conformation of the sugar moiety is fixed to N-type by cross-linking 2 'oxygen and 4' carbon. , T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides Nucleotides Nucleicides Nucleicides NucleicidesAcids , 22, 1619-21, 2003) can also be preferably used.
　本発明のアンチセンスオリゴヌクレオチドは、GPR120のcDNA配列もしくはゲノミックDNA配列に基づいてmRNAもしくは初期転写産物の標的配列を決定し、市販のDNA/RNA自動合成機（アプライド・バイオシステムズ社、ベックマン社等）を用いて、これに相補的な配列を合成することにより調製することができる。また、上記した各種修飾を含むアンチセンス核酸も、いずれも自体公知の手法により、化学的に合成することができる。 The antisense oligonucleotide of the present invention determines the target sequence of mRNA or initial transcript based on the cDNA sequence or genomic DNA sequence of GPR120, and is a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman, etc.) ) To synthesize a sequence complementary thereto. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a method known per se.
(ii) GPR120のmRNAに対するsiRNA
　本明細書においては、GPR120のmRNAに相補的なオリゴRNAとその相補鎖とからなる二本鎖RNA、いわゆるsiRNAもまた、GPR120のmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸に包含されるものとして定義される。短い二本鎖RNAを細胞内に導入するとそのRNAに相補的なmRNAが分解される、いわゆるRNA干渉（RNAi）と呼ばれる現象は、以前から線虫、昆虫、植物等で知られていたが、この現象が動物細胞でも広く起こることが確認されて以来［Nature, 411(6836): 494-498 (2001)］、リボザイムの代替技術として汎用されている。siRNAは標的となるmRNAの塩基配列情報に基づいて、市販のソフトウェア（例：RNAi Designer; Invitrogen）を用いて適宜設計することができる。 (ii) siRNA against GPR120 mRNA
In the present specification, a double-stranded RNA consisting of an oligo RNA complementary to GPR120 mRNA and its complementary strand, so-called siRNA, is also complementary or substantially complementary to the base sequence of GPR120 mRNA. Or defined as encompassed by a nucleic acid containing a portion thereof. When a short double-stranded RNA is introduced into a cell, the mRNA complementary to that RNA is degraded. So-called RNA interference (RNAi) has long been known in nematodes, insects, plants, etc. Since this phenomenon has been confirmed to occur widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative to ribozyme. siRNA can be appropriately designed using commercially available software (eg, RNAi Designer; Invitrogen) based on the base sequence information of the target mRNA.
　siRNAを構成するリボヌクレオシド分子もまた、安定性、比活性などを向上させるために、上記のアンチセンス核酸の場合と同様の修飾を受けていてもよい。但し、siRNAの場合、天然型RNA中のすべてのリボヌクレオシド分子を修飾型で置換すると、RNAi活性が失われる場合があるので、RISC複合体が機能できる最小限の修飾ヌクレオシドの導入が必要である。 The ribonucleoside molecule constituting siRNA may also be modified in the same manner as in the above-described antisense nucleic acid in order to improve stability, specific activity and the like. However, in the case of siRNA, if all ribonucleoside molecules in natural RNA are replaced with a modified form, RNAi activity may be lost, so the introduction of the minimum modified nucleoside that allows the RISC complex to function is necessary. .
　siRNAは、mRNA上の標的配列のセンス鎖及びアンチセンス鎖をDNA/RNA自動合成機でそれぞれ合成し、適当なアニーリング緩衝液中、約90～約95℃で約1分程度変性させた後、約30～約70℃で約1～約8時間アニーリングさせることにより調製することができる。また、siRNAの前駆体となるショートヘアピンRNA（shRNA）を合成し、これをダイサー（dicer）を用いて切断することにより調製することもできる。 The siRNA is synthesized by synthesizing a sense strand and an antisense strand of a target sequence on mRNA with a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. Alternatively, it can be prepared by synthesizing a short hairpin RNA (shRNA) that is a precursor of siRNA and cleaving it with a dicer.
(iii) GPR120のmRNAに対するsiRNAを生成し得る核酸
　本明細書においては、生体内で上記のGPR120のmRNAに対するsiRNAを生成し得るようにデザインされた核酸もまた、GPR120のmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸に包含されるものとして定義される。そのような核酸としては、上記したshRNAやそれを発現するように構築された発現ベクターなどが挙げられる。shRNAは、mRNA上の標的配列のセンス鎖およびアンチセンス鎖を適当なループ構造を形成しうる長さ（例えば15から25塩基程度）のスペーサー配列を間に挿入して連結した塩基配列を含むオリゴRNAをデザインし、これをDNA/RNA自動合成機で合成することにより調製することができる。shRNAの発現カセットを含む発現ベクターは、上記shRNAをコードする二本鎖DNAを常法により作製した後、適当な発現ベクター中に挿入することにより調製することができる。shRNAの発現ベクターとしては、U6やH1などのPol III系プロモーターを有するものが用いられ得る。この場合、該発現ベクターを導入された動物細胞内で転写されたshRNAは、自身でループを形成した後に、内在の酵素ダイサー（dicer）などによってプロセシングされることにより成熟siRNAが形成される。 (iii) Nucleic acid capable of generating siRNA for GPR120 mRNA In the present specification, a nucleic acid designed to generate siRNA for GPR120 mRNA in vivo is also complementary to the base sequence of GPR120 mRNA. Defined as being encompassed by a nucleic acid comprising a base sequence that is complementary or substantially complementary or a portion thereof. Examples of such a nucleic acid include the above-mentioned shRNA and an expression vector constructed so as to express it. shRNA is an oligo containing a base sequence in which the sense and antisense strands of the target sequence on mRNA are linked by inserting a spacer sequence (for example, about 15 to 25 bases) long enough to form an appropriate loop structure. It can be prepared by designing RNA and synthesizing it with an automatic DNA / RNA synthesizer. An expression vector containing an shRNA expression cassette can be prepared by preparing a double-stranded DNA encoding the above shRNA by a conventional method and then inserting it into an appropriate expression vector. As an shRNA expression vector, one having a Pol III promoter such as U6 or H1 can be used. In this case, the shRNA transcribed in the animal cell into which the expression vector has been introduced forms a loop by itself, and then is processed by an endogenous enzyme dicer or the like to form a mature siRNA.
　GPR120のmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸の他の好ましい例としては、該mRNAをコード領域の内部で特異的に切断し得るリボザイムが挙げられる。「リボザイム」とは、狭義には、核酸を切断する酵素活性を有するRNAをいうが、本明細書では配列特異的な核酸切断活性を有する限りDNAをも包含する概念として用いるものとする。リボザイムとして最も汎用性の高いものとしては、ウイロイドやウイルソイド等の感染性RNAに見られるセルフスプライシングRNAがあり、ハンマーヘッド型やヘアピン型等が知られている。ハンマーヘッド型は約40塩基程度で酵素活性を発揮し、ハンマーヘッド構造をとる部分に隣接する両端の数塩基ずつ（合わせて約10塩基程度）をmRNAの所望の切断部位と相補的な配列にすることにより、標的mRNAのみを特異的に切断することが可能である。このタイプのリボザイムは、RNAのみを基質とするので、ゲノムDNAを攻撃することがないというさらなる利点を有する。GPR120のmRNAが自身で二本鎖構造をとる場合には、RNAヘリカーゼと特異的に結合し得るウイルス核酸由来のRNAモチーフを連結したハイブリッドリボザイムを用いることにより、標的配列を一本鎖にすることができる［Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)］。さらに、リボザイムを、それをコードするDNAを含む発現ベクターの形態で使用する場合には、転写産物の細胞質への移行を促進するために、tRNAを改変した配列をさらに連結したハイブリッドリボザイムとすることもできる［Nucleic Acids Res., 29(13): 2780-2788 (2001)］。 Another preferred example of a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of GPR120 mRNA or a part thereof is a ribozyme capable of specifically cleaving the mRNA within the coding region. It is done. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acids in a narrow sense, but in this specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleavage activity. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends (about 10 bases in total) adjacent to the part having the hammerhead structure are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate. When GPR120 mRNA itself has a double-stranded structure, the target sequence should be made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when ribozymes are used in the form of expression vectors containing the DNA that encodes them, they should be hybrid ribozymes in which tRNA-modified sequences are further linked in order to promote the transfer of transcripts to the cytoplasm. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
　GPR120のmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸は、リポソーム、ミクロスフェアのような特殊な形態で供与されたり、遺伝子治療に適用されたり、付加された形態で与えられることができうる。こうして付加形態で用いられるものとしては、リン酸基骨格の電荷を中和するように働くポリリジンのようなポリカチオン体、細胞膜との相互作用を高めたり、核酸の取込みを増大せしめるような脂質（例、ホスホリピド、コレステロールなど）などの疎水性のものが挙げられる。付加するに好ましい脂質としては、コレステロールやその誘導体（例、コレステリルクロロホルメート、コール酸など）が挙げられる。こうしたものは、核酸の3'端または5'端に付着させることができ、塩基、糖、分子内ヌクレオシド結合を介して付着させることができうる。その他の基としては、核酸の3'端または5'端に特異的に配置されたキャップ用の基で、エキソヌクレアーゼ、RNaseなどのヌクレアーゼによる分解を阻止するためのものが挙げられる。こうしたキャップ用の基としては、ポリエチレングリコール、テトラエチレングリコールなどのグリコールをはじめとした当該分野で知られた水酸基の保護基が挙げられるが、それに限定されるものではない。 Nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of GPR120 mRNA or a part thereof is provided in a special form such as liposome or microsphere, applied to gene therapy, or added Can be given in the form of In this way, the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( Examples include hydrophobic ones such as phospholipid and cholesterol. Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.). Such can be attached to the 3 ′ or 5 ′ end of the nucleic acid and can be attached via a base, sugar, intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid for preventing degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
　これらの核酸のGPR120蛋白質発現抑制活性は、GPR120遺伝子を導入した形質転換体、生体内や生体外のGPR120遺伝子発現系、または生体内や生体外のGPR120蛋白質翻訳系を用いて調べることができる。 The GPR120 protein expression inhibitory activity of these nucleic acids can be examined using a transformant into which the GPR120 gene has been introduced, an in vivo or in vitro GPR120 gene expression system, or an in vivo or in vitro GPR120 protein translation system.
　GPR120のmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸を上述の予防・治療剤として使用する場合、上記GPR120またはその部分ペプチドをコードする塩基配列を含む核酸と同様に製剤化することができ、同様の投与経路および投与量で、ヒトまたは哺乳動物（例えば、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ウマ、ネコ、イヌ、サル、チンパンジーなど）に対して、経口的にまたは非経口的に投与することができる。 When a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of GPR120 mRNA or a part thereof is used as the above preventive / therapeutic agent, the base sequence encoding GPR120 or a partial peptide thereof is included. It can be formulated in the same way as a nucleic acid, with a similar route and dose of human or mammal (eg, mouse, rat, rabbit, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc.) Can be administered orally or parenterally.
（４）EPCの選別試薬／方法
　GPR120は、骨髄細胞由来および末梢血中単核球由来のsmall及びlarge EPCで特異的に発現し、EPCの前駆細胞である骨髄細胞やEPCから分化した血管内皮細胞等の他の血管系細胞では、ほとんどあるいは全く発現していない。また、マウスにおいては、GPR120は、small EPCに比べて、分化のさらに進んだlarge EPCにおいてより高発現している。従って、GPR120を表面抗原マーカーとして利用することにより、血管系細胞集団からEPCを選別することが可能であり、さらに、EPCを分化段階の異なるlarge EPCとsmall EPCとにさらに選別することもできる。 (4) EPC screening reagent / method GPR120 is specifically expressed in bone marrow cells and small and large EPCs derived from peripheral blood mononuclear cells, and is differentiated from bone marrow cells and EPCs that are precursor cells of EPC. It is hardly or not expressed in other vascular cells such as cells. Moreover, in mice, GPR120 is highly expressed in large EPC that is further differentiated compared to small EPC. Therefore, by using GPR120 as a surface antigen marker, EPC can be selected from a vascular cell population, and EPC can be further selected into large EPC and small EPC having different differentiation stages.
　EPCの細胞表面に発現するGPR120を検出・定量し得る物質としては、好ましくは前記した本発明の検出用抗体が挙げられる。一方、EPC内でのGPR120遺伝子の発現をRNAレベルで検出・定量し得る物質としては、好ましくは前記した本発明の検出用核酸が挙げられる。
　従って、本発明はまた、（a）GPR120またはその部分ペプチドに対する抗体、あるいは（b）GPR120をコードする塩基配列を含む核酸またはその部分ポリヌクレオチドを含有してなるEPCの選別試薬を提供する。
　上記抗体は、水もしくは適当な緩衝液（例、リン酸緩衝生理食塩水等）中に溶解した状態で提供することができる。また、上記核酸は、乾燥した状態もしくはアルコール沈澱の状態で、固体として提供することもできるし、水もしくは適当な緩衝液（例、TE緩衝液等）中に溶解した状態で提供することもできる。
　標識抗体、標識プローブとして用いられる場合、該抗体、該核酸は予め標識物質で標識した状態で提供することもできるし、標識物質とそれぞれ別個に提供され、用時標識して用いることもできる。 The substance capable of detecting and quantifying GPR120 expressed on the cell surface of EPC is preferably the above-described detection antibody of the present invention. On the other hand, the substance capable of detecting and quantifying the expression of the GPR120 gene in EPC at the RNA level is preferably the above-described detection nucleic acid of the present invention.
Accordingly, the present invention also provides an EPC selection reagent comprising (a) an antibody against GPR120 or a partial peptide thereof, or (b) a nucleic acid containing a base sequence encoding GPR120 or a partial polynucleotide thereof.
The antibody can be provided in a dissolved state in water or a suitable buffer (eg, phosphate buffered saline). In addition, the nucleic acid can be provided as a solid in a dry state or in a state of alcohol precipitation, or can be provided in a state dissolved in water or a suitable buffer (eg, TE buffer). .
When used as a labeled antibody or a labeled probe, the antibody and the nucleic acid can be provided in a state of being previously labeled with a labeling substance, or can be provided separately from the labeling substance, and can be used after labeling.
　細胞含有試料としては血管系細胞集団を含むものであれば特に限定されず、例えば、末梢血、臍帯血、骨髄液等が挙げられる。EPCの選別は、上記選別試薬を常法により標識（例、FITCなどによる蛍光標識等）し、セルソータを用いてGPR120陽性細胞と陰性細胞とを分離することにより行うことができる。また、large EPCとsmall EPCとの選別は、GPR120の発現量を比較することにより行うことができる。
　選別されたEPCのフラクションを回収し、例えば、EPCの移植治療などに用いることができる。 The cell-containing sample is not particularly limited as long as it contains a vascular system cell population, and examples thereof include peripheral blood, umbilical cord blood, and bone marrow fluid. The EPC can be selected by labeling the above-described selection reagent by a conventional method (eg, fluorescent labeling using FITC, etc.), and separating GPR120 positive cells and negative cells using a cell sorter. Further, large EPC and small EPC can be selected by comparing the expression levels of GPR120.
The selected EPC fraction can be collected and used, for example, for EPC transplantation therapy.
（５）EPC量の測定／診断
　上述の通り、GPR120は、骨髄細胞由来および末梢血中単核球由来のsmall及びlarge EPCで特異的に発現しているので、生体から採取した血管系細胞集団を含有する試料中のGPR120の蛋白質量もしくはmRNA量を測定することにより、該試料中に含まれるEPC量を評価することができる。
　試料となる被験動物由来の細胞含有試料は血管系細胞集団を含有する限り特に限定されないが、例えば、末梢血、臍帯血、骨髄液などが挙げられる。
　検出対象がGPR120をコードするmRNAである場合には、採取した末梢血等から常法を用いて全RNAもしくはポリ(A)+RNA画分を抽出・精製しておいてもよい。 (5) Measurement / diagnosis of EPC amount As described above, GPR120 is specifically expressed in small and large EPCs derived from bone marrow cells and peripheral blood mononuclear cells. The amount of EPC contained in the sample can be evaluated by measuring the amount of protein or mRNA of GPR120 in the sample containing.
The cell-containing sample derived from the subject animal as the sample is not particularly limited as long as it contains a vascular system cell population, and examples thereof include peripheral blood, umbilical cord blood, and bone marrow fluid.
When the detection target is mRNA encoding GPR120, the total RNA or poly (A) + RNA fraction may be extracted and purified from the collected peripheral blood or the like using a conventional method.
　試料中のGPR120の発現量は、該蛋白質もしくはそれをコードするmRNAを、従来公知の方法を用いてそれぞれ検出することにより調べることができる。例えば、GPR120蛋白質を検出する場合は、ゲル電気泳動（例：SDS-PAGE、二次元ゲル電気泳動など）や、各種の分離精製法（例：イオン交換クロマトグラフィー、疎水性クロマトグラフィー、ゲル濾過クロマトグラフィー、アフィニティークロマトグラフィー、逆相クロマトグラフィー、等電点クロマトグラフィー、キャピラリー電気泳動など）、イオン化法（例：電子衝撃イオン化法、フィールドディソープション法、二次イオン化法、高速原子衝突法、マトリックス支援レーザー脱離イオン化（MALDI）法、エレクトロスプレーイオン化法など）、質量分析計（例：二重収束質量分析計、四重極型分析計、飛行時間型質量分析計、フーリエ変換質量分析計、イオンサイクロトロン質量分析計など）を組み合わせる方法等に供することにより行うことができる。
　好ましくは、GPR120蛋白質の検出・定量は、前記した本発明の検出用抗体を用いて、前記したGPR120の発現を調節する物質のスクリーニングにおいて詳述した各種イムノアッセイやウェスタンブロット分析により行われる。また、蛍光標識した抗体を用いて、試料中の細胞をホモジナイズすることなく、免疫組織染色を行い、蛍光顕微鏡下で染色細胞数を計測することによっても、試料中のEPC数を測定することができる。 The expression level of GPR120 in a sample can be examined by detecting the protein or mRNA encoding the same using a conventionally known method. For example, when GPR120 protein is detected, gel electrophoresis (eg, SDS-PAGE, two-dimensional gel electrophoresis, etc.) and various separation and purification methods (eg, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography). Chromatography, affinity chromatography, reverse phase chromatography, isoelectric focusing, capillary electrophoresis, etc.), ionization methods (eg, electron impact ionization, field desorption, secondary ionization, fast atom collision, matrix) Assisted laser desorption ionization (MALDI) method, electrospray ionization method, etc.), mass spectrometers (eg, double-focusing mass spectrometer, quadrupole analyzer, time-of-flight mass spectrometer, Fourier transform mass spectrometer, Ion cyclotron mass spectrometer etc.) You can.
Preferably, the GPR120 protein is detected and quantified by various immunoassays and Western blot analysis described in detail in the above-described screening for substances that regulate the expression of GPR120, using the detection antibody of the present invention. It is also possible to measure the number of EPCs in a sample by performing immunohistochemical staining using a fluorescently labeled antibody without homogenizing the cells in the sample and measuring the number of stained cells under a fluorescence microscope. it can.
　あるいは、本発明の検出用抗体を用いる別の測定方法として、該抗体を後述の質量分析計に適合し得るプローブの表面上に固定化し、該プローブ上の該抗体に被験試料を接触させ、該抗体に捕捉された成分を質量分析にかけ、GPR120蛋白質のピークを検出する方法が挙げられる。 Alternatively, as another measurement method using the detection antibody of the present invention, the antibody is immobilized on the surface of a probe that can be adapted to a mass spectrometer described later, the test sample is brought into contact with the antibody on the probe, Examples include a method in which a component captured by an antibody is subjected to mass spectrometry to detect a peak of GPR120 protein.
　あるいは、GPR120蛋白質の検出において、他の好ましい測定法として、クロマトグラフィー技術と飛行時間型質量分析（TOF-MS）を組み合わせて、クロマト担体（例：カチオン交換体、アニオン交換体、疎水性クロマト担体、金属イオンなど）に一定条件下で捕捉される被験試料中のすべての成分の質量を一括して測定する方法、とりわけ飛行時間型質量分析に使用するプローブの表面に該クロマト担体を固定し、このプローブ表面と被験試料を接触させ、適当な条件で洗浄した後、該プローブ表面に捕捉された成分の質量を飛行時間型質量分析計で測定する方法などが挙げられる。飛行時間型質量分析計に適合可能なプローブとしては、サイファージェン・バイオシステムズ社製の各種プロテインチップ等が挙げられるが、それらに限定されない。洗浄は水または緩衝液を用いて行うことができるが、例えば、pHは、ターゲット蛋白質の等電点に合わせて適宜選択される。例えば、pH 4～8のトリス塩酸緩衝液、リン酸緩衝液、ホウ酸緩衝液、酢酸緩衝液等が用いられる。質量分析は適当なマトリックスを用いてMALDI法で行うことが好ましく（MALDI-TOFMS）、使用するマトリックス分子としては、例えばシナピン酸（SPA）、飽和2,5-ジヒドロキシ安息香酸（DHB）、インドールアクリル酸（IAA）、桂皮酸などが挙げられる。 Alternatively, in the detection of GPR120 protein, as another preferred measurement method, a chromatographic carrier (eg, cation exchanger, anion exchanger, hydrophobic chromatographic carrier) is combined with chromatography technology and time-of-flight mass spectrometry (TOF-MS). A method for measuring the mass of all components in a sample to be captured under a certain condition in a batch, in particular, fixing the chromatographic carrier on the surface of a probe used for time-of-flight mass spectrometry, Examples include a method in which the probe surface and a test sample are brought into contact with each other and washed under appropriate conditions, and then the mass of a component captured on the probe surface is measured with a time-of-flight mass spectrometer. Examples of probes that can be adapted to a time-of-flight mass spectrometer include, but are not limited to, various protein chips manufactured by Cyphergen Biosystems. The washing can be performed using water or a buffer solution. For example, the pH is appropriately selected according to the isoelectric point of the target protein. For example, a Tris-HCl buffer solution having a pH of 4 to 8, a phosphate buffer solution, a borate buffer solution, an acetate buffer solution, or the like is used. Mass spectrometry is preferably performed by a MALDI method using an appropriate matrix (MALDI-TOFMS). Examples of matrix molecules used include sinapinic acid (SPA), saturated 2,5-dihydroxybenzoic acid (DHB), and indole acrylic. Examples include acid (IAA) and cinnamic acid.
　被験試料中のGPR120の発現量を、該蛋白質をコードするmRNAを検出することにより行う場合、該mRNAとハイストリンジェントな条件下でハイブリダイズし得る核酸（プローブ）や、該mRNAの一部もしくは全部を増幅するプライマーとして機能し得るオリゴヌクレオチドのセット（即ち、前記した本発明の検出用核酸）を用いて、ノーザンハイブリダイゼーションやRT-PCRなどにより行うことができる。プローブとして用いられる核酸は、DNAであってもRNAであってもよく、あるいはDNA/RNAキメラであってもよい。好ましくはDNAが挙げられる。また、該核酸は二本鎖であっても一本鎖であってもよい。二本鎖の場合は、二本鎖DNA、二本鎖RNAまたはDNA:RNAのハイブリッドでもよい。該核酸の長さは標的mRNAと特異的にハイブリダイズし得る限り特に制限はなく、例えば約15塩基以上、好ましくは約30塩基以上である。該核酸は、標的mRNAの検出・定量を可能とするために、標識剤により標識されていることが好ましい。標識剤としては、例えば、放射性同位元素、酵素、蛍光物質、発光物質などが用いられる。放射性同位元素としては、例えば、〔32Ｐ〕、〔3Ｈ〕、〔14Ｃ〕などが用いられる。酵素としては、安定で比活性の大きなものが好ましく、例えば、β－ガラクトシダーゼ、β－グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素などが用いられる。蛍光物質としては、例えば、フルオレスカミン、フルオレッセンイソチオシアネートなどが用いられる。発光物質としては、例えば、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなどが用いられる。さらに、プローブと標識剤との結合にビオチン－（ストレプト）アビジンを用いることもできる。 When the expression level of GPR120 in the test sample is detected by detecting mRNA encoding the protein, a nucleic acid (probe) that can hybridize with the mRNA under highly stringent conditions, a part of the mRNA, It can be carried out by Northern hybridization, RT-PCR, etc., using a set of oligonucleotides (that is, the above-described detection nucleic acid of the present invention) that can function as a primer for amplifying the whole. The nucleic acid used as the probe may be DNA or RNA, or may be a DNA / RNA chimera. Preferably, DNA is used. The nucleic acid may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid. The length of the nucleic acid is not particularly limited as long as it can specifically hybridize with the target mRNA, and is, for example, about 15 bases or more, preferably about 30 bases or more. The nucleic acid is preferably labeled with a labeling agent in order to enable detection and quantification of the target mRNA. As the labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ 32 P], [ 3 H], [ 14 C] and the like are used. As the enzyme, those which are stable and have high specific activity are preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, biotin- (strept) avidin can also be used for binding between the probe and the labeling agent.
　プライマーとして用いられるオリゴヌクレオチドのセットとしては、GPR120をコードするmRNAの塩基配列（センス鎖）およびそれに相補的な塩基配列（アンチセンス鎖）とそれぞれ特異的にハイブリダイズすることができ、それらに挟まれるDNA断片を増幅し得るものであれば特に制限はなく、例えば、各々約15～約100塩基、好ましくは各々約15～約50塩基の長さを有し、約100bp～数kbpのDNA断片を増幅するようにデザインされたオリゴDNAのセットが挙げられる。 The set of oligonucleotides used as primers can specifically hybridize with the base sequence (sense strand) of mRNA encoding GPR120 and the base sequence complementary to it (antisense strand). The DNA fragment is not particularly limited as long as it can amplify the DNA fragment. For example, each DNA fragment has a length of about 15 to about 100 bases, preferably about 15 to about 50 bases, and about 100 bp to several kbp. A set of oligo DNAs designed to amplify.
　微量RNA試料を用いてGPR120の遺伝子発現を定量的に解析するためには、競合RT-PCRまたはリアルタイムRT-PCRを用いることが好ましい。競合RT-PCRとは、目的のDNAを増幅し得るプライマーのセットにより増幅され得る既知量の他の鋳型核酸をcompetitorとして反応液中に共存させて競合的に増幅反応を起こさせ、増幅産物の量を比較することにより、目的DNAの量を算出する方法をいう。従って、競合RT-PCRを用いる場合、上記プライマーセットに加えて、該プライマーセットにより増幅され、目的DNAと区別することができる増幅産物（例えば、目的のDNAとはサイズの異なる増幅産物、制限酵素処理により異なる泳動パターンを示す増幅産物など）を生じる核酸をさらに含有することができる。このcompetitor核酸はDNAであってもRNAであってもよい。DNAの場合、RNA試料から逆転写反応によりcDNAを合成した後にcompetitorを添加してPCRを行えばよく、RNAの場合は、RNA試料に最初から添加してRT-PCRを行うことができる。後者の場合、逆転写反応の効率も考慮に入れているので、元のmRNAの絶対量を推定することができる。
　一方、リアルタイムRT-PCRは、PCRの増幅量をリアルタイムでモニタリングできるので、電気泳動が不要で、より迅速にGPR120の遺伝子発現を解析可能である。通常、モニタリングは種々の蛍光試薬を用いて行われる。これらの中には、SYBR Green I、エチジウムブロマイド等の二本鎖DNAに結合することにより蛍光を発する試薬（インターカレーター）の他、上記プローブとして用いることができる核酸（但し、該核酸は増幅領域内で標的核酸にハイブリダイズする）の両端をそれぞれ蛍光物質（例：FAM、HEX、TET、FITC等）および消光物質（例：TAMRA、DABCYL等）で修飾したもの等が含まれる。 In order to quantitatively analyze the gene expression of GPR120 using a trace RNA sample, it is preferable to use competitive RT-PCR or real-time RT-PCR. Competitive RT-PCR is a method in which a known amount of another template nucleic acid that can be amplified by a set of primers that can amplify the target DNA is used as a competitor in the reaction solution to cause an amplification reaction competitively. A method for calculating the amount of target DNA by comparing the amounts. Therefore, when using competitive RT-PCR, in addition to the above primer set, an amplification product that is amplified by the primer set and can be distinguished from the target DNA (for example, an amplification product having a different size from the target DNA, a restriction enzyme) Nucleic acids that produce amplification products that exhibit different migration patterns upon processing) can be further included. The competitor nucleic acid may be DNA or RNA. In the case of DNA, PCR may be performed by adding a competitor after synthesizing cDNA from an RNA sample by a reverse transcription reaction. In the case of RNA, RT-PCR can be performed by adding it to the RNA sample from the beginning. In the latter case, the efficiency of the reverse transcription reaction is taken into consideration, so that the absolute amount of the original mRNA can be estimated.
On the other hand, since real-time RT-PCR can monitor the amount of PCR amplification in real time, electrophoresis is unnecessary and GPR120 gene expression can be analyzed more quickly. Usually, monitoring is performed using various fluorescent reagents. Among these, in addition to a reagent (intercalator) that emits fluorescence by binding to double-stranded DNA such as SYBR Green I and ethidium bromide, a nucleic acid that can be used as the probe (provided that the nucleic acid is an amplification region) In which both ends of the target nucleic acid are modified with a fluorescent substance (eg, FAM, HEX, TET, FITC, etc.) and a quencher (eg, TAMRA, DABCYL, etc.).
　上記プローブとして用いる核酸は、GPR120をコードするcDNAやその断片であってよく、あるいはその塩基配列情報（例えば、ヒトGPR120の場合、配列番号１に示される塩基配列）に基づいて、市販のDNA/RNA自動合成機等を用いて化学的に合成することによって得られるものであってもよい。また、上記プライマーとして用いるオリゴヌクレオチドのセットは、上記塩基配列情報に基づいて、該塩基配列およびその相補鎖配列の一部を市販のDNA/RNA自動合成機等を用いて化学的に合成することによって得ることができる。 The nucleic acid used as the probe may be a cDNA encoding GPR120 or a fragment thereof, or based on the base sequence information (for example, the base sequence shown in SEQ ID NO: 1 in the case of human GPR120), It may be obtained by chemically synthesizing using an automatic RNA synthesizer or the like. In addition, the oligonucleotide set used as the primer may be chemically synthesized using a commercially available DNA / RNA automatic synthesizer or the like based on the base sequence information. Can be obtained by:
　種々の既知細胞数のEPCを用いて、上記いずれかの測定方法によりGPR120の蛋白質量またはmRNA量を測定して検量線を作成し、未知細胞数のEPC含有試料における測定値をこれと比較することにより、該試料中のEPCを定量することができる。 Using EPC with various known cell numbers, measure the protein amount or mRNA amount of GPR120 by any of the above measurement methods, create a calibration curve, and compare the measured values in EPC-containing samples with unknown cell numbers Thus, EPC in the sample can be quantified.
　上記のEPC測定方法を用いて、被験動物の糖尿病、動脈硬化、脳・心血管系疾患などのEPC量の異常が関連する疾患への罹病の有無、あるいはその重症度等を診断することができる。即ち、被験動物から採取した試料中のGPR120の発現料を測定、EPC量を算出し、正常動物のそれと比較した結果、正常動物に比べてEPC量が有意に減少していた場合、該被験動物は、EPC量の異常が関連する疾患、例えば、糖尿病およびその合併症（例、神経障害、腎症、足病変、血流障害）、肥満症、動脈硬化、閉塞性動脈硬化症、バージャー病、肺高血圧、心筋梗塞、脳梗塞、肝炎、肝硬変、慢性閉塞性肺疾患、創傷、骨折、骨粗鬆症、歯周病、認知症などに罹患しているか、将来罹患するおそれが高いと診断することができる。 Using the above EPC measurement method, it is possible to diagnose the presence or severity of a disease associated with abnormalities in EPC amount such as diabetes, arteriosclerosis, brain / cardiovascular disease, etc. in a test animal. . That is, when the expression charge of GPR120 in a sample collected from a test animal is measured, the EPC amount is calculated, and compared with that of a normal animal, the EPC amount is significantly reduced compared to that of a normal animal. Are diseases associated with abnormal EPC levels, such as diabetes and its complications (eg, neuropathy, nephropathy, foot lesions, blood flow disorders), obesity, arteriosclerosis, obstructive arteriosclerosis, Buerger's disease, It can be diagnosed that the patient is suffering from pulmonary hypertension, myocardial infarction, cerebral infarction, hepatitis, cirrhosis, chronic obstructive pulmonary disease, wound, fracture, osteoporosis, periodontal disease, dementia, etc. .
　以下に実施例を挙げて、本発明をさらに具体的に説明する。 The present invention will be described more specifically with reference to the following examples.
参考例１　マウス骨髄細胞のEPCコロニーフォーミングアッセイ法によるマウス骨髄細胞由来EPCの培養
　原著論文（Tamarat R et al., Am J Pathol. 2004; 164: 457-466.）に従ってマウスより骨髄細胞を単離した。雄性C57BL/6Jマウスの左下肢の大腿骨と脛骨から1% FBSを含むHBSS培地（Gibco）で骨髄細胞を押し出した。骨髄細胞を遠心回収し、EDTA-PBSで再懸濁後、セルストレーナーを通して塊を除き、再度遠心した。0.8% 塩化アンモニウムで再懸濁することで溶血し、遠心後、細胞をIMDM (Gibco) で懸濁して細胞数をカウントした。培養方法は原著論文（Masuda H et al., Circ Res. 2007; 101(6): 598-606., 第71回日本循環器学会総会要旨集OJ-063-H）の方法を改変して行った。コロニーフォーミングアッセイ法では培地は10% FBSと栄養因子を含むMethoCult SF M3236 (Stemcell technologies)を用いた（20 ng/ml 幹細胞因子 (SCF), 50 ng/ml 血管内皮増殖因子 (VEGF), 20 ng/ml インターロイキン-3 (IL-3), 50 ng/ml 塩基性線維芽細胞増殖因子 (bFGF), 50 ng/ml 上皮増殖因子レセプター (EGFR), 50 ng/ml インスリン様増殖因子-1, (IGF-1) 2 U/ml ヘパリン）。細胞密度が3×104cells/dishになるように細胞懸濁液をMethoCult SF培地に入れてよく混ぜ、35 mm dishに1 mLずつ播種し、8日間培養した。8日後に培養された細胞に0.4 μg/mL acLDL-DiIと2 μg/mL FITC-conjugated BS-1 lectinを反応させ、2% PFAで固定後にPBSで洗浄して蛍光顕微鏡下で観察したところ、図１に示すように、DiI-LDLの取り込みとBS-1 lectinの染色性が確認され、EPCであることが確認された。顕微鏡下（×40レンズ）で観察を行うと、ディッシュに2種類のコロニーが形成されており、接着細胞により構成されているコロニーをlarge EPC、接着性がなく、large EPCに比べて細胞サイズの小さい細胞により構成されているコロニーをsmall EPCと判定した。 Reference Example 1 Mouse bone marrow cell EPC culture by mouse bone marrow cell EPC colony forming assay Bone marrow cells were isolated from mice according to the original paper (Tamarat R et al., Am J Pathol. 2004; 164: 457-466.) did. Bone marrow cells were extruded from the femur and tibia of the left lower limb of male C57BL / 6J mice with HBSS medium (Gibco) containing 1% FBS. Bone marrow cells were collected by centrifugation, resuspended in EDTA-PBS, removed the mass through a cell strainer, and centrifuged again. Hemolysis was performed by resuspension with 0.8% ammonium chloride. After centrifugation, the cells were suspended in IMDM (Gibco) and the number of cells was counted. The culture method was modified from the original paper (Masuda H et al., Circ Res. 2007; 101 (6): 598-606., 71st Annual Meeting of the Japanese Circulation Society OJ-063-H). It was. In the colony forming assay, MethoCult SF M3236 (Stemcell technologies) containing 10% FBS and nutrient factors was used (20 ng / ml stem cell factor (SCF), 50 ng / ml vascular endothelial growth factor (VEGF), 20 ng / ml interleukin-3 (IL-3), 50 ng / ml basic fibroblast growth factor (bFGF), 50 ng / ml epidermal growth factor receptor (EGFR), 50 ng / ml insulin-like growth factor-1, (IGF-1) 2 U / ml heparin). The cell suspension was placed in MethoCult SF medium and mixed well so that the cell density was 3 × 10 4 cells / dish, seeded in 1 mL each in a 35 mm dish, and cultured for 8 days. Cells cultured after 8 days were reacted with 0.4 μg / mL acLDL-DiI and 2 μg / mL FITC-conjugated BS-1 lectin, fixed with 2% PFA, washed with PBS, and observed under a fluorescence microscope. As shown in FIG. 1, DiI-LDL uptake and BS-1 lectin staining were confirmed, confirming that it was EPC. When observing under a microscope (× 40 lens), two types of colonies are formed in the dish, and colonies composed of adherent cells are large EPC, non-adhesive, and have a cell size that is larger than that of large EPC. A colony composed of small cells was determined as small EPC.
参考例２　マウス末梢血中単核球のEPCコロニーフォーミングアッセイ法によるマウス末梢血中単核球由来EPCの培養
　原著論文（Kalka C et al., Proc Natl Acad Sci U S A. 2000; 97: 3422-3427.）に従ってマウスより末梢血中単核球を単離した。雄性C57BL/6Jマウスの頚動脈から採取した血液を生理食塩水で二倍希釈した後、1/2 volの血球分離用Histopaque 1088 (Sigma) に二倍希釈血液を重層し、濃度勾配遠心を行った。界面に集まった単核球細胞を回収してPBS/EDTAで懸濁し、遠心して回収後、0.8% 塩化アンモニウムで再懸濁することで溶血して再度遠心し、細胞をIMDMで懸濁して細胞数をカウントした。コロニーフォーミングアッセイ法による培養は参考例１に従って行った。なお、末梢血中単核球の播種密度は7×105cells/dishとした。 Reference Example 2: Culture of mouse peripheral blood mononuclear cells by EPC colony forming assay of mouse peripheral blood mononuclear cells Original paper (Kalka C et al., Proc Natl Acad Sci US A. 2000; 97: 3422- 3427.) peripheral blood mononuclear cells were isolated from mice. Blood collected from the carotid artery of male C57BL / 6J mice was diluted 2-fold with physiological saline, then double-diluted blood was layered on 1/2 vol of Histopaque 1088 (Sigma) for blood cell separation, and concentration gradient centrifugation was performed. . Mononuclear cells collected at the interface were collected, suspended in PBS / EDTA, collected by centrifugation, then hemolyzed by resuspension with 0.8% ammonium chloride, centrifuged again, and the cells were suspended in IMDM. Counted the number. The culture by the colony forming assay was performed according to Reference Example 1. The seeding density of mononuclear cells in peripheral blood was 7 × 10 5 cells / dish.
参考例３　ヒトCD133陽性骨髄細胞のEPCコロニーフォーミングアッセイ法によるヒトCD133陽性骨髄細胞由来EPCの培養
　ヒトCD133陽性骨髄細胞（CAMBREX社）の培養は原著論文（Masuda H et al., Circ Res. 2007; 101(6): 598-606.）の方法を改変して行った。ヒト細胞用培地は30% FBSと栄養因子を含むMethoCult SF H4236 (Stemcell technologies)を用いた（20 ng/ml 幹細胞因子 (SCF), 50 ng/ml 血管内皮増殖因子 (VEGF), 20 ng/ml インターロイキン-3 (IL-3), 50 ng/ml 塩基性線維芽細胞増殖因子 (bFGF), 50 ng/ml 上皮増殖因子レセプター (EGFR), 50 ng/ml インスリン様増殖因子-1, (IGF-1) 2 U/ml ヘパリン）。ヒトCD133陽性骨髄細胞はIMDMで懸濁し、細胞密度が1×103 cells/dishになるようにMethoCult SF 培地にいれてよく混ぜ、35 mm dishに1 mLずつ播種し、21日間培養した。21日後、顕微鏡下（×40レンズ）で観察を行うと、ディッシュに2種類のコロニーが形成されており、接着細胞により構成されているコロニーをlarge EPC、接着性がなく、large EPCに比べて細胞サイズの小さい細胞により構成されているコロニーをsmall EPCと判定した。 Reference Example 3 Culture of human CD133-positive bone marrow cell-derived EPC by EPC colony forming assay of human CD133-positive bone marrow cells Human CD133-positive bone marrow cells (CAMBREX) were cultured in the original paper (Masuda H et al., Circ Res. 2007; 101 (6): 598-606.). Human cell culture medium was MethoCult SF H4236 (Stemcell technologies) containing 30% FBS and nutrient factors (20 ng / ml stem cell factor (SCF), 50 ng / ml vascular endothelial growth factor (VEGF), 20 ng / ml Interleukin-3 (IL-3), 50 ng / ml basic fibroblast growth factor (bFGF), 50 ng / ml epidermal growth factor receptor (EGFR), 50 ng / ml insulin-like growth factor-1, (IGF -1) 2 U / ml heparin). Human CD133 positive bone marrow cells were suspended in IMDM, mixed well in MethoCult SF medium so that the cell density would be 1 × 10 3 cells / dish, seeded in 1 mL each in a 35 mm dish, and cultured for 21 days. After 21 days, when observing under a microscope (× 40 lens), two types of colonies are formed in the dish. A colony composed of cells having a small cell size was determined as small EPC.
参考例４　マウス骨髄細胞、マウス末梢血中単核球、およびヒトCD133陽性骨髄細胞由来large EPCとsmall EPCのRNA抽出
　コロニーフォーミングアッセイ法を用いてマウスの骨髄細胞または末梢血中単核球、およびヒトCD133陽性骨髄細胞を培養し、分化したsmall EPCを顕微鏡下でピペットを用いて回収した。small EPCはPBSでwash後、RLT bufferで溶解させた。培養ディッシュをPBSで洗浄し、接着しているlarge EPCにRLT bufferを加えてセルスクレーバーでかき集めて溶解させ、各細胞のRNAをRNeasy Mini Kit (Qiagen)を用いて抽出した。 Reference Example 4 Mouse bone marrow cells, mouse peripheral blood mononuclear cells, and human CD133-positive bone marrow derived large EPC and small EPC RNA extraction Using colony forming assay, mouse bone marrow cells or peripheral blood mononuclear cells, and Human CD133 positive bone marrow cells were cultured, and differentiated small EPCs were collected using a pipette under a microscope. Small EPC was washed with PBS and then dissolved with RLT buffer. The culture dish was washed with PBS, RLT buffer was added to large EPC that was adhered, and the cells were scraped and lysed with a cell scraper, and RNA of each cell was extracted using RNeasy Mini Kit (Qiagen).
参考例５　TaqMan PCRを用いたGPR120の発現解析系の設定
　マウスの細胞におけるGPR120の詳細な発現解析を行うために、TaqMan PCR条件を設定した。用いたプライマーはForward Primer（5’-TCCGAGTGTCCCAACAAGACTAC-3’；配列番号７）とReverse Primer（5’-GACTCCACATGATGAAGAAGGAAA-3’；配列番号８）で、検出に用いたプローブは5’-Fam-CCGCACGCTCTTCCTGCTCATG-Tamra（配列番号９）であった。TaqMan PCRには、TaqMan Universal MixtureとForward Primer 900 nM, Reverse Primer 900 nM, Probe 250nM, 及びtotal RNA 25 ngを鋳型とし合成したcDNAを混合し、95℃ 15秒と60℃ 60秒の40サイクルのリアルタイムPCRによってマウスGPR120遺伝子を定量した。スタンダードオリゴ（CCAGATCCGAGTGTCCCAACAAGACTACCGACTCTTCCGCACGCTCTTCCTGCTCATGGTTTCCTTCTTCATCATGTGGAGTCCCATC；配列番号１０）を用いて検量線を引いて定量化し、典型的な検量線パターンを図２Ａに示した。
　ヒトの細胞におけるGPR120の詳細な発現解析を行うために、TaqMan PCR条件を設定した。用いたプライマーはForward Primer（5’-GCGCCGACCAGGAAATTT-3’；配列番号１１）とReverse Primer（5’-CAAAAGAGACATCCCACGAGATC-3’；配列番号１２）で、検出に用いたプローブは5’-Fam-TTGCACACTGATTTGGCCCACCATT-Tamra（配列番号１３）であった。TaqMan PCRには、TaqMan Universal MixtureとForward Primer 900 nM, Reverse Primer 900 nM, Probe 250nM, 及びtotal RNA 25 ngを鋳型とし合成したcDNAを混合し、95℃ 15秒と60℃ 60秒の40サイクルのリアルタイムPCRによってヒトGPR120遺伝子を定量した。スタンダードオリゴ（CCCCGGCGCCGACCAGGAAATTTCGATTTGCACACTGATTTGGCCCACCATTCCTGGAGAGATCTCGTGGGATGTCTCTTTTGTTACT；配列番号１４）を用いて検量線を引いて定量化し、典型的な検量線パターンを図２Ｂに示した。 Reference Example 5 Setting up an expression analysis system for GPR120 using TaqMan PCR TaqMan PCR conditions were set in order to perform detailed expression analysis of GPR120 in mouse cells. The primers used were Forward Primer (5'-TCCGAGTGTCCCAACAAGACTAC-3 '; SEQ ID NO: 7) and Reverse Primer (5'-GACTCCACATGATGAAGAAGGAAA-3'; SEQ ID NO: 8), and the probe used for detection was 5'-Fam-CCGCACGCTCTTCCTGCTCATG- Tamra (SEQ ID NO: 9). TaqMan PCR is mixed with TaqMan Universal Mixture and cDNA synthesized using Forward Primer 900 nM, Reverse Primer 900 nM, Probe 250 nM, and 25 ng of total RNA as a template, and 40 cycles of 95 ° C for 15 seconds and 60 ° C for 60 seconds. The mouse GPR120 gene was quantified by real-time PCR. Standard calibration (CCAGATCCGAGTGTCCCAACAAGACTACCGACTCTTCCGCACGCTCTTCCTGCTCATGGTTTCCTTCTTCATCATGTGGAGTCCCATC; SEQ ID NO: 10) was used to draw a standard curve and quantified, and a typical standard curve pattern is shown in FIG. 2A.
TaqMan PCR conditions were set for detailed expression analysis of GPR120 in human cells. The primers used were Forward Primer (5'-GCGCCGACCAGGAAATTT-3 '; SEQ ID NO: 11) and Reverse Primer (5'-CAAAAGAGACATCCCACGAGATC-3'; SEQ ID NO: 12), and the probe used for detection was 5'-Fam-TTGCACACTGATTTGGCCCACCATT- Tamra (SEQ ID NO: 13). TaqMan PCR is mixed with TaqMan Universal Mixture and cDNA synthesized using Forward Primer 900 nM, Reverse Primer 900 nM, Probe 250 nM, and 25 ng of total RNA as a template, and 40 cycles of 95 ° C for 15 seconds and 60 ° C for 60 seconds. The human GPR120 gene was quantified by real-time PCR. A standard oligo (CCCCGGCGCCGACCAGGAAATTTCGATTTGCACACTGATTTGGCCCACCATTCCTGGAGAGATCTCGTGGGATGTCTCTTTTGTTACT; SEQ ID NO: 14) was drawn and quantified, and a typical calibration curve pattern is shown in FIG. 2B.
実施例１　GPR120 mRNAのマウス骨髄細胞由来および末梢血中単核球由来EPCにおける発現の検討
　参考例１、４、５の要領でマウス骨髄細胞をEPCコロニーフォーミングアッセイ法で培養し、分化したlarge EPCとsmall EPCを単離することで、マウス骨髄細胞由来EPCにおけるGPR120 mRNAの発現量をTaqMan PCR法を用いて定量した。その結果、図３に示すように、GPR120はマウスの骨髄細胞には発現が低く、骨髄細胞由来EPCにおいて高く発現していることが判明した。また、マウスにおいては、small EPCに比べてlarge EPCでGPR120の高い発現が認められた。
　参考例２、４、５の要領でマウス末梢血中単核球をEPCコロニーフォーミングアッセイ法で培養し、分化したlarge EPCとsmall EPCを単離することで、マウス末梢血中単核球由来EPCにおけるGPR120 mRNAの発現量をTaqMan PCR法を用いて定量した。その結果、図３に示すように、GPR120はマウスの末梢血中単核球には発現がなく、末梢血中単核球由来EPCにおいて発現していることが判明した。また、骨髄細胞由来EPCと同様に、マウス末梢血中単核球においても、small EPCに比べてlarge EPCでGPR120の高い発現が認められた。 Example 1 Examination of GPR120 mRNA Expression in Mouse Bone Marrow Cell-Derived and Peripheral Blood Mononuclear Cell-Derived EPC Mouse bone marrow cells were cultured by EPC colony forming assay as in Reference Examples 1, 4, and 5 and differentiated large EPC And small EPC were isolated, and the expression level of GPR120 mRNA in mouse bone marrow cell-derived EPC was quantified using the TaqMan PCR method. As a result, as shown in FIG. 3, GPR120 was found to be low in mouse bone marrow cells and highly expressed in bone marrow cell-derived EPCs. In mice, higher expression of GPR120 was observed in large EPC than in small EPC.
Mouse peripheral blood mononuclear cells are cultured by EPC colony forming assay as in Reference Examples 2, 4 and 5, and differentiated large EPC and small EPC are isolated. The expression level of GPR120 mRNA in was quantified using the TaqMan PCR method. As a result, as shown in FIG. 3, it was found that GPR120 was not expressed in mouse peripheral blood mononuclear cells, but was expressed in peripheral blood mononuclear cell-derived EPCs. In addition, as with bone marrow cell-derived EPCs, mouse peripheral blood mononuclear cells showed higher expression of GPR120 in large EPCs than in small EPCs.
実施例２　GPR120 mRNAのヒトCD133陽性骨髄細胞由来EPCにおける発現の検討
　参考例３、４、５の要領でヒトCD133陽性骨髄細胞をEPCコロニーフォーミングアッセイ法で培養し、分化したlarge EPCとsmall EPCを単離することで、ヒトCD133陽性骨髄細胞由来EPCにおけるGPR120 mRNAの発現量をTaqMan PCR法を用いて定量した。その結果、図４に示すように、GPR120はヒトのCD133陽性骨髄細胞や大動脈内皮細胞（human aortic endothelial cell: HAEC, KURABO社）には発現がなく、ヒトCD133陽性骨髄細胞由来EPCにおいてのみ特異的に発現していることが判明した。 Example 2 Examination of expression of GPR120 mRNA in EPC derived from human CD133-positive bone marrow cells Human CD133-positive bone marrow cells were cultured by EPC colony forming assay as in Reference Examples 3, 4 and 5, and differentiated large EPC and small EPC By isolation, the expression level of GPR120 mRNA in human CD133-positive bone marrow cell-derived EPC was quantified using the TaqMan PCR method. As a result, as shown in FIG. 4, GPR120 is not expressed in human CD133-positive bone marrow cells or aortic endothelial cells (HAEC, KURABO), and is specific only in EPC derived from human CD133-positive bone marrow cells. It was found that
参考例６　GPR120 KOマウスにおけるGPR120の発現量確認
　GPR120 KOマウスのGPR120の発現を検討するために、参考例１、４、５の要領で雄性C57BL/6JマウスとGPR120 KOマウスそれぞれの骨髄細胞をEPCコロニーフォーミングアッセイ法で培養し、マウス骨髄細胞由来EPCを単離し、GPR120 mRNAの発現量をTaqMan PCR法を用いて定量した。その結果、図５に示すように、C57BL/6Jマウスの骨髄細胞由来large EPCにはGPR120が発現していた。一方、GPR120 KOマウスの骨髄細胞由来large EPCにおいては、GPR120が発現しておらず、GPR120 KOマウスではGPR120の遺伝子がノックアウトされていることが確認された。 Reference Example 6 Confirmation of GPR120 expression level in GPR120 KO mice In order to examine the expression of GPR120 in GPR120 KO mice, the bone marrow cells of male C57BL / 6J mice and GPR120 KO mice were EPCed as described in Reference Examples 1, 4 and 5. Cultured by colony forming assay, mouse bone marrow cell-derived EPC was isolated, and the expression level of GPR120 mRNA was quantified using TaqMan PCR method. As a result, as shown in FIG. 5, GPR120 was expressed in bone marrow cell-derived large EPC of C57BL / 6J mice. On the other hand, GPR120 was not expressed in bone marrow cell-derived large EPC of GPR120 KO mice, and it was confirmed that GPR120 gene was knocked out in GPR120 KO mice.
実施例３　GPR120 KOマウスにおける骨髄細胞のlarge EPCへの分化能の検討
　GPR120 KOマウスを用いて、骨髄細胞のEPCコロニーフォーミングアッセイを行い、分化したlarge EPCとsmall EPCのコロニー数をカウントしてGPR120 KOマウスにおける骨髄細胞のlarge EPCへの分化能の評価を行った。
　参考例１の要領で12週齢の雄性GPR120 KOマウスより骨髄細胞を採取し、EPCコロニーフォーミングアッセイを行った。なお、判定は顕微鏡下（×40レンズ）で行い、接着細胞により構成されているコロニーをlarge EPC、接着性がなく、large EPCに比べて細胞サイズの小さい細胞により構成されているコロニーをsmall EPCと判定し、１ディッシュあたりのコロニー数をカウントした。その結果、図６に示すように、Wild typeであるC57BL/6Jの骨髄細胞に比べて、GPR120 KOマウスではlarge EPCのコロニー形成数が有意に少なく、GPR120 KOマウスでは骨髄細胞からlarge EPCへの分化能が低下していることが判明した。これらの結果からGPR120はEPCへの分化に関連する受容体であることが示された。 Example 3 Examination of differentiation ability of bone marrow cells into large EPC in GPR120 KO mice GPR120 KO mice were subjected to EPC colony forming assay of bone marrow cells, and the number of differentiated large EPC colonies and small EPC colonies were counted. We evaluated the ability of bone marrow cells to differentiate into large EPCs in KO mice.
Bone marrow cells were collected from 12-week-old male GPR120 KO mice as described in Reference Example 1 and subjected to EPC colony forming assay. Judgment is made under a microscope (× 40 lens), colonies composed of adherent cells are large EPC, colonies that are not adherent and are composed of cells smaller in size than large EPC are small EPC And the number of colonies per dish was counted. As a result, as shown in FIG. 6, the number of large EPC colonies formed was significantly smaller in the GPR120 KO mouse than in the bone type C57BL / 6J bone marrow, and from the bone marrow cell to the large EPC in the GPR120 KO mouse. It was found that the differentiation potential was reduced. These results indicated that GPR120 is a receptor related to differentiation into EPC.
実施例４　GPR120 KOマウスにおける末梢血中のEPC数の検討
　GPR120 KOマウスを用いて、末梢血中単核球のEPC分化アッセイを行い、GPR120 KOマウスにおける末梢血中単核球中のEPC数の評価を行った。
　参考例２の要領で16週齢の雄性GPR120 KOマウスより末梢血中単核球を採取し、細胞をEGM2 (EBM-2/5％FCS/EGM-2添加因子セットを含む培地、Cambrex社)で懸濁してフィブロネクチンコート96穴プレートに1×105cells/wellで播種した。培養4日目に浮遊細胞を除き、DiI-AcLDL(1μg/mL, Molecular probe)とFITCラベルBS-1 Lectin（Sigma L-9381）2μg/mLで染色後、2％ PFAを用いて細胞を固定した。PBSで洗浄後、IN Cell Analyzer 1000で画像を取り込み、解析ソフト（Developer）を用いて二重染色された細胞をEPCとしてカウントした。その結果、図７に示すように、Wild typeであるC57BL/6Jに比べて、GPR120 KOマウスでは末梢血中単核球中のEPC数が有意に増加していることが判明した。これらの結果からGPR120は末梢血中へのEPCの動員やEPCの分化に関連する受容体であることが示された。 Example 4 Examination of EPC Count in Peripheral Blood in GPR120 KO Mouse Using GPR120 KO mouse, EPC differentiation assay of peripheral blood mononuclear cells was performed, and EPC count in peripheral blood mononuclear cells in GPR120 KO mice Evaluation was performed.
Peripheral blood mononuclear cells were collected from 16-week-old male GPR120 KO mice as described in Reference Example 2, and the cells were collected as EGM2 (EBM-2 / 5% FCS / EGM-2 supplemented factor medium, Cambrex). And suspended in a fibronectin-coated 96-well plate at 1 × 10 5 cells / well. Remove floating cells on the 4th day of culture, stain with DiI-AcLDL (1 μg / mL, Molecular probe) and FITC-labeled BS-1 Lectin (Sigma L-9381) 2 μg / mL, then fix the cells with 2% PFA did. After washing with PBS, images were taken with IN Cell Analyzer 1000, and double-stained cells were counted as EPC using analysis software (Developer). As a result, as shown in FIG. 7, it was found that the number of EPCs in the mononuclear cells in peripheral blood was significantly increased in the GPR120 KO mouse as compared to the wild type C57BL / 6J. These results indicate that GPR120 is a receptor related to EPC mobilization into peripheral blood and EPC differentiation.
実施例５　GPR120 KOマウスの糖尿病に関する血中指標の検討
　GPR120 KOマウスを用いて、体重、血漿中のグルコース濃度、糖化ヘモグロビンを測定し、GPR120 KOマウスの糖尿病に関する血中指標の検討を行った。
　5週齢の雄性C57BL/6J (Wild type) およびGPR120 KOマウスを、個別飼育で1週間Low fat (10%) 固形飼料 (Research diet社, D12450B) にて馴化した後、実験を開始した (n=9-15)。実験開始時 (6週齢) および実験開始後3週間毎に体重を測定し、ヘパリン採血を行って、糖化ヘモグロビンはTOSO Glycohemoglobin analyzer HLC-723 GHb V (A1c 2.2) を用いて測定した。また、遠心により得られた血漿成分のグルコース濃度について、日立自動分析装置7080型を用いて酵素法により測定した。
　その結果、図８に示すように、Wild typeであるC57BL/6Jに比べて、GPR120 KOマウスでは体重に変化はなかったものの、GHBは有意に増加していた。血漿中グルコース濃度はGPR120 KOマウスで増加傾向にあり、12週齢と18週齢で有意な増加が認められた。以上の結果から、GPR120は糖尿病に関連する受容体である可能性が示唆された。
Example 5 Examination of blood index related to diabetes in GPR120 KO mice Using GPR120 KO mice, body weight, plasma glucose concentration, and glycated hemoglobin were measured, and blood indices related to diabetes in GPR120 KO mice were examined.
5 weeks old male C57BL / 6J (Wild type) and GPR120 KO mice were habituated to Low fat (10%) solid diet (Research diet, D12450B) for 1 week in individual breeding, and then the experiment was started (n = 9-15). Body weight was measured at the start of the experiment (6 weeks old) and every 3 weeks after the start of the experiment, heparin blood was collected, and glycated hemoglobin was measured using TOSO Glycohemoglobin analyzer HLC-723 GHb V (A1c 2.2). Further, the glucose concentration of the plasma component obtained by centrifugation was measured by an enzymatic method using a Hitachi automatic analyzer type 7080.
As a result, as shown in FIG. 8, GHB was significantly increased in the GPR120 KO mice although there was no change in body weight as compared to the wild type C57BL / 6J. Plasma glucose concentration tended to increase in GPR120 KO mice, and a significant increase was observed at 12 and 18 weeks of age. These results suggest that GPR120 may be a receptor related to diabetes.
　本発明のスクリーニング方法は、EPCの分化・増殖を調節する物質および末梢血中のEPC数を増加させる物質を選択することができるので、EPCを介した血管形成能を調節することにより予防・治療効果が得られうる各種疾患の予防・治療薬の候補化合物の探索に有用である。
　またGPR120はEPCの表面抗原マーカーとして利用することができるので、EPCの選別・定量、EPC量の異常が関連する疾患の診断に有用である。 In the screening method of the present invention, a substance that regulates EPC differentiation and proliferation and a substance that increases the number of EPCs in peripheral blood can be selected. Therefore, prevention and treatment can be achieved by regulating angiogenic ability via EPC. It is useful for searching for candidate compounds for prophylactic / therapeutic agents for various diseases that can be effective.
Since GPR120 can be used as a surface antigen marker for EPC, it is useful for EPC selection / quantification and diagnosis of diseases associated with abnormal EPC levels.
　本発明を、好ましい態様を強調して説明してきたが、好ましい態様が変更され得ることは当業者にとって自明であろう。本発明は、本発明が本明細書に詳細に記載された以外の方法で実施され得ることを意図する。したがって、本発明は添付の「請求の範囲」の精神および範囲に包含されるすべての変更を含むものである。
　ここで述べられた特許および特許出願明細書を含む全ての刊行物に記載された内容は、ここに引用されたことによって、その全てが明示されたと同程度に本明細書に組み込まれるものである。
　本出願は、日本で出願された特願2008-101961（出願日：平成20年4月9日）を基礎としており、そこに開示される内容は本明細書にすべて包含されるものである。 Although the present invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments can be modified. The present invention contemplates that the present invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.
The contents of all publications, including the patents and patent application specifications mentioned herein, are hereby incorporated by reference herein to the same extent as if all were expressly cited. .
This application is based on Japanese Patent Application No. 2008-101961 filed in Japan (filing date: April 9, 2008), the contents of which are incorporated in full herein by this reference.
〔配列番号７〕プライマー
〔配列番号８〕プライマー
〔配列番号９〕プローブ
〔配列番号１０〕スタンダードオリゴ
〔配列番号１１〕プライマー
〔配列番号１２〕プライマー
〔配列番号１３〕プローブ
〔配列番号１４〕スタンダードオリゴ [SEQ ID NO: 7] Primer [SEQ ID NO: 8] Primer [SEQ ID NO: 9] Probe [SEQ ID NO: 10] Standard Oligo [SEQ ID NO: 11] Primer [SEQ ID NO: 12] Primer [SEQ ID NO: 13] Probe [SEQ ID NO: 14] Standard Oligo

Claims (20)

  1. 　配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチド、あるいは該蛋白質またはその部分ペプチドを産生する細胞を用いることを特徴とする、血管内皮前駆細胞の分化・増殖調節または末梢血中の血管内皮細胞増加物質のスクリーニング方法。 A blood vessel characterized by using a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof, or a cell producing the protein or the partial peptide. A method of screening for a differentiation / proliferation regulation of endothelial progenitor cells or a substance that increases vascular endothelial cells in peripheral blood.
  2. 　配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質をコードする核酸またはその部分ポリヌクレオチド、あるいは該蛋白質またはその部分ペプチドに対する抗体をさらに用いることを特徴とする、請求項１記載の方法。 A nucleic acid encoding a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial polynucleotide thereof, or an antibody against the protein or a partial peptide thereof, The method of claim 1.
  3. 　脂肪酸もしくは脂肪酸と前記蛋白質との結合性を変化させる低分子化合物またはその塩をさらに用いることを特徴とする、請求項１記載の方法。 The method according to claim 1, further comprising using a fatty acid or a low molecular weight compound that changes the binding property between the fatty acid and the protein, or a salt thereof.
  4. 　細胞が血管内皮前駆細胞またはその前駆細胞である、請求項１記載の方法。 The method according to claim 1, wherein the cell is a vascular endothelial progenitor cell or a progenitor cell thereof.
  5. 　血管形成能の調節が予防・治療効果を示し得る疾患の予防・治療物質を選択するための、請求項１記載の方法。 The method according to claim 1, for selecting a substance for preventing / treating a disease whose regulation of angiogenic ability can exhibit a preventive / therapeutic effect.
  6. 　以下のいずれかの物質を含有してなる血管内皮前駆細胞の分化・増殖促進剤。
    （ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチド
    （ｂ）上記（ａ）の蛋白質またはその部分ペプチドをコードする塩基配列を含む核酸
    （ｃ）上記（ａ）の蛋白質の発現を増強する化合物
    （ｄ）上記（ａ）の蛋白質の活性を増強する化合物     An agent for promoting differentiation / proliferation of vascular endothelial progenitor cells, comprising any of the following substances:
    (A) a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof (b) a base sequence encoding the protein (a) or a partial peptide thereof (C) a compound that enhances the expression of the protein of (a) above (d) a compound that enhances the activity of the protein of (a) above
  7. 　血管形成能の増強が予防・治療効果を示し得る疾患の予防・治療のための、請求項６記載の剤。 [7] The agent according to claim 6, for the prevention / treatment of a disease for which enhancement of angiogenic ability may exhibit a prevention / treatment effect.
  8. 　以下のいずれかの物質を含有してなる血管内皮前駆細胞の分化増殖抑制剤。
    （ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する中和抗体
    （ｂ）上記（ａ）の蛋白質をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸
    （ｃ）上記（ａ）の蛋白質の発現を阻害する化合物
    （ｄ）上記（ａ）の蛋白質の活性を阻害する化合物     An agent for inhibiting differentiation and proliferation of vascular endothelial precursor cells, comprising any of the following substances.
    (A) a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a partial peptide thereof (b) a base sequence encoding the protein of (a) above (C) a compound that inhibits expression of the protein of (a) above (d) a compound that inhibits the activity of the protein of (a) above
  9. 　血管形成能の抑制が予防・治療効果を示し得る疾患の予防・治療のための、請求項７記載の剤。 The agent according to claim 7, for the prevention / treatment of a disease for which inhibition of angiogenic ability can exhibit a prevention / treatment effect.
  10. 　以下のいずれかの物質を含有してなる末梢血中の血管内皮前駆細胞増加剤。
    （ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する中和抗体
    （ｂ）上記（ａ）の蛋白質をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸
    （ｃ）上記（ａ）の蛋白質の発現を阻害する化合物
    （ｄ）上記（ａ）の蛋白質の活性を阻害する化合物     An agent for increasing vascular endothelial progenitor cells in peripheral blood, comprising any of the following substances:
    (A) a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof (b) a base sequence encoding the protein of (a) above (C) a compound that inhibits expression of the protein of (a) above (d) a compound that inhibits the activity of the protein of (a) above
  11. 　以下のいずれかの物質を含有してなる血管内皮前駆細胞の選別試薬。
    （ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する抗体
    （ｂ）上記（ａ）の蛋白質をコードする核酸またはその部分ポリヌクレオチド     A screening reagent for vascular endothelial progenitor cells comprising any of the following substances:
    (A) an antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof; (b) a nucleic acid encoding the protein of (a) above or a portion thereof Polynucleotide
  12. 　細胞含有試料に請求項１１記載の試薬を接触させることを特徴とする、該試料中の血管内皮前駆細胞の選別方法。 A method for selecting vascular endothelial progenitor cells in a sample, which comprises contacting the reagent according to claim 11 with a cell-containing sample.
  13. 　哺乳動物から採取した細胞含有試料における、配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその遺伝子の発現を測定することを特徴とする、該試料中の血管内皮前駆細胞量の測定方法。 Measuring the expression of a protein containing the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a gene thereof in a cell-containing sample collected from a mammal, A method for measuring the amount of vascular endothelial progenitor cells in a sample.
  14. 　血管内皮前駆細胞量の異常が関連する疾患の診断のための、請求項１３記載の方法。 The method according to claim 13, for diagnosis of a disease associated with an abnormality in the amount of vascular endothelial precursor cells.
  15. 　哺乳動物に対して、以下のいずれかの物質を投与することを特徴とする血管内皮前駆細胞の分化・増殖促進方法。
    （ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチド
    （ｂ）上記（ａ）の蛋白質またはその部分ペプチドをコードする塩基配列を含む核酸
    （ｃ）上記（ａ）の蛋白質の発現を増強する化合物
    （ｄ）上記（ａ）の蛋白質の活性を増強する化合物     A method for promoting differentiation / proliferation of vascular endothelial progenitor cells, comprising administering any of the following substances to a mammal:
    (A) a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof (b) a base sequence encoding the protein of (a) or a partial peptide thereof (C) a compound that enhances the expression of the protein of (a) above (d) a compound that enhances the activity of the protein of (a) above
  16. 　血管内皮前駆細胞の分化・増殖促進剤を製造するための、以下のいずれかの物質の使用。
    （ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチド
    （ｂ）上記（ａ）の蛋白質またはその部分ペプチドをコードする塩基配列を含む核酸
    （ｃ）上記（ａ）の蛋白質の発現を増強する化合物
    （ｄ）上記（ａ）の蛋白質の活性を増強する化合物     Use of any of the following substances for producing an agent for promoting differentiation / proliferation of vascular endothelial precursor cells.
    (A) a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6, or a partial peptide thereof (b) a base sequence encoding the protein of (a) or a partial peptide thereof (C) a compound that enhances the expression of the protein of (a) above (d) a compound that enhances the activity of the protein of (a) above
  17. 　哺乳動物に対して、以下のいずれかの物質を投与することを特徴とする血管内皮前駆細胞の分化増殖抑制方法。
    （ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する中和抗体
    （ｂ）上記（ａ）の蛋白質をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸
    （ｃ）上記（ａ）の蛋白質の発現を阻害する化合物
    （ｄ）上記（ａ）の蛋白質の活性を阻害する化合物     A method for inhibiting differentiation and proliferation of vascular endothelial progenitor cells, comprising administering any of the following substances to a mammal:
    (A) a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a partial peptide thereof (b) a base sequence encoding the protein of (a) above (C) a compound that inhibits expression of the protein of (a) above (d) a compound that inhibits the activity of the protein of (a) above
  18. 　血管内皮前駆細胞の分化・増殖抑制剤を製造するための、以下のいずれかの物質の使用。
    （ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する中和抗体
    （ｂ）上記（ａ）の蛋白質をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸
    （ｃ）上記（ａ）の蛋白質の発現を阻害する化合物
    （ｄ）上記（ａ）の蛋白質の活性を阻害する化合物     Use of any of the following substances for producing a differentiation / proliferation inhibitor of vascular endothelial progenitor cells.
    (A) a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a partial peptide thereof (b) a base sequence encoding the protein of (a) above (C) a compound that inhibits expression of the protein of (a) above (d) a compound that inhibits the activity of the protein of (a) above
  19. 　哺乳動物に対して、以下のいずれかの物質を投与することを特徴とする末梢血中の血管内皮前駆細胞増加方法。
    （ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する中和抗体
    （ｂ）上記（ａ）の蛋白質をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸
    （ｃ）上記（ａ）の蛋白質の発現を阻害する化合物
    （ｄ）上記（ａ）の蛋白質の活性を阻害する化合物     A method for increasing vascular endothelial precursor cells in peripheral blood, comprising administering any of the following substances to a mammal:
    (A) a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a partial peptide thereof (b) a base sequence encoding the protein of (a) above (C) a compound that inhibits expression of the protein of (a) above (d) a compound that inhibits the activity of the protein of (a) above
  20. 　末梢血中の血管内皮前駆細胞増加剤を製造するための、以下のいずれかの物質の使用。
    （ａ）配列番号２、４または６に示されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含む蛋白質またはその部分ペプチドに対する中和抗体
    （ｂ）上記（ａ）の蛋白質をコードする塩基配列に相補的な塩基配列またはその一部を含む核酸
    （ｃ）上記（ａ）の蛋白質の発現を阻害する化合物
    （ｄ）上記（ａ）の蛋白質の活性を阻害する化合物     Use of any of the following substances for producing an agent for increasing vascular endothelial progenitor cells in peripheral blood.
    (A) a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence as shown in SEQ ID NO: 2, 4 or 6 or a partial peptide thereof (b) a base sequence encoding the protein of (a) above (C) a compound that inhibits expression of the protein of (a) above (d) a compound that inhibits the activity of the protein of (a) above
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WO2007026874A1 (en) * 2005-09-02 2007-03-08 Eisai R & D Management Co., Ltd. Method for screening of substance effective on disease using gpr120 and phospholipase

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016192967A (en) * 2010-12-31 2016-11-17 ゼウス サイエンティフィック、インク． Methods for microbe-specific filter-in situ analysis for blood samples
WO2014011926A1 (en) 2012-07-11 2014-01-16 Elcelyx Therapeutics, Inc. Compositions comprising statins, biguanides and further agents for reducing cardiometabolic risk
JP2016530487A (en) * 2013-06-14 2016-09-29 マース インコーポレーテッドＭａｒｓ Ｉｎｃｏｒｐｏｒａｔｅｄ Method
US10222387B2 (en) 2013-06-14 2019-03-05 Mars, Incorporated Methods for increasing palatability of pet foodstuff
US10473673B2 (en) 2013-06-14 2019-11-12 Mars, Incorporated Methods for increasing palatability of pet foodstuff
EP3008082B1 (en) * 2013-06-14 2020-01-01 Mars, Incorporated Methods
US10768184B2 (en) 2013-06-14 2020-09-08 Mars, Incorporated Methods for increasing palatability of pet foodstuff
WO2021241659A1 (en) * 2020-05-29 2021-12-02 株式会社 Numt Fat cell hyper-expressing ffar4 and use thereof
JP7411180B2 (en) 2020-05-29 2024-01-11 株式会社Numt Adipocytes with high expression of FFAR4 and use thereof

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