CN103361307B - The method of inducing differentiation of a kind of C3H10T1/2 mesoderm pluripotency embryonic stem cell strain - Google Patents
The method of inducing differentiation of a kind of C3H10T1/2 mesoderm pluripotency embryonic stem cell strain Download PDFInfo
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Abstract
The present invention relates to the method for inducing differentiation of cell, in particular to the method for inducing differentiation of a kind of C3H10T1/2 mesoderm pluripotency embryonic stem cell strain, its step comprises: C3H10T1/2 cell cultures cultivated in nutrient solution, cell adds 0.25% pancreatin 5mL after reaching 80%-90% fusion to be digested, trysinization reaction is stopped when cellular contraction is rounded, centrifugal 3-5 minute, precipitation, supernatant discarded, then to blow and beat after adding nutrient solution; Cultivate to 6 orifice plates or 12 orifice plates in 1:4 ratio Secondary Culture, within every 2-4 days, change nutrient solution 1 time; The cell treating on culture plate covers with and is placed in induced liquid A, removes induced liquid A and changes induced liquid B into, remove induced liquid B and change nutrient solution into after 2 days after 2 days, within every 2-4 days, changes nutrient solution 1 time.The adipocyte of the differentiation-inducing one-tenth of the present invention, can apply in follow-up experiment well.
Description
Technical field
The present invention relates to the method for inducing differentiation of cell, the method for inducing differentiation of particularly a kind of C3H10T1/2 mesoderm pluripotency embryonic stem cell strain.
Background technology
Along with the raising of economic level, the continuing to increase of life stress, the change of people's dietary structure, momental minimizing, the morbidity of disease of causeing fat rises rapidly, and the overweight or population of being obese in the whole world about reaches 1,000,000,000 at present according to estimates.Obesity is as one of the main ingredient of metabolism syndrome, and as closely related in diabetes B, hyperlipemia, hypertension, coronary heart disease with various diseases, the health of the serious harm mankind, affects and annoying daily life.Recent study finds, autophagy plays an important role in the lipid metabolism of liver and fatty tissue, autophagy is one and engulfs own cells matter albumen or organoid and make it wrap and entered vesica, and form autophagy lysosome with lysosome fusion, degrade the process of its content wrapped up, the metabolism realizing cell itself by this needs and the renewal of some organoid.So autophagy phenomenon is that the treatment of obesity provides new thinking.
Berberine is a kind of isoquinoline alkaloid, it is the main component of Chinese Drug Rhizomes of Coptis, studied in the past and confirmed that Berberine had a lot of pharmacological action and comprises antibacterial, antitumor, the expression of the inflammation-inhibiting factor TNF-α, IL-6 etc. especially can there is 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, separate from the C3H mouse embryonic cell of 14 to 17 days first, these cells show inoblast form in culturing process, functionally be similar to mescenchymal stem cell, if C3H10T1/2 induced orientation is divided into PECTORAL LIMB SKELETON, and then be divided into mature fat cell according to the induction scheme of standard, show the characteristic of fat, and then apply C3H10T1/2 mature fat cell to study the impact of Berberine on autophagy, in monitoring mature fat cell, autophagy is to the regulation and control of lipid metabolism, treatment for obesity provides new drug target and methods for the treatment of.
Summary of the invention
The object of this invention is to provide the method for inducing differentiation of a kind of C3H10T1/2 mesoderm pluripotency embryonic stem cell strain, pass through the method, C3H10T1/2 cell induction is divided into mature fat cell, show as the accumulation of triglyceride level and the expression of adipocyte marker protein, making C3H10T1/2 cell can apply to follow-up Berberine well suppresses in the research experiment of C3H10T1/2 adipocyte autophagy aspect, for the treatment that more reasonably Berberine can be applied to obese patient clinically provides foundation.
Object of the present invention can be achieved through the following technical solutions:
A method of inducing differentiation for C3H10T1/2 mesoderm pluripotency embryonic stem cell strain, its step comprises:
(1), cultivate and go down to posterity: by C3H10T1/2 cell cultures in nutrient solution, and being placed in 5%CO
2incubator in cultivate, reach after 80%-90% merges until cell and add 0.25% pancreatin 5mL and digest, add nutrient solution when cellular contraction is rounded and stop trysinization reaction, centrifugal 3-5 minute, precipitate, supernatant discarded, then to blow and beat after adding nutrient solution; In 1:4 ratio Secondary Culture to 6 orifice plates or 12 orifice plates, be placed in 5%CO
2incubator in cultivate, within every 2-4 days, change nutrient solution 1 time.Preferably, within every 2 days, nutrient solution 1 time is changed.
(2), differentiation-inducing: the cell treating on the culture plate in step (1) covers with and is placed in 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, within every 2-4 days, change nutrient solution 1 time, treat that more than 90% cell is rounded and occur that a large amount of fat drips, namely representing that C3H10T1/2 cell has been induced successfully.Preferably, within every 2 days, nutrient solution 1 time is changed.
Nutrient solution in described step (1) or step (2) is be the DMEM nutrient solution of 10%FBS containing volume percent.
In described step (1), 5%CO
2the culture temperature of incubator be 35 DEG C-40 DEG C.Preferably, 5%CO
2the culture temperature of incubator be 37 DEG C.
In described step (2), induced liquid A is be the DMEM nutrient solution of 10%FBS containing volume percent, also containing Regular Insulin 4-5 μ g/mL in this nutrient solution, 3-isobutyl-1-methylxanthine 0.5-0.7mmol/L, dexamethasone 1-2 μm ol/L, indomethacin 120-130nmol/L, triiodothyronine 1-2nmol/L, rosiglitazone 1-2 μm ol/L.Preferably, described induced liquid A is be the DMEM nutrient solution of 10%FBS containing volume percent, also containing Regular Insulin 5 μ g/mL in this nutrient solution, 3-isobutyl-1-methylxanthine 0.5mmol/L, dexamethasone 1 μm of ol/L, indomethacin 125nmol/L, triiodothyronine 1nmol/L, rosiglitazone 1 μm of ol/L.
In described step (2), induced liquid B is be the DMEM nutrient solution of 10%FBS containing volume percent, goes back insulin-containing 5-10 μ g/ml, triiodothyronine 1-3nmol/L, rosiglitazone 1-3 μm ol/L in this nutrient solution.Preferably, described induced liquid B is be the DMEM nutrient solution of 10%FBS containing volume percent, goes back insulin-containing 5 μ g/ml, triiodothyronine 1nmol/L, rosiglitazone 1 μm of ol/L in this nutrient solution.
The adipocyte of having induced in step (2) the DMEM nutrient solution containing volume percent being 0.2%BSA is hatched 10-14 hour.Preferably, the adipocyte of having induced in step (2) the DMEM nutrient solution containing volume percent being 0.2%BSA is hatched 12 hours.
The present invention has the following advantages:
1, the present invention first successfully induces the strain of C3H10T1/2 mesoderm pluripotency embryonic stem cell to be divided into adipocyte, the cells show of this maturation is the accumulation of triglyceride level and the expression of adipocyte marker protein, and C3H10T1/2 cell can be applied in follow-up experiment well.
2, the C3H10T1/2 cell that the present invention induces can be used for Berberine and suppresses in the research experiment of C3H10T1/2 adipocyte autophagy aspect, for the treatment more reasonably Berberine being used for obese patient clinically provides experimental data and theoretical foundation.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
The reagent used in experiment: mesoblastic pluripotent stem cell strain C3H10T1/2 cell, purchased from American ATCC (American Type Culture Collection).Dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, triiodothyronine and rosiglitazone equal purchased from American Sigma company; Regular Insulin purchased from American Li Lai company; Bovine serum albumin (Bovine serum albumin, BSA) is purchased from German Roche company; 10%FBS/DMEM nutrient solution purchased from American Gibco BRL company.
Embodiment 1
The cultivation of C3H10T1/2 mesoderm pluripotency embryonic stem cell strain with go down to posterity:
It is in the DMEM nutrient solution of 10%FBS that C3H10T1/2 cell routine is incubated at containing per-cent, puts 5%CO
2, 37 DEG C of incubators are hatched, reach after 80%-90% fusion until cell, in super clean bench with transfer pipet by nutrient solution sucking-off, add 0.25% pancreatin 5mL to digest, see that cellular contraction is rounded under microscope, adding containing per-cent is stop trysinization reaction in the DMEM nutrient solution of 10%FBS, cell residual on bottle wall is is repeatedly blown and beaten 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, after centrifugal, supernatant discarded, adding containing per-cent is the DMEM nutrient solution of 10%FBS again, blow and beat with transfer pipet, cell dispersal is opened.In 1:4 ratio Secondary Culture to 6 orifice plates or 12 orifice plates, put 5%CO
2incubator is cultivated, and culture temperature is 37 DEG C, then, within every 2 days, changes nutrient solution (being the DMEM nutrient solution of 10%FBS containing per-cent) 1 time.
Embodiment 2
C3H10T1/2 mesoderm pluripotency embryonic stem cell strain differentiation-inducing:
The cell treating on culture plate covers with and is placed on induced liquid A(and contains the DMEM nutrient solution that per-cent is 10%FBS, also containing Regular Insulin 5ug/ml in this nutrient solution, 3-isobutyl-1-methylxanthine 0.5mmol/L, dexamethasone 1 μm of ol/L, indomethacin 125nmol/L, triiodothyronine 1nmol/L, rosiglitazone 1 μm of ol/L), remove induced liquid A after 2 days to change induced liquid B(into and contain the DMEM nutrient solution that per-cent is 10%FBS, also containing Regular Insulin 5 μ g/ml in this nutrient solution, triiodothyronine 1nmol/L, rosiglitazone 1 μm of ol/L), removing after 2 days that induced liquid B changes into containing per-cent is the DMEM nutrient solution of 10%FBS, change a nutrient solution every other day later.Treat that more than 90% cell is rounded and occur that a large amount of fat drips, namely representing that C3H10T1/2 cell has been induced successfully, become ripe adipocyte.The adipocyte of the having induced DMEM containing volume percent being 0.2%BSA is hatched 12 hours, follow-up Berberine can be carried out 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 content disclosed in this embodiment.The equivalence completed under not departing from spirit disclosed in this invention so every or amendment, all fall into the scope of protection of the invention.
Claims (4)
1. a method of inducing differentiation for C3H10T1/2 mesoderm pluripotency embryonic stem cell strain, its step comprises:
(1), cultivate and go down to posterity: by C3H10T1/2 cell cultures in nutrient solution, and being placed in 5%CO
2incubator in cultivate, reach after 80%-90% merges until cell and add 0.25% pancreatin 5mL and digest, add nutrient solution when cellular contraction is rounded and stop trysinization reaction, centrifugal 3-5 minute, precipitate, supernatant discarded, then to blow and beat after adding nutrient solution; In 1:4 ratio Secondary Culture to 6 orifice plates or 12 orifice plates, be placed in 5%CO
2incubator in cultivate, within every 2-4 days, change nutrient solution 1 time;
(2), differentiation-inducing: the cell treating on the culture plate in step (1) covers with and is placed in 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, within every 2-4 days, change nutrient solution 1 time, treat that more than 90% cell is rounded and occur that a large amount of fat drips, namely representing that C3H10T1/2 cell has been induced successfully;
Induced liquid A is be the DMEM nutrient solution of 10%FBS containing volume percent, also containing Regular Insulin 5 μ g/mL in this nutrient solution, 3-isobutyl-1-methylxanthine 0.5mmol/L, dexamethasone 1 μm of ol/L, indomethacin 125nmol/L, triiodothyronine 1nmol/L, rosiglitazone 1 μm of ol/L;
Described induced liquid B is be the DMEM nutrient solution of 10%FBS containing volume percent, insulin-containing 5 μ g/ml, triiodothyronine 1nmol/L in this nutrient solution, rosiglitazone 1 μm of ol/L.
2. the method for inducing differentiation of C3H10T1/2 mesoderm pluripotency embryonic stem cell according to claim 1 strain, is characterized in that: the nutrient solution in described step (1) or step (2) is be the DMEM nutrient solution of 10%FBS containing volume percent.
3. the method for inducing differentiation of C3H10T1/2 mesoderm pluripotency embryonic stem cell according to claim 1 strain, is characterized in that: in described step (1), 5%CO
2the culture temperature of incubator be 35 DEG C-40 DEG C.
4. the method for inducing differentiation of C3H10T1/2 mesoderm pluripotency embryonic stem cell according to claim 1 strain, is characterized in that: the C3H10T1/2 cell of having induced in step (2) the DMEM nutrient solution containing volume percent being 0.2%BSA is hatched 10-14 hour.
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| 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 |
| CN110747163B (en) * | 2019-11-13 | 2021-06-11 | 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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| CN1912110A (en) * | 2005-08-09 | 2007-02-14 | 中国人民解放军军事医学科学院野战输血研究所 | Method and application of induced differentiating fat originate stem cells to adipocyte |
| CN103040821A (en) * | 2012-12-31 | 2013-04-17 | 上海市内分泌代谢病研究所 | Application of berberine in pharmacy |
| CN103060267A (en) * | 2012-12-26 | 2013-04-24 | 上海市内分泌代谢病研究所 | An induced differentiation method for 3T3-L1 preadipocytes |
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| CN1912110A (en) * | 2005-08-09 | 2007-02-14 | 中国人民解放军军事医学科学院野战输血研究所 | Method and application of induced differentiating fat originate stem cells to adipocyte |
| CN103060267A (en) * | 2012-12-26 | 2013-04-24 | 上海市内分泌代谢病研究所 | An induced differentiation method for 3T3-L1 preadipocytes |
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