CN1673390B - Human high-density lipoprotein receptor expression up regulating agent screening model - Google Patents
Human high-density lipoprotein receptor expression up regulating agent screening model Download PDFInfo
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Abstract
The present invention relates to one kind of screening human high-density lipoprotein acceptor expression up regulator, and the method is used in screening human high-density lipoprotein acceptor expression up regulator in high flux from combined chemical library, secondary microbe metabolic product library and natural product library.
Description
Invention field
The present invention relates to a kind of method of adjusting on the human hdl expression of receptor of screening, it is used for adjusting from combinatorial chemical library, microbial secondary meta-bolites storehouse and natural product storehouse high flux screening human hdl expression of receptor.
Background of invention
Coronary heart disease (coronary heart disease, cardiovascular disorder serious harm human health such as CHD), and atherosclerosis (atherosclerosis AS) is its main pathological basis.Epidemiology and clinical study show that the sickness rate of (LDL-C) level of low density lipoprotein cholesterol in the blood plasma and AS is proportionate, and high density lipoprotein cholesterol (HDL-C) level then is negative correlation with the sickness rate of AS.The ldl receptor of SUV picked-up regulates the discovery of path and the widespread use of statins makes developed country's cardiovascular disorder lethality rate descend more than 50%; However, cardiovascular disorder is still the main healthy killer of developed country and most of developing countries.
For more effectively reducing the harm of cardiovascular disorder, in utilizing existing medicine reduction blood plasma, in the LDL-C, also must seek medicine from the new treatment target spot of prevention and/or reverse AS with new role mechanism.
Research in recent years shows; Reverse cholesterol transport (the reverse cholesterol transport that the anti-AS effect of HDL is mainly participated in based on HDL; RCT) process; And confirm that the scavenger receptor SR-BI that found in the past is functional HDL acceptor [1], and in the RCT process, play keying action, be considered to find the potential new target drone [2] of novel cardiovascular agent.The enhancing of reverse cholesterol transport will help the reverse of minimizing of atherosclerotic lesion accumulative SUV and pathology, therefore can express the RCT process of quickening through rising SR-BI, promote the removing of body cholesteryl ester more than needed.
From the above, can the raise compound of SR-BI genetic expression is expected to develop the newtype drug [2,6,7,8] that becomes effective treatment ACVD.
Along with the fast development of combinatorial chemistry, natural product chemistry and drug screening technology, (high throughput screening HTS) has become the important means of new drug early discovery to the high flux screening of medicine.Making up the novel medicaments sifting model that is suitable for high flux screening is the basis that new drug is found.Obtained bigger progress in recent years and used [9] widely based on the drug screening method of examining report gene.Utilization has screened receptor stimulant [10,11] and human ldl receptor up-regulated agent [12,13,14] of cytokine, hormone etc. etc. based on the drug screening method of examining report gene.
The inventor is a target spot with human hdl acceptor SR-BI; Successfully made up the high flux screening model of novel Antiatherosclerosis medicine; Be used for from the adjustment of a large amount of microbial metabolites and testing compound screening human hdl acceptor SR-BI, and utilize said model to obtain to have the meta-bolites that raises the human hdl receptor active.
Summary of the invention
In people and some animal models, HDL SUV in the blood plasma (being commonly called " good SUV ") is inversely proportional to the sickness rate of AS.Research to HDL functional receptor SR-BI and AS relation in recent years shows that SR-BI has the effect of anti-AS.Trigatti etc. [4] find in the ApoE knock-out mice, if SR-BI is knocked out more being prone to take place AS.Arai etc. [5] find that overexpression SR-BI will obviously reduce the AS damage in the mouse that the ldl receptor in high fat diet knocks out.Zooperal result shows, to improve pharmacological agent that SR-BI expression level in the liver is a purpose or gene therapy might in atherosclerotic control, make a breakthrough [2,6,7,8].
The present invention has successfully made up the model that is suitable for the agent of high flux screening human hdl acceptor SR-BI up-regulated on this basis, is applied to from microbial metabolites and combinatorial chemical library, seek the active substance of the SR-BI gene expression dose of can transferring person.
The regulation and control of human hdl acceptor SR-BI gene transcription are (like C/EBP through many transcription factors; SF-1; SREBP-1 (sterol regulatory element conjugated protein-1, sterolregulatory element binding protein-1) and its upstream regulatory sequence interact and realize.The about 1kb in the transcriptional start point upper reaches of human hdl acceptor gene CLA-1 is known promoter sequence, contains and different transcription factor bonded cis elements [3].
For making up human hdl acceptor screening model of the present invention, the cell that can select to participate in the reverse cholesterol transport process includes but not limited to liver cell as test cell, liver cancer BEL-7402 cell for example, liver cancer HepG2 cell strain; Peripheral cells, for example scavenger cell or cells of vascular wall (vascular endothelial cell and VSMC).For above-mentioned test cell, the compound that can cause people SR-BI gene promoter activity state to increase is the active compound with rise SR-BI genetic expression.Wherein, the reporter gene replacement target gene SR-BI that can select suitable this area to know, thus can be so that the mode that detects reflects the activity change of said promotor.Particularly, can testing compound be added the test cell culture system, compare with the contrast test cell that does not add said compound; If it can the Impact Report gene; The for example expression of luciferase then utilizes and detects the for example method of uciferase activity of said reporter gene, detects said reporter gene; The for example change of luciferase gene expression, thus confirm whether said compound is the agent of human hdl SR-BI up-regulated.
In one embodiment of the invention; The inventor utilizes the method for PCR; From the total DNA of human hepatocellular carcinoma BEL-7402 cell, amplify the regulating and controlling sequence (SEQ ID NO:1) of people SR-BI gene C LA-1 (CD36 andLIMPII Analogous-1), be cloned into the luciferase reporter gene upper reaches.Obtain being used for the recombinant expression vector of transfection BEL-7402 cell.Behind gained recombinant expression vector transfection BEL-7402 cell, be used to detect on the human hdl expression of receptor and adjust.Wherein, the expression of luciferase gene receives the control of people SR-BI upstream region of gene regulating and controlling sequence.
What adopt in the model according to the invention is that the system of reporter gene has and do not use isotropic substance with the firefly luciferase gene, advantage such as susceptibility is good, and linearity range is wide, and the endogenous activity is minimum in mammalian cell, and is relatively inexpensive.Utilization luciferase assay kit adopts the fluorophotometer that is applicable to 96 holes even 384 well culture plates to detect, and has flux height, little, the automatable characteristics of systematic error again.Compare with traditional receptor-ligand combined techniques, can directly provide the function information of allied compound based on the drug screening method of examining report gene, the positive compound that sifts out more is prone to get into the composition optimizes process.
The human hdl up-regulated agent screening model that utilizes the inventor to set up; Characteristics to small molecules class medicine; Can be on cell levels extensive screening can influence the micromolecular compound of human hdl expression of receptor level, is suitable for high flux screening is carried out in natural product, synthetic compound, combination of compounds storehouse etc.
Description of drawings
Fig. 1 desoxyneohydroxyaspergillic acid (2101C) suppresses the microscopy result (oil red O stain method) of macrophage foam cell formationization.
The structure of embodiment 1 recombinant expression plasmid pGL3-CLAP
Bacterial strain and cell strain
E.coli DH5 α is used for the genetic manipulation of DNA.Liver cancer BEL-7402 cell strain (available from upright Bioisystech Co., Ltd of Shanghai noon) is used for the structure of adjustment screening model on the human hdl expression of receptor.
Plasmid
PGEM-T (available from Promega company) is used for the clone of PCR product.PGL3-Basic (available from Promega company) is the reporter gene carrier, contains promoterless Lampyridea (Photinus pyralis) luciferase encoding sox, is used for the clone of human hdl receptor modulators sequence.
PGL3-control (available from Promega company) contains SV40 promotor and enhancer sequence, is used for the positive control of luciferase expression.PRL-TK (available from Promega company) comprises the cDNA of coding sea pansy (Renilla reniformis) luciferase, with experiment carrier cotransfection mammalian cell, is used for the internal reference of two luciferase reporter gene systems.
The acquisition of HDL receptor gene regulating sequence
According to the CLA-1 gene order of document [3] report, utilize software Primer Premier5.0 design PCR primer P1 (5 '-C
CTCGAGTGG AGC CAT TGT GTG CAAAG-3 ') (SEQ ID NO:2) and P2 (5 '-C
AAGCTTCGG CGA CAG AGACGA CAC AG-3 ') (SEQ ID NO:3); People SR-BI upstream region of gene 1055bp 62bp (A with SR-BI gene start codon ATG is+1) length that is used to increase is the fragment of 996bp; Wherein P1 has introduced the XhoI restriction enzyme site, and P2 has introduced the HindIII restriction enzyme site.
With the total DNA of human liver cancer cell BEL-7402 is that template is set up 25 μ l PCR reaction systems: 1 * PCR damping fluid, 0.5 μ mol/L P1 and P2,2.5 μ mol/L dNTPs, 0.5U TaqDNA polysaccharase.Reaction conditions is 94 ℃ of sex change 5min; 94 ℃ of sex change 40s, 54 ℃ of annealing 40s, 72 ℃ are extended 1min 20s (30 circulations); 72 ℃ are extended 10min.The purpose fragment of pcr amplification with the pGEM-T carrier cloning after enzyme cut the sequence of identifying and measuring the gained regulating and controlling sequence, shown in SEQ ID NO:1.
According to molecular cloning: the method described in the lab guide, the PCR product is connected with the pGEM-T carrier, transformed into escherichia coli DH5 α recipient bacterium selects transformant with blue hickie method.Transformant is carried out carrying out enzyme after rapid plasmid extracts cut evaluation, use XhoI, HindIII is two to contain the segmental transformant of purpose after cutting has the segmental recon of purpose, called after pGEM-CLAP for cloning.
The structure of recombinant plasmid pGL3-CLAP
Use XhoI; HindIII is two cut recombinant plasmid pGEM-CLAP and obtain the purpose fragment after; The purpose fragment is continuous with the pGL3-basic plasmid through identical double digestion; Thereby people SR-BI upstream region of gene regulating and controlling sequence orientation is inserted into the luciferase gene upper reaches of pGL3-basic, is built into recombinant plasmid pGL3-CLAP.
Embodiment 2 human hdl acceptor SR-BI go up the screening of adjusting
Cell cultures and transfection
RPMI-1640 Medium (Hyclone) is used for (containing 10% standard foetal calf serum (Hyclone), 100U/ml penicillium mould, 100 μ g/ml Streptomycin sulphates) cultivation of liver cancer cell BEL-7402.Transfection is adopted Wizard with the extraction of DNA
RPureFection Plasmid DNAPurification System test kit (Promega).Adopt LipofectAMINE
TM2000Reagent (Invitrogen) transfection reagent box carries out cell transfecting.
The active mensuration of luciferase expression
The active mensuration of the plain expression of enzymes of cell fluorescence adopts fluorescence detection reagent kit LuciferaseAssay System and Dual-Luciferase Assay System (Promega).Detect uciferase activity with the desk-top culture plate of BMGPolarstar Galaxy ultraviolet-visible-fluorescence with spectrophotometer.
Screening sample
Compound to be sieved is dissolved in DMSO.Microbial fermentation solution is dissolved in DMSO with equal-volume acetone or ethyl acetate extraction after drying.
Confirming of primary dcreening operation condition
With the negative control plasmid of pGL3-basic; The positive control plasmid of pGL3-control; Distinguish transient transfection liver cancer BEL-7402 cell simultaneously with the plasmid pGL3-CLAP that makes up; Transfection conditions is optimized, confirms following: every hole inoculation 5 * 10 with the condition of 96 well culture plate preliminary screening compounds and microbial secondary meta-bolites
4Individual cell, the 200ng DNA, 0.5 μ l liposome, the transfection time is 6 hours, transfection is changed to serum-free antibiotic-free substratum after 6 hours.Add sample continued incubation to be screened after 18 hours, measure uciferase activity in the liver cancer BEL-7402 cell, the result sees table 1.
Experimental result shows that the variation coefficient (CV) in different holes is no more than 10%, and consistence is better between the hole, and significant difference between pGL3-CLAP and pGL3-basic group.
The hole differences of table 1 96 well culture plate readings (x ± s, n=10)
DMSO is to the influence of The selection result
Wait to sieve samples using DMSO dissolving, therefore measured the influence of different concns DMSO uciferase activity, the result shows, and is less to the uciferase activity influence when DMSO concentration is 0.1%-0.01%, adopts this scope concentration during screening and the solvent control wells is set.
Testing sample is to the calculating of luciferase expression activity change rate
With following Equation for Calculating testing sample to the active change rate of luciferase expression:
Change rate (%)=(A-B)/B * 100
Wherein, A is for adding the luciferase expression activity of measuring behind the testing sample (or corresponding flat fluorescent), and B is active for the luciferase expression that adds negative control sample (blank) back mensuration.
For the change rate the testing sample more than+20% can tentatively regard as have on the human hdl expression of receptor adjust active.
The primary dcreening operation result
Use above-mentioned definite screening conditions to 124 compounds, 800 microbial secondary meta-bolitess have carried out preliminary screening, and wherein 1 compound 2101C and 4 bacterial strains are positive, and the primary dcreening operation positive rate is 5.4 ‰.
The selection result
Use above-mentioned definite screening conditions and measure desoxyneohydroxyaspergillic acid according to the invention, the result sees table 2, and desoxyneohydroxyaspergillic acid wherein according to the invention makes uciferase activity change rate greater than+60%, is shown as on strong man's HDL receptor and adjusts.
Table 2. desoxyneohydroxyaspergillic acid (2101 C) raises the active result of RHDL
(fluorescence report gene approach, fluorescence intensity)
| Sample | Uciferase activity (average) | Amplification (%) |
| Blank | 26335.3 | ? |
| Contrast (0.1%DMSO) | 17949.1 | ?-7.6048381 |
| ?2101C(5μg/ml) | 40685.3 | ?126.67042 |
| ?2101C(2.5μg/ml) | 38647.6 | ?115.31776 |
| ?2101C(1.25μg/ml) | 29757.6 | ?65.788814 |
Multiple sieve
Compound and fermented liquid to positive findings is arranged adopt two luciferase detection kit further to detect.In the time of with plasmid pGL3-CLAP transfection liver cancer BEL-7402 cell, another kind of luciferase (Renilla reniformis, sea pansy) the reporter gene plasmid pRL-TK of transfection, this plasmid consumption is the former 1/10th.The SV40 promotor is contained at the Renilla luciferase reporter gene upper reaches, in dissimilar mammalian cells, express stable, and its expression activity medicine irritation is insensitive to external world, can be used for proofreading and correct the error that the transfection efficiency difference causes.Active as the relative expression with the Firefly uciferase activity to the relative value of Renilla uciferase activity.
Testing sample is to the calculating of two luciferase expression activity change rates
With following Equation for Calculating testing sample to the active change rate of two luciferase expressions:
Change rate (%)=(A-B)/B * 100
Wherein, A is active for adding the luciferase relative expression who measures behind the testing sample, and the luciferase relative expression that B measures for adding negative control sample (blank) back is active.
For the change rate the testing sample more than+10% can tentatively regard as have on the human hdl expression of receptor adjust active.
Sieve the result again
1 compound 2101C and 3 bacterial strains sieve the positive again.
The active result of 2101C: the luciferase relative expression is active during 2 μ g/ml raises 23.01%.
Embodiment 4 desoxyneohydroxyaspergillic acids suppress the active mensuration of macrophage foam cell formationization
A) preparation of the low-density lipoprotein of oxidation (OX-LDL)
(LDL) is dissolved in 0.15mol/L with low-density lipoprotein, in the NaCl solution of pH7.4, puts into dialysis tubing then and places the PBS that contains 10 μ mol/L CuSO4; 37 ℃ of dialysis are after 12 hours; Dialysis is 24 hours in containing the PBS of 0.1%EDTA, and 0.2 μ m filtering with microporous membrane degerming is subsequent use.Identify the LDL degree of oxidation with thiobarbituric acid reaction thing content.With the tetraethoxypropane is standard substance, presses mda (MDA) and measures the operation of test kit specification sheets, measures the mda content of every milligram of LDL after this batch oxidation.
B), place 37 ℃ to contain 0.5%CO with human monocyte's strain U937
2In the incubator, the doubling time is 24～48 hours, suspension growth in containing the RPMI-1640 nutrient solution of 10% foetal calf serum, add in the nutrient solution penicillium mould and Streptomycin sulphate each 1.0 * 10
5IU/L.Buddhist ripple ester (PMA) with 100nmol/L before the U937 cell experiment stimulated 72 hours, made it be divided into scavenger cell by monocyte.Change serum-free medium and be added on 96 well culture plates, every hole 150 μ l, cell density is 1 * 10
6About individual/milliliter, be divided into control group, foam cell group and application of sample group with the U937 cell this moment.Control group only adds serum-free medium, and it is 80mg/L to every hole final concentration that the foam cell group adds OX-LDL in addition, and the application of sample group adds testing sample (5 μ l microbial fermentation solution) again on the basis of foam group, cultivate after 48 hours, dyes.
C) U937 cell oil red O stain
1. the compound method of oil red O stain liquid: oil red 0.3 gram is dissolved in 30 milliliters of Virahols, stirs following 60 ℃ of incubated overnight, and then adds 20 ml distilled water stirred overnight, filters.Must use the good filtrating of fresh filtration during use.
2. the method for the oil red O stain method of U937 cell neutral lipid: set by step 2) mentioning will add excellent 96 well culture plates and from the carbonic acid gas incubator, take out, and 10% LUTARALDEHYDE stationary liquid is fixed (15 μ l/ hole); Discard solution after 10 minutes; Washing twice adds 60% Virahol (150 μ l/ hole) again and placed 5 minutes, discards solution; With oil red O stain liquid (150 μ l/ hole) dyeing 1 hour; Discard solution with 60% Virahol (150 μ l/ hole) hole flushing, water (150 μ l/ hole) washes twice then, and last every hole adds 150 μ l water and is put into the microscopically observation.
D) microscopy result
Behind oil red O stain; Observation does not add the interior no obvious red oil droplet shape particle of cellular control unit of OX-LDL under the mirror; And the cell of foam cell group can be seen more red olesome is arranged in the cytoplasm; And because the oil droplet engulfed of individual cells too much makes these cell volumes increase, in the sample sets if there is pair macrophage foam cell formationization that inhibiting sample is arranged, redfree olesome almost in its cell.Shown in accompanying drawing, the adding of desoxyneohydroxyaspergillic acid according to the invention makes it possible to effectively suppress the foamed to scavenger cell.
Reference
1?Acton?S，Rigotti?A，Landschulz?KT，et?al.Identification?ofscavenger?receptor?SR-BI?as?a?high?density?lipoprotein?receptor.Science，1996，271：518-520
2?Acton?SL，Kozarsky?KF，Rigotti?A.The?HDL?receptor?SR-BI：anew?therapeutic?target?for?atherosclerosis?Mol?Med?Today，1999，5(12)：518-524
3?Cao?G，Garcia?CK，Wyne?KL，et?al.Structure?and?localizationof?the?human?gene?encoding?SR-BI/CLA-1.J?Biol?Chem，1997，272(52)：33068-33076
4?Trigatti?B，Rayburn?H，Vinals?M，et?al.Influence?of?the?highdensity?lipoprotein?receptor?SR-BI?on?reproductive?andcardiovascular?pathophysiology.Proc?Natl?Acad?Sci?USA，1999，96(16)：9322-9327
5?Arai?T，Wang?N，Bezouevski?M，et?al.Decreased?atherosclerosisin?heterozygous?low?density?lipoprotein?receptor-deficient?miceexpressing?the?scavenger?receptor?BI?transgene.J?Biol?Chem，1999，22，274(4)：2366-2371
6?Krieger?M.Scavenger?receptor?class?B?type?I?is?a?multiligandHDL?receptor?that?influences?diverse?physiologic?systems.J?ClinInvest，2001，108(6)：793-797
7?Krause?BR，Auerbach?BJ.Reverse?cholesterol?transport?andfuture?pharmacological?approaches?to?the?treatment?of?atherosclerosis.Curr?Opin?Investig?Drugs，2001，2(3)：375-381
8 Liu Xiao brightness, Hong Bin.Antiatherogenic new target drone HDL receptor SR-BI.Foreign medical science microbiotic fascicle, 2003,24 (2): 70-73
9 Sun Zhe, Lv Qiujun.Drug screening method progress based on the examining report gene.Chinese Pharmaceutical Journal, 2000,35 (8): 507-510
10 Liu Wei capital, Lv Qiujun, Wen Liqing, etc.Foundation based on the sieve medicine model of STAT3 transcriptional regulatory.The Chinese Pharmacological circular, 2002,18 (1): 103-106
11 Wang Ling are skilful, Lv Qiujun, and Niu Jianzhao, etc.Foundation based on the medicaments sifting model of oestrogenic hormon response element transcriptional regulatory.CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2003,28 (6): 536-540
12 China supply Tian Yang, big village intelligence.The research of the active substance of the increase LDLR genetic expression that fungi KS-1995 produces.China's microbiotic magazine, 1998,23 (4): 248-252
13?Koguchi?Y，Nishio?M，Kotera?J，et?al.Trichostatin?A?andherboxidiene?up-regulate?the?gene?expression?of?low?densitylipoprotein?receptor.J?Antibiot，1997，50(11)：970-971
14?Murakami?S，Nitanai?I，Uchida?S，et?al.Up-regulation?of?lowdensity?lipoprotein?receptor?by?a?novel?isobenzofranone?derivative，MD-700.Atherosclerosis，1999，146：281-290
Sequence table
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< 120>adjust screening model on the human hdl expression of receptor
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Claims (5)
1. method of screening the agent of human hdl acceptor SR-BI up-regulated, it comprises
The test cell that 1) will contain the recombinant expression vector of the human hdl acceptor SR-BI regulating and controlling sequence that is positioned at the reporter gene upper reaches is mixed with testing compound;
2) be suitable for cultivating the gained mixture under the condition of reporter gene expression;
3) change of reporter gene expression level behind the detection adding testing compound.
2. the described method of claim 1, wherein said test cell is liver cell and/or the peripheral cells relevant with the reverse cholesterol transport process.
3. the method for claim 2, wherein said liver cell is selected from liver cancer BEL-7402 cell strain, liver cancer HepG2 cell strain, peripheral cells is selected from scavenger cell, vascular endothelial cell and VSMC.
4. the described method of claim 1, wherein said human hdl acceptor SR-BI regulating and controlling sequence is SEQ ID NO:1.
5. the described method of claim 1, wherein said reporter gene is a luciferase reporter gene.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5846720A (en) * | 1989-07-18 | 1998-12-08 | Oncogene Science, Inc. | Methods of determining chemicals that modulate expression of genes associated with cardiovascular disease |
| CN1350061A (en) * | 2000-10-20 | 2002-05-22 | 上海医药(集团）总公司 | Method of estalishing ApoAI target cell screening medicine system and screening HDL raising medicine |
| CN1368551A (en) * | 2001-02-07 | 2002-09-11 | 龚邦强 | Process for creating medicine screening system with ABC1 gene cell target to screen 3G medicine of cardiovascular disease |
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2004
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5846720A (en) * | 1989-07-18 | 1998-12-08 | Oncogene Science, Inc. | Methods of determining chemicals that modulate expression of genes associated with cardiovascular disease |
| CN1350061A (en) * | 2000-10-20 | 2002-05-22 | 上海医药(集团）总公司 | Method of estalishing ApoAI target cell screening medicine system and screening HDL raising medicine |
| CN1368551A (en) * | 2001-02-07 | 2002-09-11 | 龚邦强 | Process for creating medicine screening system with ABC1 gene cell target to screen 3G medicine of cardiovascular disease |
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| Title |
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| .基于检测报告基因的药物筛选方法研究进展.中国药学杂志 8.2000,(8),全文. |
| 刘晓辉.抗动脉粥样硬化的新靶位─高密度脂蛋白受体SR-BI.国外医药.抗生素分册 2.2003,(8),全文. |
| 刘晓辉.抗动脉粥样硬化的新靶位─高密度脂蛋白受体SR-BI.国外医药.抗生素分册 2.2003,(8),全文. * |
| 吕秋军 |
| 孙哲 |
| 孙哲;吕秋军;.基于检测报告基因的药物筛选方法研究进展.中国药学杂志 8.2000,(8),全文. * |
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