CN103361307A - Induced differentiation method of C3H10T1/2 mesodermal multipotential embryonic stem cell stain - Google Patents
Induced differentiation method of C3H10T1/2 mesodermal multipotential embryonic stem cell stain Download PDFInfo
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Abstract
The invention relates to an induced differentiation method of cells, and in particular relates to an induced differentiation method of a C3H10T1/2 mesodermal multipotential embryonic stem cell stain. The induced differentiation method comprises the following steps of: culturing C3H10T1/2 cells in a culture solution, adding 5 mL of 0.25% pancreatin when cells are fused 80%-90%, and terminating trypsinization reaction when the cells are contracted into circles; centrifuging for 3-5 minutes, precipitating, abandoning the supernatant, and adding the culture solution and then performing blowing and beating; culturing onto a 6-hole plate or a 12-hole plate for continuous cell culture in a ratio of 1: 4, and changing the culture solution every 2-4 days; after cells fully grow on the culture plate, putting the culture plate in induction solution A, and replacing the induction solution A with induction solution B two days later, and further replacing the induction solution B with the culture solution two days later; and changing the culture solution every 2-4 days. Adipose cells induced and differentiated by the method provided by the invention can be well applied to subsequent environments.
Description
Technical field
The present invention relates to the method for inducing differentiation of cell, particularly the method for inducing differentiation of a kind of C3H10T1/2 mesoderm pluripotency embryonic stem cell strain.
Background technology
Along with the continuing to increase of the raising of economic level, life stress, the change of people's dietary structure, momental minimizing, the morbidity of the disease of causeing fat rises rapidly, and the overweight or population of being obese in the whole world approximately reaches 1,000,000,000 at present according to estimates.Obesity is as one of main ingredient of metabolism syndrome, and is closely related with various diseases such as diabetes B, hyperlipemia, hypertension, coronary heart disease, and the serious harm mankind's health affects and perplexing daily life.Recent study is found, autophagy is playing an important role aspect the lipid metabolism of liver and fatty tissue, autophagy is one and engulfs self cytoplasm protein or organoid and make its coated vesica that enters, and merge to form the autophagy lysosome with lysosome, the degrade process of its content that wraps up, the metabolism that realizes by this cell itself need and the renewal of some organoid.So the autophagy phenomenon provides new thinking for the treatment of obesity.
Berberine is a kind of isoquinoline alkaloid, it is the main component of Chinese Drug Rhizomes of Coptis, former studies has confirmed that Berberine has a lot of pharmacological actions and comprises antibiotic, antitumor, expression that can inflammation-inhibiting factor TNF-α, IL-6 etc. especially has obvious anti-inflammatory effect at the individuality of obesity, nearest research finds that Berberine can be hypoglycemic, improves Insulin Resistance.
C3H10T1/2 clone is in 1973, from 14 to 17 days C3H mouse embryonic cell is separated first, these cells show the inoblast form in culturing process, be similar to mescenchymal stem cell in function, if the C3H10T1/2 induced orientation is divided into front adipocyte, and then be divided into mature fat cell according to the induction scheme of standard, show the characteristic of fat, and then use the C3H10T1/2 mature fat cell and study Berberine to the impact of autophagy, autophagy is to the regulation and control of lipid metabolism in the monitoring mature fat cell, for the treatment of obesity provides new drug target and methods for the treatment of.
Summary of the invention
The method of inducing differentiation that the purpose of this invention is to provide the strain of a kind of C3H10T1/2 mesoderm pluripotency embryonic stem cell, pass through the method, the C3H10T1/2 cell induction is divided into mature fat cell, show as the expression with the adipocyte marker protein of accumulating of triglyceride level, the C3H10T1/2 cell can be applied in the research experiment that follow-up Berberine suppresses C3H10T1/2 adipocyte autophagy aspect well, for the treatment that clinically can be more rational Berberine be applied to the obese patient provides foundation.
Purpose of the present invention can be achieved through the following technical solutions:
The method of inducing differentiation of a kind of C3H10T1/2 mesoderm pluripotency embryonic stem cell strain, its step comprises:
(1), cultivates and go down to posterity: the C3H10T1/2 cell cultures in nutrient solution, and is placed 5%CO
2Incubator in cultivate, after cell reaches 80%-90% and merges, add 0.25% pancreatin 5mL digestion, add nutrient solution when cellular contraction is rounded and stop the trysinization reaction, centrifugal 3-5 minute, precipitation, supernatant discarded are blown and beaten after adding nutrient solution again; Go down to posterity cultivation to 6 orifice plates or 12 orifice plates in the 1:4 ratio, place 5%CO
2Incubator in cultivate, changed nutrient solution 1 time in every 2-4 days.Preferably, changed nutrient solution 1 time in per 2 days.
(2), induce differentiation: treating that cell on the culture plate in the step (1) covers with is placed among the induced liquid A, remove induced liquid A after 2 days and change induced liquid B into, remove induced liquid B after 2 days and change nutrient solution into, changed nutrient solution 1 time in every 2-4 days, treating that 90% above cell is rounded and a large amount of fat occur drips, and represents that namely the C3H10T1/2 cell induces successfully.Preferably, changed nutrient solution 1 time in per 2 days.
Nutrient solution in described step (1) or the step (2) is to contain the DMEM nutrient solution that volume percent is 10%FBS.
In the described step (1), 5%CO
2The culture temperature of incubator be 35 ℃-40 ℃.Preferably, 5%CO
2The culture temperature of incubator be 37 ℃.
In the described step (2), induced liquid A contains the DMEM nutrient solution that volume percent is 10%FBS, also contain Regular Insulin 4-5 μ g/mL in this nutrient solution, 3-isobutyl-1-methylxanthine 0.5-0.7mmol/L, dexamethasone 1-2 μ mol/L, indomethacin 120-130nmol/L, triiodothyronine 1-2nmol/L, rosiglitazone 1-2 μ mol/L.Preferably, described induced liquid A contains the DMEM nutrient solution that volume percent is 10%FBS, also contain Regular Insulin 5 μ g/mL in this nutrient solution, 3-isobutyl-1-methylxanthine 0.5mmol/L, dexamethasone 1 μ mol/L, indomethacin 125nmol/L, triiodothyronine 1nmol/L, rosiglitazone 1 μ mol/L.
In the described step (2), induced liquid B contains the DMEM nutrient solution that volume percent is 10%FBS, goes back insulin-containing 5-10 μ g/ml in this nutrient solution, triiodothyronine 1-3nmol/L, rosiglitazone 1-3 μ mol/L.Preferably, described induced liquid B contains the DMEM nutrient solution that volume percent is 10%FBS, goes back insulin-containing 5 μ g/ml in this nutrient solution, triiodothyronine 1nmol/L, rosiglitazone 1 μ mol/L.
Be that the DMEM nutrient solution of 0.2%BSA was hatched 10-14 hour with the adipocyte of inducing in the step (2) with containing volume percent.Preferably, be that the DMEM nutrient solution of 0.2%BSA was hatched 12 hours with the adipocyte of inducing in the step (2) with containing volume percent.
The present invention has the following advantages:
1, the present invention at first successfully induces the strain of C3H10T1/2 mesoderm pluripotency embryonic stem cell to be divided into adipocyte, this ripe cell shows as the expression with the adipocyte marker protein of accumulating of triglyceride level, and the C3H10T1/2 cell can be applied in the follow-up experiment well.
2, the C3H10T1/2 cell that induces of the present invention can be used in the research experiment that Berberine suppresses C3H10T1/2 adipocyte autophagy aspect, for the more rational treatment that Berberine is used for the obese patient clinically provides experimental data and theoretical foundation.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
The reagent of using in the experiment: mesoblastic pluripotent stem cell strain C3H10T1/2 cell, available from U.S. ATCC (American Type Culture Collection).Dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, triiodothyronine and rosiglitazone are all available from U.S. Sigma company; Regular Insulin is available from Lilly Co., Eli.; Bovine serum albumin (Bovine serum albumin, BSA) is available from German Roche company; The 10%FBS/DMEM nutrient solution is available from U.S. Gibco BRL company.
Embodiment 1
The cultivation of C3H10T1/2 mesoderm pluripotency embryonic stem cell strain with go down to posterity:
The C3H10T1/2 cell routine is incubated at and contains in the DMEM nutrient solution that per-cent is 10%FBS, puts 5%CO
237 ℃ of incubators are hatched, after cell reaches the 80%-90% fusion, in super clean bench with transfer pipet with the nutrient solution sucking-off, add 0.25% pancreatin 5mL digestion, microscopically sees that cellular contraction is rounded, add and contain termination trysinization reaction in the DMEM nutrient solution that per-cent is 10%FBS, repeatedly blow and beat cell residual on bottle wall with transfer pipet, make cell detachment culturing bottle wall, suck and be equipped with in the centrifuge tube of nutrient solution, the centrifugal 3min of 1000rpm, sedimentation cell, centrifugal after, supernatant discarded, add again and contain the DMEM nutrient solution that per-cent is 10%FBS, with transfer pipet piping and druming, cell is scatter.Go down to posterity cultivation to 6 orifice plates or 12 orifice plates in the 1:4 ratio, put 5%CO
2Incubator is cultivated, and culture temperature is 37 ℃, then, changes nutrient solution (containing per-cent is the DMEM nutrient solution of 10%FBS) 1 time in per 2 days.
Embodiment 2
The strain of C3H10T1/2 mesoderm pluripotency embryonic stem cell induce differentiation:
Treating that cell on the culture plate covers with is placed on induced liquid A(and contains the DMEM nutrient solution that per-cent is 10%FBS, also contain Regular Insulin 5ug/ml in this nutrient solution, 3-isobutyl-1-methylxanthine 0.5mmol/L, dexamethasone 1 μ mol/L, indomethacin 125nmol/L, triiodothyronine 1nmol/L, rosiglitazone 1 μ mol/L), removing induced liquid A after 2 days changes induced liquid B(into and contains the DMEM nutrient solution that per-cent is 10%FBS, also contain Regular Insulin 5 μ g/ml in this nutrient solution, triiodothyronine 1nmol/L, rosiglitazone 1 μ mol/L), remove induced liquid B after 2 days and change into and contain the DMEM nutrient solution that per-cent is 10%FBS, change every other day later on nutrient solution one time.Treating that 90% above cell is rounded and a large amount of fat occur drips, and represents that namely the C3H10T1/2 cell induced successfully, becomes ripe adipocyte.Be that the DMEM of 0.2%BSA was hatched 12 hours with the adipocyte of inducing with containing volume percent, can carry out follow-up Berberine and suppress in the research experiment of C3H10T1/2 adipocyte autophagy aspect.
The above is preferred embodiment of the present invention, but the present invention should not be confined to the disclosed content of this embodiment.So everyly do not break away from the equivalence of finishing under the spirit disclosed in this invention or revise, all fall into the scope of protection of the invention.
Claims (8)
1. the method for inducing differentiation of C3H10T1/2 mesoderm pluripotency embryonic stem cell strain, its step comprises:
(1), cultivates and go down to posterity: the C3H10T1/2 cell cultures in nutrient solution, and is placed 5%CO
2Incubator in cultivate, after cell reaches 80%-90% and merges, add 0.25% pancreatin 5mL digestion, add nutrient solution when cellular contraction is rounded and stop the trysinization reaction, centrifugal 3-5 minute, precipitation, supernatant discarded are blown and beaten after adding nutrient solution again; Go down to posterity cultivation to 6 orifice plates or 12 orifice plates in the 1:4 ratio, place 5%CO
2Incubator in cultivate, changed nutrient solution 1 time in every 2-4 days;
(2), induce differentiation: treating that cell on the culture plate in the step (1) covers with is placed among the induced liquid A, remove induced liquid A after 2 days and change induced liquid B into, remove induced liquid B after 2 days and change nutrient solution into, changed nutrient solution 1 time in every 2-4 days, treating that 90% above cell is rounded and a large amount of fat occur drips, and represents that namely the C3H10T1/2 cell induces successfully.
2. the method for inducing differentiation of C3H10T1/2 mesoderm pluripotency embryonic stem cell according to claim 1 strain, it is characterized in that: the nutrient solution in described step (1) or the step (2) is to contain the DMEM nutrient solution that volume percent is 10%FBS.
3. the method for inducing differentiation of C3H10T1/2 mesoderm pluripotency embryonic stem cell according to claim 1 strain is characterized in that: in the described step (1), and 5%CO
2The culture temperature of incubator be 35 ℃-40 ℃.
4. the method for inducing differentiation of C3H10T1/2 mesoderm pluripotency embryonic stem cell according to claim 1 strain, it is characterized in that: in the described step (2), induced liquid A contains the DMEM nutrient solution that volume percent is 10%FBS, also contain Regular Insulin 4-5 μ g/mL in this nutrient solution, 3-isobutyl-1-methylxanthine 0.5-0.7mmol/L, dexamethasone 1-2 μ mol/L, indomethacin 120-130nmol/L, triiodothyronine 1-2nmol/L, rosiglitazone 1-2 μ mol/L.
5. the method for inducing differentiation of C3H10T1/2 mesoderm pluripotency embryonic stem cell according to claim 4 strain, it is characterized in that: described induced liquid A contains the DMEM nutrient solution that volume percent is 10%FBS, also contain Regular Insulin 5 μ g/mL in this nutrient solution, 3-isobutyl-1-methylxanthine 0.5mmol/L, dexamethasone 1 μ mol/L, indomethacin 125nmol/L, triiodothyronine 1nmol/L, rosiglitazone 1 μ mol/L.
6. the method for inducing differentiation of C3H10T1/2 mesoderm pluripotency embryonic stem cell according to claim 1 strain, it is characterized in that: in the described step (2), induced liquid B contains the DMEM nutrient solution that volume percent is 10%FBS, go back insulin-containing 5-10 μ g/ml in this nutrient solution, triiodothyronine 1-3nmol/L, rosiglitazone 1-3 μ mol/L.
7. the method for inducing differentiation of C3H10T1/2 mesoderm pluripotency embryonic stem cell according to claim 6 strain, it is characterized in that: described induced liquid B contains the DMEM nutrient solution that volume percent is 10%FBS, go back insulin-containing 5 μ g/ml in this nutrient solution, triiodothyronine 1nmol/L, rosiglitazone 1 μ mol/L.
8. the method for inducing differentiation of C3H10T1/2 mesoderm pluripotency embryonic stem cell according to claim 1 strain is characterized in that: be that the DMEM nutrient solution of 0.2%BSA was hatched 10-14 hour with the adipocyte of inducing in the step (2) with containing volume percent.
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| CN104651298A (en) * | 2013-11-21 | 2015-05-27 | 上海交通大学医学院附属第九人民医院 | Inducing agent and medium for inducing directional differentiation of embryonic stem cell into keratinocytes |
| CN109097324A (en) * | 2018-08-30 | 2018-12-28 | 丰泽康生物医药（深圳）有限公司 | A kind of cultural method for improving monocyte and being converted to multipotential cell |
| CN110747163A (en) * | 2019-11-13 | 2020-02-04 | 暨南大学 | Method for improving adipogenic differentiation of human adipose-derived mesenchymal stem cells and special culture medium thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110747163A (en) * | 2019-11-13 | 2020-02-04 | 暨南大学 | Method for improving adipogenic differentiation of human adipose-derived mesenchymal stem cells and special culture medium thereof |
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