CN101173247B - Method for culturing osteoclast with mesenchyma stem cell combined with cell factor - Google Patents
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- CN101173247B CN101173247B CN200710120072XA CN200710120072A CN101173247B CN 101173247 B CN101173247 B CN 101173247B CN 200710120072X A CN200710120072X A CN 200710120072XA CN 200710120072 A CN200710120072 A CN 200710120072A CN 101173247 B CN101173247 B CN 101173247B
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Abstract
The invention discloses a culturing method of osteoclasts utilizing mesenchymal stem cells and cytikines, mainly utilizing the mesenchymal stem cells from mouse bone substance and the mouse splenic mononuclear cells and then adding nuclear factor kB receptor activator ligand and macrophage-colony to stimulate the factors. The invention has the advantages: the invention has the advantages of simple operation, convenience and practicality, capability of getting osteoclasts at the earliest time, and saving the culture time; the invention gets a large quantity of osteoclasts, solves the problem that osteoclasts are difficult to prepare in large scale, and can meet the need of a general cell biology experiment; the osteoclasts gotten through the invention has high maturity and strong bone substance absorptive capability, which is favorable for the function study; the cell factors needed by the invention is few, which saves expenditure.
Description
Technical field
The invention belongs to the cultural method of osteoclast, relate in particular to a kind of method of utilizing mesenchyma stem cell combined cytokine to cultivate osteoclast.
Background technology
(osteoclasts is the main cell that absorbs osseous tissue in the humans and animals body OC) to osteoclast, and under physiological conditions, osteoclast and scleroblast are assisted mutually, participate in running through the bony remodeling activity of life whole process, keep the running balance of Skeletal system; Under pathologic conditions such as tumour, inflammation, osteoporosis and autoimmune disorder, the generation of osteoclast involved in diseases, progress influence lapsing to and prognosis of disease.
Osteoclast is by the hemopoietic stem cell differentiation and development, can decompose osseous tissue in vivo, osteoclast in vitro culture can be at osteocomma, form the absorptivity lacuna on ivory or the artificial bone tissue culture, become when ripe and contain a plurality of nuclear giant cells, the principal character of osteoclast is positive for being specific anti-tartaric acid phosphatase (TRAP+) groupization, and F-actin stained positive, express Calcitonin Receptor, histone enzyme K (cathepsin-k), integrin alpha v beta 3 (Walsh MC etc., AnnuRev Immunol.2006 such as (integrins α v β 3); 24:33-63).
In osteoclasts cultured in vitro is the basis of the multiple hormone of research, cytokine and osteoclast and bone resorption relation, also is the basis of research tumour, inflammation, osteoporosis and autoimmune disorder pathogenesis and drug treatment.In recent years, according to the discovery that obtains from osteoclast research, people use its regulatory factor as mobilization agent, as one of the means of gathering hemopoietic stem cell before the hematopoietic stem cell transplantation (KolletO, Nat Med.2006 Jun; 12 (6): 657-64.Epub 2006 May 21); Developed antibody, antagonist and the inhibitor of osteoclast excreted factor, be used for the treatment of osteopathia such as osteoporosis and cancer such as malignant tumour and shift (Abe M etc., Leukemia.2006 Jul; 20 (7): 1313-5.Epub 2006 Apr 13); Osteoclast is introduced field of tissue engineering technology, itself and scleroblast are planted jointly on biomaterial, improve three-dimensional structure of tissue-engineered bone or the like (Tianjin biomedical engineering such as Han Daqing 2006 academic nd Annual Meeting summaries).In sum, set up the extracorporeal culturing method of osteoclast, the growth, differentiation, maturation of further investigation osteoclast and the rule of bringing into play function with and molecular regulation mechanism, help seeking hematopoiesis, autoimmune disease, tumour and bone metabolism disease aspect new diagnostic techniques and medicine.
Up to the present, cultivate method (the international osteology magazine such as Lv Mingbo of osteoclast, in September, 2006, the 27th volume, the 5th phase) mainly contain: (1), mature osteoclast isolated culture Chamber in 1984 etc. take the lead in from rabbit long bone of limbs Central Plains for having isolated osteoclast, yet quantity is few in animal body, volume big for osteoclast, be close to the growth of medullary space internal surface, cell space damages when separating easily, separates the back and is difficult for cultivating; In addition, partition method obtains is osteoclast in whole latter stage, be difficult to use in grow, the research of propagation and differentiation aspect; (2), giant cell tumor of bone osteoclast partition method giant cell tumor of bone is a kind of bone tumor that is feature with molten bone osteoclasia, Zambonin-ZallonA etc. isolate a large amount of osteoclasts from giant cell tumor of bone knurl body.But, the giant cell tumor of bone sickness rate is low, and is tissue-derived few, can't long-term stability provide the osteoclast source; And the osteoclast that this method obtains has the tumour correlation properties, also has any different with normal osteoclast, can not be as conventional cell source; (3), report such as marrow inducing culture method Burger adds various inductors (Vitamin D3 500,000 I.U/GM, interleukin 6, tumour necrosis factor, granulocyte-macrophage colony stimutaing factor, dexamethasone or the like) with marrow and comes inducing culture to go out osteoclast.This method adopts medullary cell as culturing cell, to a certain extent can the simulate bone marrow microenvironment to the influence of osteoclast differentiation and development.But, the actual multiple components such as endotheliocyte, adipocyte, inoblast that comprise of marrow that this method is used, be unfavorable for carrying out accurate developmental regulation Study on Mechanism, in addition, the acquisition of medullary cell need be peeled off animal muscle, isolate long bone, wash marrow then repeatedly, operation is wasted time and energy; (4), the peripheral blood of personnel selection such as peripheral blood lymphocytes culture method Fujikawa isolates monocyte, inducing culture obtains osteoclast then.But the hematopoietic stem/progenitor cells cell number is few in the peripheral blood, and hematopoietic stem/progenitor cells is the early development source of osteoclast, and this method is only cultivated with monocyte, and the acquisition efficient of osteoclast is lower; (5), spleen cell inducing culture method is an osteoclast cultural method commonly used at present.Udagawa at first cultivates with rat spleen cells and obtains a large amount of osteoclasts and confirm to have the bone resorption ability.Compare with the medullary cell culture method, spleen cell inducing culture method is drawn materials conveniently, opens the animal belly and can extract spleen, and need not separate many long bones through peeling off muscle, repeated multiple times flushing marrow; And got rid of the influence of cellular environment complicated in the marrow; Spleens such as muroid have lifelong hematopoietic potential, exist hemopoietic stem cell in the spleen, adopt spleen cell than adopting monocyte and have better growing multiplication potentiality.Set up a kind of stable practicality, economical and convenient, the osteoclast cultural method is to carry out pressing for of osteoclast correlative study efficiently.
Except selected inducing cell difference, the selection of inductor is also very important for the vitro culture of osteoclast, discover: the sub-part of nf κ B receptor activation (RANKL) and its antagonistic molecule osteoprotegerin (OPG) realize that by the RANKL-RANK-TRAF6 signal path osteoclast is grown differentiation and maturation carries out decisive regulation and control, macrophage colony stimulating factor (M-CSF) then plays synergy (Lacey DL etc., Cell.1998 Apr 17 for path; 93 (2): 165-76.Kong YY etc., Nature.1999 Jan28; 397 (6717): 315-23).Therefore, sub-part of nf κ B receptor activation and macrophage colony stimulating factor induce the method for osteoclast to set up rapidly jointly, and become the main inductor of osteoclast vitro culture.
The inventor is at (Guo Z etc., Stem Cells.2006Apr such as the patent method of separating mesenchymal ancestral cells " a kind of from bony process matter " (patent No.: ZL 200510055261.4) and Guo Z; 24 (4): 992-1000), disclose a kind of from mouse bony process matter the method for separating mesenchymal ancestral cells.The mescenchymal stem cell in mouse bony process matter source has the ability to multiple tissue differentiation, can be in vivo and the function of external adjusting immunocyte.Mescenchymal stem cell with mouse bony process matter source is done trophoderm, can support the hematopoietic stem cell expansion of derived from bone marrow.
By retrieval, up to the present, also find to provide more than the quantity and the cultural method of the osteoclast that purity is high.
Summary of the invention
The purpose of this invention is to provide a kind of mescenchymal stem cell and mouse spleen cell co-cultivation of utilizing mouse bony process matter source, and add the method that cytokine is cultivated osteoclast, utilize present method can obtain the osteoclast that quantity is many, purity is high.
The present invention is achieved through the following technical solutions:
The invention provides a kind of method of utilizing mesenchyma stem cell combined cytokine to cultivate osteoclast, comprise the steps:
(1) separate in the mouse bony process matter, cultivate mescenchymal stem cell, and cultivations of going down to posterity, the cultivation of will going down to posterity then the 4th～6 generation mescenchymal stem cell be laid in the culture plate, at 35～38 ℃, 5%CO
2Cultivated under the condition 12～24 hours;
(2) separate mononuclearcell from mouse spleen, and plant in the culture plate of step (1);
(3) the substratum co-cultivation of interpolation factor-containing in the culture plate in step (2), half amount is changed liquid every three days, at 35～38 ℃, 5%CO
2Cultivated under the condition 3～15 days, and can obtain osteoclast;
Wherein, the described substratum of step (3) is for containing 1～2mmol/L glutamine, 10～15% volume foetal calf serums, 10～20ng/ml recombined small-mouse macrophage stimulation factor, the a-MEM substratum of the sub-part of 10～20ng/ml recombined small-mouse nf κ B receptor activation.
The mescenchymal stem cell in the described mouse bony process of above-mentioned steps (1) matter source can be according to the inventor at (Stem Cells.2006 Apr such as the patent method of separating mesenchymal ancestral cells " a kind of from bony process matter " (patent No. ZL200510055261.4) or Guo Z; 24 (4): 992-1000) disclosed method separation and Culture obtains.
Above-mentioned cytokine is meant recombined small-mouse macrophage stimulation factor and the sub-part of recombined small-mouse nf κ B receptor activation, all can buy on market.
The used culture plate of above-mentioned steps (1) can be 48 well culture plates, makes with the biological plastics material that is fit to cell cultures.
The used mescenchymal stem cell quantity of above-mentioned steps (1) shop culture plate is 2000～10000/hole.
The described mouse spleen mononuclearcell of above-mentioned steps (2) obtains from mouse separation in 2～3 ages in week.
The mouse spleen mononuclearcell that above-mentioned steps (2) is planted is 1800～2200/hole.
The used cytokine source of above-mentioned steps (3) preparation substratum is concentration (weight percent): the recombined small-mouse macrophage stimulation factor is 10～20ng/ul, the sub-part of recombined small-mouse nf κ B receptor activation is 10～20ng/ul, and solvent is the phosphate buffered saline buffer that contains 0.1% bovine serum albumin.
A kind of method of utilizing mesenchyma stem cell combined cytokine to cultivate osteoclast of the present invention, its detailed culturing step comprises:
(1) healthy C57BL/6 female mice in 2～3 ages in week is got in separation of the mescenchymal stem cell of mouse bony process matter and cultivation, taking off neck puts to death, 75% after alcohol-pickled 3～5 minutes, aseptic condition separates mouse femur and shin bone down, go clean surface structure, draw the phosphate buffered saline buffer that contains 5～15% volume foetal calf serums with syringe and wash down medullary space, shredding is big or small 1～2mm
2Tiny osteocomma, place that concentration expressed in percentage by weight is 0.1～0.15%, solvent is the two Collagen Type VI enzyme solution that contain the a-MEM substratum of 15%～20% volume foetal calf serum, at 37 ℃, digest and plant osteocomma after 1 hour in containing 1～2mmol/L glutamine, 10～15% volume foetal calf serum a-MEM substratum, at 35～38 ℃, 5%CO
2Cultivate under the condition after 72 hours and change liquid first, remove suspension cell, keep osteocomma, had digestive transfer culture after the cell that grows in the osteocomma covers with 70～80% culturing bottle areas, continuous passage, get the 4th～6 generation cell do to cultivate and use;
(2) according to 2000～10000 in every hole step (1) gained mescenchymal stem cell is layered in the 48 porocyte culture plates, every hole adds the a-MEM substratum that 500～800 μ l contain 1～2mmol/L glutamine, 10% volume foetal calf serum, at 35～38 ℃, 5%CO
2Cultivated under the condition 12～24 hours;
(3) separation of mouse spleen mononuclearcell with 2～3 ages in week healthy C57BL/6 female mice take off neck and put to death, 75% after alcohol-pickled 3～5 minutes, aseptic condition takes out spleen down, spleen is placed on 200 mesh filter screens, mill with the glass syringe handle, the phosphate buffered saline buffer flushing obtains the spleen single cell suspension; Get 4～5 milliliters of mouse lymphocyte parting liquids in vitro, draw 4～5 milliliters of careful test tubes that add of spleen single cell suspension, note keeping the interface complete, centrifugal 15～20 minutes with 1500～1800 rev/mins, draw the mononuclearcell layer, add phosphate buffered saline buffer again with 1000～1200 rev/mins of washings twice in 8～10 minutes, cell counting is continued to employ;
(4) the spleen mononuclearcell kind that separation is obtained goes into to spread in advance in the culture plate of mouse bony process matter derived mesenchymal stem cell, and the density of each 48 well culture plate single hole is 1800～2200 spleen mononuclearcells; Every hole adds substratum 500 μ l, and half amount is changed liquid every three days, at 35～38 ℃, 5%CO
2Cultivated under the condition 3～15 days, and can get osteoclast;
Wherein the described substratum of step (4) is for containing 1～2mmol/L glutamine, 10～15% volume foetal calf serums, 10～20ng/ml recombined small-mouse macrophage stimulation factor, the a-MEM substratum of the sub-part of 10～20ng/ml recombined small-mouse nf κ B receptor activation.
Cytokine solution composition that above-mentioned preparation substratum is used and concentration (weight percent) are: the recombined small-mouse macrophage stimulation factor is 10～20ng/ul, the sub-part of recombined small-mouse nf κ B receptor activation is 10～20ng/ul, and solvent is the phosphate buffered saline buffer that contains 0.1% bovine serum albumin.
The liquid that above-mentioned half amount is every three days changed liquid is meant and contains 1～2mmol/L glutamine, 10～15% volume foetal calf serums, 10～20ng/ml recombined small-mouse macrophage stimulation factor, the a-MEM substratum of the sub-part of 10～20ng/ml recombined small-mouse nf κ B receptor activation.
The component of the phosphate buffered saline buffer described in the above-mentioned steps (1) and concentration (weight percent) are: sodium-chlor 8g/L, and Sodium phosphate dibasic 2.9g/L, Repone K 0.2g/L, potassium primary phosphate 0.24g/L, solvent are deionized water.
Above-mentioned go down to posterity to cultivate be meant that the cells in vitro molecular marker for increased proliferation is to satisfy the needs of general Cell Biology Experiment with the cultivation of going down to posterity of the low serum system of gained mescenchymal stem cell utilization.
The component of above-mentioned culture plate, reagent and substratum all can be bought from the market.
The detection method of gained osteoclast mainly contains: (1), resulting osteoclast is carried out specific anti-tartaric acid phosphatase (TRAP+) groupization dyeing, and the result should be strong positive; (2), opticmicroscope observes down, unicellular have a plurality of nucleus (〉=3 nucleus); (3), the ivory film absorption test shows to have the bone absorption ability.
The inventive method can be cultivated the osteoclast that obtains more than 400 from per 2000 spleen mononuclearcells, can satisfy general Cell Biology Experiment quantity demand fully.
Advantage that the present invention had and beneficial effect: (1) the inventive method is simple to operate, and is convenient and practical, can obtain osteoclast in earliest time, can shorten incubation time 2～3 days; (2) the inventive method gained amount of osteoclast is big, has solved the problem that osteoclast is difficult to a large amount of preparations, can satisfy the needs of relevant cell biology and molecular biology experiment; (3) the inventive method is cultivated the osteoclast ripening degree height of gained, and the bone absorption ability is strong, helps carrying out functional study; (4) the needed cytokine of the inventive method is few, and required expense is low, can reduce expenses.
Description of drawings
Anti-tartaric acid phosphatase (TRAP+) groupization of osteoclast that the different culture systems of Fig. 1 obtain is photo as a result.(A, B, C, D, E, embodiment 1 is seen in the F grouping)
Mature osteoclast quantity that the different culture systems of Fig. 2 obtain and incubation time concern synoptic diagram.(A, B, C, D, E, embodiment 1 is seen in the F grouping))
The ratio column diagram of Fig. 3 mature osteoclast in the TRAP+ cell.(A, B, C, D, E, embodiment 1 is seen in the F grouping)
The bone resorption lacuna photo that Fig. 4 mature osteoclast forms on the ivory osteocomma.(figure A (D group) and figure B (F group))
Embodiment
Embodiment 1
(1) separation and the cultivation of mouse bony process matter mescenchymal stem cell
Obtain (Stem Cells.2006 Apr such as mescenchymal stem cell (patent No. ZL 200510055261.4) and Guo Z according to inventor's patent separation and Culture in the mouse bony process matter; 24 (4): 992-1000) disclosed method is carried out the separation and the cultivation of mouse bony process matter mescenchymal stem cell, healthy C57BL/6 female mice (providing by military medicine science animal center) in 2 ages in week is provided, taking off neck puts to death, 75% after alcohol-pickled 5 minutes, aseptic condition separates mouse femur and shin bone down, go clean surface structure, (weightmeasurement ratio is: sodium-chlor 8g/L to draw the phosphate buffered saline buffer that contains 10% volume foetal calf serum (buying from Hyclone company) with syringe, Sodium phosphate dibasic 2.9g/L, Repone K 0.2g/L, potassium primary phosphate 0.24g/L, solvent are deionized water) wash down medullary space, carefully shred for size be 2mm
2Tiny osteocomma, put concentration expressed in percentage by weight and be 0.15%, solvent is to contain two Collagen Type VI enzymes (buying from the Sigma company) solution of a-MEM substratum of 15% volume foetal calf serum, at 37 ℃, digest and plant osteocomma after 1 hour in containing 2mmol/L glutamine (buying) from Sigma company, in the a-MEM substratum of 10% volume foetal calf serum (buying), be 37 ℃, 5%CO from Hyclone company
2Condition is changed liquid after following 72 hours first, removes suspension cell, keeps osteocomma, and the cell that grows from osteocomma covers with had digestive transfer culture behind the 80% culturing bottle area, continuous passage, get the 4th generation cell do to cultivate and use.
(2) with step (1) mouse bony process matter derived mesenchymal stem cell kind in 48 porocyte culture plates (buying) from Corning company, each single hole area 0.8cm
2, 2000 or 10000 mouse bony process matter of every hole kind derived mesenchymal stem cell, every hole adds 500 μ l and contains the 2mmol/L glutamine, in the a-MEM substratum of 10% volume foetal calf serum, at 37 ℃, 5%CO
2Cultivated 24 hours under the condition.
(3) the mouse spleen mononuclearcell separates
Healthy C57BL/6 female mice (being provided by Military Medical Science Institute's Experimental Animal Center) in 2 ages in week is provided, and the strict respect of all experimentation on animalies of the present invention military medicine scientific experiment animal guide carries out.Mouse is taken off neck put to death, 75% after alcohol-pickled 5 minutes, and aseptic condition takes out spleen down, mills on 200 mesh filter screens with the glass syringe handle and produces single cell suspension.4 milliliters of mouse lymphocyte parting liquids (Tianjin Hao ocean biology buy) are in vitro, draw 4 milliliters of careful test tubes that add of spleen single cell suspension, note keeping the interface complete, centrifugal 15 minutes with 1500 rev/mins, draw the mononuclearcell layer, add phosphate buffered saline buffer again with 1000 rev/mins of washings twice in centrifugal 10 minutes, cell counting is continued to employ.
(4) spleen mononuclearcell kind is in culture plate
The mescenchymal stem cell bed board in mouse bony process matter source is after 24 hours, and it is adherent good to examine under a microscope mescenchymal stem cell, and the single hole of sucking-off contains the 2mmol/L glutamine, and the a-MEM substratum 500ul of 10% volume foetal calf serum abandons it.To contain the 2mmol/L glutamine, 10% volume foetal calf serum, recombined small-mouse macrophage stimulation factor 10ng/ml or 20ng/ml, the resuspended spleen mononuclearcell of a-MEM substratum of sub-ligand 1 0ng/ml of recombined small-mouse nf κ B receptor activation or 20ng/ml, density according to 2000 spleen mononuclearcells of mononuclearcell in each 48 well culture plate single hole, substratum 500ul plants spleen mononuclearcell down.Be divided into 6 groups, be respectively:
A group: Sp MNC (2000/hole)+M-CSF (10ng/ml)+RANKL (10ng/ml);
B group: Sp MNC (2000/hole)+MSC (2000/hole)+M-CSF (10ng/ml)+RANKL (10ng/ml);
C group: Sp MNC (2000/hole)+MSC (10000/hole)+M-CSF (10ng/ml)+RANKL (10ng/ml);
D group: Sp MNC (2000/hole)+M-CSF (20ng/ml)+RANKL (20ng/ml);
E group: Sp MNC (2000/hole)+MSC (2000/hole)+M-CSF (20ng/ml)+RANKL (20ng/ml);
F group: Sp MNC (2000/hole)+MSC (10000/hole)+M-CSF (20ng/ml)+RANKL (20ng/ml);
(annotate: Sp MNC: mouse spleen mononuclearcell, MSC: the mescenchymal stem cell in mouse bony process matter source, M-CSF: reorganization macrophage colony stimulating factor, RANKL: the sub-part of recombined small-mouse nf κ B receptor activation).
Establish altogether 3 days, 6 days, 9 days, 12 days, 15 days five detection time point, every group of each detection time, point was all established 3 multiple holes.At 37 ℃, 5%CO
2Cultivate under the condition, half amount is changed liquid every three days, can get osteoclast.
Embodiment 2 anti-tartaric acid phosphatase enzymatic determinations
Respectively at the 3rd day that cultivates, the 6th day, the 9th day, the 12nd day and the 15th day, according to the operating recommendation that anti-tartaric acid phosphatase enzymatic determination test kit (Sigma-Aldrich product) specification sheets provides the cell in each group culture hole is dyeed, every group is dyed 3 holes at every turn, the cell of burgundy look is judged to TRAP (+) cell, 〉=3 above TRAP of nucleus (+) cell is judged to be mature osteoclast, opticmicroscope is observed form down and is taken pictures, and respectively TRAP (+) cell and mature osteoclast counting is carried out statistical analysis.
The osteoclast that the osteoclast of (see figure 1) the inventive method acquisition as a result and cytokine method obtain compares, and the former form is very active, and nucleus can reach tens, and cell space is huge, anti-tartaric acid phosphatase engrain.
The earliest time that the inventive method obtains mature osteoclast is 3 days, and early than the earliest time that mature osteoclast in the cytokine method occurs, the latter is 6 days (see figure 2)s;
The absolute quantity of the mature osteoclast that the inventive method obtains is cultivated the absolute quantity of the mature osteoclast of osteoclast method acquisition more than cytokine, respectively to mature osteoclast between A group and B group, A group and C group, D group and E group, D group and the F group the 6th, 9,12,15 days absolute quantity is carried out statistics T test, each organizes P＜0.05 (seeing Fig. 2, table 1);
The results of statistical analysis of mature osteoclast quantity variance between each group of table 1
| P value between each |
6 |
9 |
12 |
15 days |
| A and B | 0.0438144541 | 0.0038182206 | 0.0001827896 | 0.0023720557 |
| A and C | 0.0000019208 | 0.0001425134 | 0.0000386341 | 0.0000183754 |
| D and E | 0.0092616968 | 0.0020274883 | 0.0000167075 | 0.0203998971 |
| D and F | 0.0000009997 | 0.0000110412 | 0.0000119039 | 0.0000172452 |
The ratio of mature osteoclast in TRAP (+) cell that (see figure 3) the present invention as a result cultivates the method acquisition of osteoclast is higher than the ratio of mature osteoclast in TRAP (+) cell that cytokine is cultivated the acquisition of osteoclast method.
With the about 15mm of diameter, the ivory film of thickness 3 μ m (professor Jin Weifang of Medical Center of Fudan University provides), 75% alcohol-pickled two hours, uviolizing sterilization in 12 hours in the super clean bench, according to above cultivation grouping, at the bottom of before the D group is planted mononuclearcell, placing 24 orifice bores in advance, at the bottom of placing 24 orifice bores in advance before the mescenchymal stem cell of F group shop, plant successively again and go up mononuclearcell or mescenchymal stem cell and mononuclearcell.Took out ivory film at the 15 day, form situation and take pictures with toluidine blue dyeing observation ivory film surface lacuna under opticmicroscope.
The bone resorption lacuna (seeing Fig. 4 A) that the mature osteoclast that the mature osteoclast of the inventive method acquisition as a result obtains than cytokine cultivation osteoclast method at the bone resorption lacuna that forms on the ivory film (seeing Fig. 4 B) forms on ivory film is big, confirm that the osteoclast that the present invention obtains has the bone resorption ability, and function is better than the osteoclast that the cytokine method obtains.
Claims (5)
1. a method of utilizing mesenchyma stem cell combined cytokine to cultivate osteoclast comprises the steps:
(1) separate in the mouse bony process matter, cultivate mescenchymal stem cell, and cultivations of going down to posterity, the cultivation of will going down to posterity then the 4th～6 generation mescenchymal stem cell be laid in the culture plate, at 35～38 ℃, 5%CO
2Cultivated under the condition 12～24 hours;
(2) separate mononuclearcell from mouse spleen, and plant in the culture plate of step (1);
(3) the substratum co-cultivation of interpolation factor-containing in the culture plate in step (2), half amount is changed liquid every three days, at 35～38 ℃, 5%CO
2Cultivated under the condition 3～15 days, and can obtain osteoclast;
Wherein, the described substratum of step (3) is for containing 1～2mmol/L glutamine, 10～15% volume foetal calf serums, 10～20ng/ml recombined small-mouse macrophage stimulation factor, the α-MEM substratum of the sub-part of 10～20ng/ml recombined small-mouse nf κ B receptor activation.
2. in accordance with the method for claim 1, it is characterized in that the mescenchymal stem cell quantity that step (1) is laid in the culture plate is 2000～10000/hole.
3. according to claim 1 or 2 described methods, it is characterized in that it was 2～3 ages in week that above-mentioned steps (2) is separated the used mouse of mouse spleen mononuclearcell.
4. in accordance with the method for claim 3, it is characterized in that the mouse spleen mononuclearcell that above-mentioned steps (2) is planted is 1800～2200/hole.
5. in accordance with the method for claim 4, it is characterized in that used culture plate can be 48 well culture plates.
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| CN102978155B (en) * | 2012-11-16 | 2015-02-04 | 哈尔滨医科大学 | Osteoclast culturing kit and preparation method and application thereof |
| CN103667182B (en) * | 2012-12-17 | 2015-11-25 | 湖州市中心医院 | A kind of mesenchymal stem cells MSCs is in vitro to induction method and the inducing culture of osteoblast differentiation |
| CN104651302A (en) * | 2015-02-09 | 2015-05-27 | 苏州大学附属第一医院 | Method for extracting myelomonocyte and differentiating to osteoclast |
| CN105505864A (en) * | 2016-01-30 | 2016-04-20 | 苏州普罗达生物科技有限公司 | Culture medium for culturing osteoclasts in vitro |
| CN109694850A (en) * | 2017-10-20 | 2019-04-30 | 许健 | A kind of induced rat mesenchymal stem cells differentiation at Osteoclast-like cells method |
| CN108753701B (en) * | 2018-05-04 | 2021-10-29 | 南京医科大学附属口腔医院 | Method for researching regulation mechanism of intracellular transport protein 80 in maxilla rapid enlargement machinery biological signal transduction |
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