CN102041245A - Kit for inducing differentiation from bone mesenchymal stem cells to osteoblasts, application of kit and method for inducing cell differentiation - Google Patents
Kit for inducing differentiation from bone mesenchymal stem cells to osteoblasts, application of kit and method for inducing cell differentiation Download PDFInfo
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Abstract
The invention provides a kit and method for inducing differentiation from bone mesenchymal stem cells to osteoblasts. The kit comprises an osteoblast inducing culture solution and an osteoblast identification solution, wherein the inducing culture solution comprises a cell culture solution A, a cell culture solution B, a cell culture solution C, a cell culture solution D and a cell culture solution E; the osteoblast identification solution comprises a cell immobilizing solution, coloring agents and a detergent; the cell culture solution A is an alpha-minimum essential medium (MEM) liquid culture medium; the cell culture solution B is fetal bovine serum; the cell culture solution C is a dexamethasone solution; the cell culture solution D is a beta-sodium glycerophosphate solution; the cell culture solution E is a vitamin C solution; the cell immobilizing solution is 4% paraformaldehyde; the coloring agents include sodium thiosulfate, silver nitrate and neutral red; and the detergent is double distilled water.
Description
Technical field
The present invention relates to a kind of test kit of inducing cell differentiation and the method for application and inducing cell differentiation thereof, particularly, relate to a kind of test kit of inducing bone mesenchymal stem cell to osteoblast direction differentiation and the method for application and the differentiation of inducing bone mesenchymal stem cell to osteoblast direction thereof.
Background technology
Mesenchymal stem cells MSCs is the adult stem cell that a class has multidirectional differentiation potential, can be divided into multiple histocytes such as scleroblast, chondrocyte, scleroblast, neurocyte, liver cell, just be applied to fields such as organizational project, gene therapy, cytokine replacement therapy clinically.The superiority that mesenchymal stem cells MSCs is difficult to compare with other stem cells becomes the ideal source of bone tissue engineer seed cell.
The major cause of fracture back disunion or delayed union is for lacking the scleroblast of sufficient amount.Utilize the autologous bone marrow cell to carry out the external evoked skeletonization that is divided into, expanded cells is transplanted to fracture, promote union of fracture, can shorten healing time.
Before mesenchymal stem cells MSCs was induced to differentiate in the osteoblastic research, inductor includes multiple materials such as newborn calf serum, penicillin, Streptomycin sulphate, glutamine, dexamethasone, sodium, vitamins C, low sugar DMEM, not only increase the complicacy of operation, and easily mesenchymal stem cells MSCs is caused interference, influence the conversion of bone mesenchymal stem cell to osteoblast.
Summary of the invention
Be induced to differentiate into osteoblastic problem in order to solve mesenchymal stem cells MSCs, according to an aspect of the present invention, the invention provides a kind of test kit of inducing cell differentiation, it is used for the differentiation of inducing bone mesenchymal stem cell to osteoblast.Further, this test kit can be used for being carried out to the osteocyte evaluation.
According to an aspect of the present invention, the present invention also provides described test kit in the application that rat bone marrow mesenchymal stem cells is induced to differentiate in the adipocyte.
According to an aspect of the present invention, the present invention also provides a kind of method of inducing cell differentiation, adopts this method can induce rat bone marrow mesenchymal stem cells to break up to the scleroblast direction.Further, adopting this method can be carried out to osteocyte identifies.
In the present invention, a kind of test kit of inducing cell differentiation comprises scleroblast inducing culture liquid, and this scleroblast inducing culture liquid comprises 0.1mM dexamethasone solution, 50mM vitamin c solution and foetal calf serum.
Described scleroblast inducing culture liquid also further comprises 1M sodium solution, and α-MEM liquid nutrient medium is as the solvent and the constant volume agent of above-mentioned materials.
In the present invention, described adipocyte inducing culture liquid can be the mixed solution of each component, also the independent package storage of each component can be faced the time spent mixed preparing.
Scleroblast inducing culture liquid main component of the present invention comprises dexamethasone, sodium and vitamins C.Dexamethasone can the inducing bone mesenchymal stem cell alkaline phosphatase activities, promote the expression of skeletonization labelled protein, rhIGF-1 and collagen mrna, the biosynthesizing of the outer collagen mechanism of irritation cell, the deposition of calcium and mineralising tubercle form, but the dexamethasone of high density can induce it to break up to adipocyte.We select to use the dexamethasone solution that concentration is 0.1mM, have both guaranteed that it stimulates the characteristic of alkaline phosphatase activities, prevented it again and breaks up to the adipocyte direction.Sodium can provide phosphate ion for scleroblast, thereby quickens the tubercle calcification.Ascorbic effect mainly promotes cell to synthesize collagen, forms calcification.Utilize scleroblast inducing culture liquid of the present invention to carry out the osteogenic induction differentiation of mesenchymal stem cells MSCs, can make things convenient for, effectively induce required scleroblast.
In an embodiment, described scleroblast inducing culture liquid comprises cell culture fluid A, cell culture fluid B, cell culture fluid C, cell culture fluid D, cell culture fluid E.Wherein, described cell culture fluid A is α-MEM liquid nutrient medium, and cell culture fluid B is a foetal calf serum, and cell culture fluid C is the 0.1mM dexamethasone solution, described cell culture fluid D is a 1M sodium solution, and described cell culture fluid E is the 50mM vitamin c solution.
Each component can preferably be prepared according to following ratio in the described scleroblast inducing culture liquid: 0.1mM dexamethasone solution 5 μ l～15 μ l, 1M sodium solution 0.5ml～1.5ml, 50mM vitamin c solution 80 μ l～120 μ l, foetal calf serum 5ml～15ml adds α-MEM liquid nutrient medium constant volume to 100ml.
In a preferred embodiment, used scleroblast inducing cell nutrient solution component ratio is as follows in the experiment: 0.1mM dexamethasone solution 10 μ l, 1M sodium solution 1ml, 50mM vitamin c solution 100 μ l, foetal calf serum 10ml adds α-MEM liquid nutrient medium constant volume to 100ml.Adopt this allocation ratio, can effectively mesenchymal stem cells MSCs be induced to differentiate into scleroblast.
Test kit of the present invention also can further comprise scleroblast evaluation liquid, and this scleroblast identifies that liquid comprises cell fixation liquid, staining agent and washing composition.Described scleroblast identifies that liquid comprises cell fixation liquid, staining agent and washing composition.Wherein, described cell fixation agent is 4% paraformaldehyde solution, and described cell dyeing liquid is that staining agent can be 5% hypo solution, 1% silver nitrate solution and 1% neutral red solution, and described cell washing agent can be distilled water.
Scleroblast of the present invention identifies that the liquid main component comprises cell fixation liquid, staining agent and washing composition.4% paraformaldehyde solution mainly plays the fixed cell effect.Mainly is to prevent to infiltrate ion component with distilled water as the cell washing agent.Stain for cell mainly comprises 5% hypo solution, 1% silver nitrate solution and 1% neutral red solution, because mesenchymal stem cells MSCs produces a large amount of acidic substance in the induced osteogenesis process, utilize hypo solution can remove hydrogen ion, add silver nitrate solution and hypo solution this moment respectively, argent is better displayed, confirmed the nodular existence of calcium indirectly, toluylene red mainly is to embody the constitutional features of cell.
The method of inducing cell differentiation provided by the present invention may further comprise the steps:
A, rat bone marrow mesenchymal stem cells obtains and increases;
B, described rat bone marrow mesenchymal stem cells changed in the scleroblast inducing culture liquid cultivated for 3～5 weeks, described scleroblast inducing culture liquid comprises that the volume in described scleroblast inducing culture liquid is 100ml, the vitamins C of the sodium of 300-330mg, 850～910 μ g and an amount of liquid nutrient medium.
Aforesaid method can further may further comprise the steps:
C, discard described scleroblast inducing culture liquid, after the stationary liquid fixed cell, add staining agent and hatch;
D, adding cell washing agent flushing, dyeing then;
The scleroblast after breaking up is observed in dyeing again after e, the adding cell washing agent flushing after the distilled water rinsing.
In steps d and e, described cell washing agent can be distilled water; In step c, described staining agent can be 5% hypo solution; In steps d, can adopt 1% silver nitrate solution to dye; In step e, can adopt 1% neutral red solution to dye.
The invention provides a kind of test kit of inducing bone mesenchymal stem cell to osteoblast differentiation, it mainly comprises scleroblast inducing culture liquid and scleroblast evaluation liquid.
The technical solution adopted in the present invention is: after utilizing the test kit for preparing mesenchymal stem cells MSCs fast to obtain a large amount of cells, add scleroblast inducing culture liquid, after external evoked 4 weeks, utilize scleroblast evaluation liquid that scleroblast is identified.
The present invention carries out inducing bone mesenchymal stem cell to osteoblast process of differentiation and comprises:
1, former generation separation and Culture and the amplification of going down to posterity of mesenchymal stem cells MSCs:
Utilize washings in medullary space, to go out in cell, utilize the test kit for preparing mesenchymal stem cells MSCs fast to obtain a large amount of cells, be the mesenchymal stem cells MSCs of fusiformis for cellular form.
2, inducing bone mesenchymal stem cell to osteoblast differentiation:
The nutrient solution of reject culturing bottle fully cleans cell with washings, is passaged in the culture dish, adds scleroblast inducing culture liquid, changes liquid weekly 2 times, is placed on 37 ℃ interior 4 weeks of cultured continuously of carbonic acid gas incubator.
3, osteoblastic evaluation:
In incubator, take out the Tissue Culture Dish of inducing for 4 weeks, discard cell induction liquid, 4% Paraformaldehyde 96 is fixed, 5% Sulfothiorine was hatched 30 minutes, and the distilled water flushing was dyeed 15 minutes down with 1% silver nitrate solution high light, distilled water rinses out the atrament that floats on cell surface, the hypo solution dyeing of continuation 5% 2 minutes, 1% toluylene red was redyed 10 minutes, observed after the distilled water rinsing.
The invention has the beneficial effects as follows: can utilize this test kit and method, mesenchymal stem cells MSCs effectively is induced to differentiate into scleroblast, both confirm the differentiation capability of mesenchymal stem cells MSCs, can obtain a large amount of scleroblasts again, be beneficial to the reparation that scleroblast lacks.
In order to understand essence of the present invention better, by description, describe in detail but do not limit the present invention better embodiment of the present invention below in conjunction with accompanying drawing.
Description of drawings
Fig. 1: cell observation figure behind mesenchymal stem cells MSCs adding scleroblast 4 week of the induced liquid back adding scleroblasts evaluation liquid.
Fig. 2: cell observation figure behind mesenchymal stem cells MSCs adding ordinary cells 4 week of the nutrient solution back adding scleroblasts evaluation liquid.
Embodiment
Further specify essentiality content of the present invention below in conjunction with the invention process, but do not limit the present invention with this.The used test materials of the present invention if no special instructions, is commercially available purchase product.
One, preparation is used for the test kit of inducing bone mesenchymal stem cell to osteoblast differentiation
(1) solution preparation
The test kit that the present invention is used to prepare mesenchymal stem cells MSCs comprises following preparation:
1, scleroblast inducing culture liquid: comprise cell culture fluid A, cell culture fluid B, cell culture fluid C, cell culture fluid D, cell culture fluid E.Wherein, described cell culture fluid A is α-MEM liquid nutrient medium, and cell culture fluid B is a foetal calf serum, and cell culture fluid C is the 0.1mM dexamethasone solution, described cell culture fluid D is a 1M sodium solution, and described cell culture fluid E is the 50mM vitamin c solution.
Scleroblast inducing cell nutrient solution component ratio is as follows: 0.1mM dexamethasone solution 10 μ l, and 1M sodium solution 1ml, 50mM vitamin c solution 100 μ l, foetal calf serum 10ml adds α-MEM liquid nutrient medium constant volume to 100ml.
2, scleroblast is identified liquid: described scleroblast identifies that liquid comprises cell fixation liquid, staining agent and washing composition.Wherein, described cell fixation agent is 4% paraformaldehyde solution, and described cell dyeing liquid is that staining agent is 5% hypo solution, 1% silver nitrate solution and 1% neutral red solution, and described cell washing agent is a distilled water.
(2) packing of test kit
The specification of test kit is 1 a time/box, the amount of each component in every box: 0.1mM dexamethasone solution 1 pipe (10 μ l/ pipe), 1M sodium solution 1 pipe (1ml/ pipe), 50mM vitamin c solution 1 pipe (100 μ l/ pipe), 1 bottle of foetal calf serum (10ml/ bottle), α-1 bottle of MEM liquid nutrient medium (88.89ml/ bottle), 1 bottle of 4% paraformaldehyde solution (20ml/ bottle), 1 bottle of 5% hypo solution (20ml/ bottle), 1 bottle of 1% silver nitrate solution (20ml/ bottle), 1 bottle of 1% neutral red solution (20ml/ bottle), 1 bottle of distilled water (20ml/ bottle).By above-mentioned dosage explanation each component in the test kit is packed, can obtain the test kit of inducing bone mesenchymal stem cell to osteoblast differentiation.
Two, bone mesenchymal stem cell to osteoblast is induced differentiation
Utilize test kit described in the step 1 to carry out bone mesenchymal stem cell to osteoblast and induce differentiation.
(1) former being commissioned to train of mesenchymal stem cells MSCs supported and the amplification of going down to posterity:
Clean 1 of level SD rat 4 ages in week, the cervical vertebra dislocation is put to death, utilize the test kit (mainly comprising phosphate buffered saline buffer, α-MEM liquid nutrient medium, foetal calf serum, trypsin solution) for preparing mesenchymal stem cells MSCs fast to cultivate, obtain the available mesenchymal stem cells MSCs in a short time.
(2) inducing bone mesenchymal stem cell to osteoblast differentiation:
The nutrient solution of reject culturing bottle fully cleans cell with washings, is passaged in the culture dish, adds scleroblast inducing culture liquid, changes liquid weekly 2 times, is placed on 37 ℃ interior 4 weeks of cultured continuously of carbonic acid gas incubator.Utilize under the same terms the mesenchymal stem cells MSCs that obtains in addition, add common nutrient solution, change liquid weekly 2 times, be placed in 37 ℃ the carbonic acid gas incubator 4 weeks of cultured continuously in contrast.
(3) osteoblastic evaluation:
In incubator, take out the Tissue Culture Dish of inducing for 4 weeks, discard cell induction liquid, 4% Paraformaldehyde 96 is fixed, 5% Sulfothiorine was hatched 30 minutes, and the distilled water flushing was dyeed 15 minutes down with 1% silver nitrate solution high light, distilled water rinses out the atrament that floats on cell surface, the hypo solution dyeing of continuation 5% 2 minutes, 1% toluylene red was redyed 10 minutes, observed after the distilled water rinsing.
Microscopically is observed, and cellular form is changed into polygon by fusiformis, and cell is multilayer, plyability is arranged, and matter includes the sedimentary calcification tubercle of a large amount of rock salts, staining agent stained positive (Fig. 1) between formation.Control group dyeing negative (Fig. 2).
More than the description of better embodiment of the present invention is not limited the present invention, those skilled in the art can make various changes or distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the scope of claims of the present invention.
Claims (10)
1. the test kit of an inducing cell differentiation is used for mesenchymal stem cells MSCs is induced to differentiate into scleroblast, comprises scleroblast inducing culture liquid, and this scleroblast inducing culture liquid comprises:
0.1mM dexamethasone solution;
The 50mM vitamin c solution; And
Foetal calf serum.
2. the described test kit of claim 1 is characterized in that, described scleroblast inducing culture liquid further comprises:
1M sodium solution; And
α-MEM liquid nutrient medium.
3. claim 1 or 2 described test kits, it is characterized in that, the component ratio of described scleroblast inducing culture liquid is: 0.1mM dexamethasone solution 5 μ l～15 μ l, 1M sodium solution 0.5ml～1.5ml, 50mM vitamin c solution 80 μ l～120 μ l, foetal calf serum 5ml～15ml adds α-MEM liquid nutrient medium constant volume to 100ml.
4. the described test kit of claim 3, it is characterized in that, the ratio of described scleroblast inducing culture fluid component is: 0.1mM dexamethasone solution 10 μ l, 1M sodium solution 1ml, 50mM vitamin c solution 100 μ l, foetal calf serum 10ml adds α-MEM liquid nutrient medium constant volume to 100ml.
5. the described test kit of claim 1 is characterized in that, described test kit further comprises scleroblast evaluation liquid, and this scleroblast identifies that liquid comprises cell fixation liquid, staining agent and cell washing agent, and wherein, described staining agent comprises:
Staining agent A:5% hypo solution,
Staining agent B:1% silver nitrate solution, and
Staining agent C:1% neutral red solution.
6. the described test kit of claim 5 is characterized in that, described cell fixation liquid is 4% paraformaldehyde solution, and described cell washing agent is a distilled water.
7. the described test kit of one of claim 1～6 is in the application that rat bone marrow mesenchymal stem cells is induced to differentiate in the scleroblast.
8. the method for an inducing cell differentiation is used for mesenchymal stem cells MSCs is induced to differentiate into scleroblast, may further comprise the steps:
A, mesenchymal stem cells MSCs obtains and increases;
B, described mesenchymal stem cells MSCs changed in the scleroblast inducing culture liquid cultivated for 3～5 weeks, described scleroblast inducing culture liquid comprises 0.1mM dexamethasone solution, 50mM vitamin c solution and foetal calf serum.
9. the described method of claim 8 is characterized in that, further may further comprise the steps:
C, discard described scleroblast inducing culture liquid, after the stationary liquid fixed cell, add staining agent and hatch;
D, adding cell washing agent flushing, dyeing then;
The scleroblast after breaking up is observed in dyeing again after e, the adding cell washing agent flushing after the distilled water rinsing.
10. the described method of claim 9 is characterized in that, described cell washing agent is a distilled water; In step c, described staining agent is 5% hypo solution; In steps d, adopt 1% silver nitrate solution to dye; In step e, adopt 1% neutral red solution to dye.
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| CN102311942A (en) * | 2011-07-26 | 2012-01-11 | 王松灵 | Vitamin C induced mesenchymal stem cell membrane and preparation method thereof |
| CN103667182A (en) * | 2012-12-17 | 2014-03-26 | 湖州市中心医院 | Inducing method and inducing culture medium for differentiation of bone marrow mesenchymal stem cells into osteoblasts in vitro |
| CN105802906A (en) * | 2016-05-27 | 2016-07-27 | 中国医学科学院医学生物学研究所 | Culture medium for osteogenic induced differentiation of mesenchymal stem cells of tupaia belangeri and preparation method thereof and application |
| CN105886461A (en) * | 2016-04-14 | 2016-08-24 | 广州赛莱拉干细胞科技股份有限公司 | Method for osteogenic differentiation of bone mesenchymal stem cells |
| CN106434539A (en) * | 2016-09-30 | 2017-02-22 | 广州赛莱拉干细胞科技股份有限公司 | Osteogenic induction medium and osteogenic differentiation method |
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| CN102311942A (en) * | 2011-07-26 | 2012-01-11 | 王松灵 | Vitamin C induced mesenchymal stem cell membrane and preparation method thereof |
| CN103667182A (en) * | 2012-12-17 | 2014-03-26 | 湖州市中心医院 | Inducing method and inducing culture medium for differentiation of bone marrow mesenchymal stem cells into osteoblasts in vitro |
| 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 |
| CN105886461A (en) * | 2016-04-14 | 2016-08-24 | 广州赛莱拉干细胞科技股份有限公司 | Method for osteogenic differentiation of bone mesenchymal stem cells |
| CN105802906A (en) * | 2016-05-27 | 2016-07-27 | 中国医学科学院医学生物学研究所 | Culture medium for osteogenic induced differentiation of mesenchymal stem cells of tupaia belangeri and preparation method thereof and application |
| CN106434539A (en) * | 2016-09-30 | 2017-02-22 | 广州赛莱拉干细胞科技股份有限公司 | Osteogenic induction medium and osteogenic differentiation method |
| CN110423722A (en) * | 2019-09-03 | 2019-11-08 | 广州赛莱拉干细胞科技股份有限公司 | A kind of culture medium and its application and induction method of the tendon stem cell to bone cell differentiation |
| CN112094807A (en) * | 2020-09-18 | 2020-12-18 | 四川省恩乐生物工程有限公司 | Differentiation culture method for directionally inducing mesenchymal stem cells |
| CN112094807B (en) * | 2020-09-18 | 2023-04-25 | 四川省恩乐生物工程有限公司 | Differentiation culture method for directional induction of bone marrow mesenchymal stem cells |
| CN112831463A (en) * | 2021-01-27 | 2021-05-25 | 中国人民解放军军事科学院军事医学研究院 | Culture medium for efficiently inducing adipose-derived stem cell bone differentiation, preparation method, induced differentiation method and application |
| CN112831463B (en) * | 2021-01-27 | 2022-08-23 | 中国人民解放军军事科学院军事医学研究院 | Culture medium for efficiently inducing adipose-derived stem cell bone differentiation, preparation method, induced differentiation method and application |
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Application publication date: 20110504 |