CN107858326B - Induction differentiation agent and induction differentiation method of mouse preadipocyte 3T3-L1 - Google Patents

Induction differentiation agent and induction differentiation method of mouse preadipocyte 3T3-L1 Download PDF

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CN107858326B
CN107858326B CN201711319732.7A CN201711319732A CN107858326B CN 107858326 B CN107858326 B CN 107858326B CN 201711319732 A CN201711319732 A CN 201711319732A CN 107858326 B CN107858326 B CN 107858326B
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李欣伦
江泉
肖泽玺
祝加和
李超
刘媛媛
张双
李秋圆
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Abstract

The invention provides an induction differentiation agent and an induction differentiation method of mouse preadipocytes 3T 3-L1. The components of the induction differentiation agent are as follows: 3-isobutyl-1-methylxanthine, insulin, dexamethasone, rosiglitazone, FBS. The induced differentiation method comprises the following steps: after the 3T3-L1 cells are cultured by conventional recovery culture, the cells are cultured by a high-glucose DMEM culture solution containing the induction differentiation agent, then cultured by a high-glucose DMEM culture solution containing insulin and 10% FBS, and then cultured by a half-liquid exchange method by using the high-glucose DMEM culture solution containing 10% FBS. The method has short differentiation period of only 6-7 days, high preadipocyte conversion rate and consistent cell differentiation, and can greatly promote the development of fat metabolism disease research.

Description

Induction differentiation agent and induction differentiation method of mouse preadipocyte 3T3-L1
Technical Field
The invention relates to the field of cell culture, in particular to an induced differentiation agent and an induced differentiation method of mouse preadipocytes 3T 3-L1.
Background
Mouse preadipocyte 3T3-L1 is a cell line with single differentiation potential separated and cloned from mouse embryo, and can be induced to differentiate into mature fat cell under certain condition. Mature adipocytes differentiated from 3T3-L1 can better simulate somatic adipocytes in morphology and function, so that 3T3-L1 is an important tool cell for researching metabolic syndromes such as obesity, diabetes and non-alcoholic fatty liver disease.
The cell differentiation process of 3T3-L1 is a process of comprehensive regulation at the gene level and the hormone level. However, 3T3-L1 preadipocytes are difficult to culture cells, and the induction of differentiation and maturation becomes a bottleneck problem of many scientific researches. The traditional 3T3-L1 preadipocyte induction method is characterized in that 3-isobutyl-1-methylxanthine and insulin with certain dosage are sequentially added into a cell culture medium as an inducer to induce the differentiation of 3T3-L1 cells, and the method is also called a cocktail method. The induction by the traditional cocktail method mainly shows that the 3T3-L1 cell has low transformation rate, long transformation period, cells in the later period are easy to float, lipid drops are small, and differentiation is uneven. Therefore, finding an induction method with short differentiation cycle, high preadipocyte conversion rate and consistent differentiation is one of the problems to be solved in the field of fat metabolism at present.
Rosiglitazone belongs to thiazolidinedione drugs, is a commonly used hypoglycemic drug, and can enhance the sensitivity of body tissues to insulin, improve the function of islet beta cells and realize effective control on blood sugar.
Patent document CN103361307A, published japanese patent No. 2013.10.23, discloses a method for inducing differentiation of a C3H10T1/2 mesodermal pluripotent embryonic stem cell line, comprising the steps of: culturing C3H10T1/2 cells in a culture solution, adding 5mL of 0.25% pancreatin for digestion after 80% -90% of the cells are fused, stopping pancreatin digestion reaction when the cells shrink to be round, centrifuging for 3-5 minutes, precipitating, discarding supernatant, adding the culture solution, and blowing; subculturing to 6-well plate or 12-well plate at a ratio of 1:4, and replacing culture solution for 1 time every 2-4 days; and (3) placing the culture plate in the inducing solution A after the cells on the culture plate are overgrown, removing the inducing solution A after 2 days and replacing the inducing solution A with the inducing solution B, removing the inducing solution B after 2 days and replacing the inducing solution B with the culture solution, and replacing the culture solution for 1 time every 2-4 days. The inducing liquid A is DMEM culture liquid containing 10% FBS by volume percentage, and the culture liquid also contains 4-5 mu g/mL of insulin, 0.5-0.7mmol/L of 3-isobutyl-1-methylxanthine, 1-2 mu mol/L of dexamethasone, 130nmol/L of indomethacin, 1-2nmol/L of triiodothyronine and 1-2 mu mol/L of rosiglitazone. The inducing liquid B is DMEM culture solution containing 10% FBS by volume percentage, and the culture solution also contains 5-10 mug/ml of insulin, 1-3nmol/L of triiodothyronine and 1-3 μmol/L of rosiglitazone.
However, no method for inducing differentiation of mouse preadipocytes 3T3-L1 with short differentiation cycle, high transformation rate and uniform differentiation is available at present.
Disclosure of Invention
The invention aims to provide a method for inducing differentiation of mouse preadipocytes 3T3-L1, which has short differentiation period, high transformation rate and consistent differentiation, aiming at the problem that the mouse preadipocytes 3T3-L1 are difficult to induce differentiation in the prior art.
The invention firstly provides an induction differentiation agent of mouse preadipocytes 3T3-L1, which comprises the following components: 3-isobutyl-1-methylxanthine, insulin, dexamethasone, rosiglitazone, FBS; the proportion of the components is as follows: (0.45-0.55) mmol/L (4.5-5.5) mu g/L (0.9-1.1) mu mol/L100 mL/L.
Preferably, the ratio of 3-isobutyl-1-methylxanthine, insulin, dexamethasone and rosiglitazone in the inducing and differentiating agent is: 0.5mmol/L, 5. mu.g/L, 1. mu. mol/L, 100 mL/L.
The invention also provides application of the induction differentiation agent in inducing mouse preadipocytes 3T3-L1 to differentiate into mature adipocytes.
The invention also provides a differentiation induction culture medium of the mouse preadipocytes 3T3-L1, and the differentiation induction culture medium contains the differentiation induction agent.
Preferably, the differentiation induction medium is a high-glucose DMEM medium.
The invention also provides a method for inducing differentiation of mouse preadipocytes 3T3-L1, which comprises the following steps:
1)3T3-L1 cells were recovered conventionally, and placed in 10% FBS-containing high-glucose DMEM medium at 37 deg.C with 5% CO2Culturing under saturated humidity condition;
2) by (4-6). times.104Inoculating cells to a cell culture plate at a density of/mL, and starting differentiation when the cells grow to 85%;
3) culturing with DMEM culture solution containing (0.45-0.55) mmol/L3-isobutyl-1-methylxanthine, (4.5-5.5) μ g/mL insulin, (0.9-1.1) μmol/L dexamethasone, (0.9-1.1) μmol/L rosiglitazone, 10% FBS and high sugar for 36-60 hr;
4) then culturing the cells in 10% FBS high-glucose DMEM culture solution containing (4.5-5.5) mu g/mL insulin for 36-60 hours;
5) then culturing with DMEM culture solution containing 10% FBS high sugar, changing the culture solution once every day, and adopting a half-liquid changing method;
6) until the cell differentiation was successful.
Preferably, the concentrations of the components in the step 3) are respectively as follows: 0.5mmol/L of 3-isobutyl-1-methylxanthine, 5 mu g/mL of insulin, 1 mu mol/L of dexamethasone and 1 mu mol/L of rosiglitazone.
Preferably, the time required until the cell differentiation is successful in step 6) is 6 to 7 days from step 3).
The invention has the beneficial effects that:
the invention selects proper inducer components, the concentration proportion of each component is reasonable, and the induction method and the steps are reasonable in design, so that the invention can play a good role in inducing the differentiation of the mouse preadipocytes 3T3-L1 to mature adipocytes, has short differentiation period of only 6-7 days, high preadipocyte conversion rate and uniform cell differentiation, and is greatly convenient for the development of fat metabolism disease research.
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FIG. 1 is a photograph of a cell stained with oil red. The left plate was from the comparative example 1 group and the left plate was from the example 7 group.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments.
In the following examples and comparative examples, mouse preadipocytes 3T3-L1 were purchased from ATCC in USA under the batch number; FBS and high-glucose DMEM media were purchased from Gibco, usa under the respective lot numbers 1453373 and 1894111; 3-Isobutyl-1-methylxanthine (IBMX), insulin, dexamethasone, and rosiglitazone were purchased from Saimer.
Example 1
The induction differentiation agent composition of the mouse preadipocyte 3T3-L1 is as follows: 3-isobutyl-1-methylxanthine, insulin, dexamethasone, rosiglitazone, FBS. The proportion of the components is as follows: 0.5mmol/L, 5. mu.g/L, 1. mu. mol/L, 100 mL/L.
Example 2
The induction differentiation agent composition of the mouse preadipocyte 3T3-L1 is as follows: 3-isobutyl-1-methylxanthine, insulin, dexamethasone, rosiglitazone, FBS. The proportion of the components is as follows: 0.45mmol/L, 5.5. mu.g/mL, 0.9. mu. mol/L, 1.1. mu. mol/L, 100 mL/L.
Example 3
The induction differentiation agent composition of the mouse preadipocyte 3T3-L1 is as follows: 3-isobutyl-1-methylxanthine, insulin, dexamethasone, rosiglitazone, FBS. The proportion of the components is as follows: 0.55mmol/L, 4.5. mu.g/mL, 1.1. mu. mol/L, 0.9. mu. mol/L, 100 mL/L.
Example 4
The induction differentiation agent composition of the mouse preadipocyte 3T3-L1 is as follows: 3-isobutyl-1-methylxanthine, insulin, dexamethasone, rosiglitazone, FBS. The proportion of the components is as follows: 0.55mmol/L, 5.5. mu.g/mL, 0.9. mu. mol/L, 100 mL/L.
Example 5
The induction differentiation agent composition of the mouse preadipocyte 3T3-L1 is as follows: 3-isobutyl-1-methylxanthine, insulin, dexamethasone, rosiglitazone, FBS. The proportion of the components is as follows: 0.45mmol/L, 4.5. mu.g/mL, 1.1. mu. mol/L, 100 mL/L.
Example 6
The induction differentiation agent composition of the mouse preadipocyte 3T3-L1 is as follows: 3-isobutyl-1-methylxanthine, insulin, dexamethasone, rosiglitazone, FBS. The proportion of the components is as follows: 0.55mmol/L, 5.5. mu.g/mL, 0.9. mu. mol/L, 1.1. mu. mol/L, 100 mL/L.
Example 7
The method for inducing the differentiation of the mouse preadipocyte 3T3-L1 comprises the following steps:
1)3T3-L1 cells were recovered conventionally, and placed in 10% FBS-containing high-glucose DMEM medium at 37 deg.C with 5% CO2Culturing under saturated humidity condition.
2) At 5X 104The cells were seeded at a density of 1 mL/mL in 12-well plates and differentiation was initiated when the cells grew to 85%；
3) Culturing with DMEM culture solution containing 0.5 mmol/L3-isobutyl-1-methylxanthine, 5 μ g/mL insulin, 1 μmol/L dexamethasone, 1 μmol/L rosiglitazone, 10% FBS, and high sugar for 2 days;
4) then culturing for 2 days by using a 10% FBS high-glucose DMEM culture solution containing 5 mu g/mL of insulin;
5) changing the culture medium with 10% FBS (fetal bovine serum) in DMEM (DMEM) culture medium once a day by adopting a half-liquid changing method, namely sucking out half of the original culture medium and adding half of fresh culture medium;
6) until the cells are successfully differentiated.
Example 8
The method for inducing the differentiation of the mouse preadipocyte 3T3-L1 comprises the following steps:
1)3T3-L1 cells were recovered conventionally, and placed in 10% FBS-containing high-glucose DMEM medium at 37 deg.C with 5% CO2Culturing under saturated humidity condition.
2) At 4X 104Inoculating cells into a 12-well plate at a density of one milliliter (mL), wherein each well is 1mL, and starting differentiation when the cells grow to 85%;
3) culturing with DMEM culture solution containing 0.45 mmol/L3-isobutyl-1-methylxanthine, 5.5 μ g/mL insulin, 0.9 μmol/L dexamethasone, 1.1 μmol/L rosiglitazone, 10% FBS, and high glucose for 60 hr;
4) then culturing the cells in a 10% FBS high-glucose DMEM culture solution containing 5.5 mu g/mL of insulin for 36 hours;
5) changing the culture medium once every 24 hours by using a DMEM culture medium containing 10% FBS and high glucose, and adopting a half-liquid changing method, namely sucking out half of the original culture medium and adding half of fresh culture medium;
6) until the cells are successfully differentiated.
Example 9
The method for inducing the differentiation of the mouse preadipocyte 3T3-L1 comprises the following steps:
1)3T3-L1 cells were recovered conventionally, and placed in 10% FBS-containing high-glucose DMEM medium at 37 deg.C with 5% CO2Culturing under saturated humidity condition.
2) At 6X 104Inoculating cells into a 12-well plate at a density of one milliliter (mL), wherein each well is 1mL, and starting differentiation when the cells grow to 85%;
3) culturing with DMEM culture solution containing 0.55 mmol/L3-isobutyl-1-methylxanthine, 4.5 μ g/mL insulin, 1.1 μmol/L dexamethasone, 0.9 μmol/L rosiglitazone, 10% FBS, and high glucose for 36 hr;
4) then culturing the cells in a 10% FBS high-glucose DMEM culture solution containing 4.5 mu g/mL of insulin for 60 hours;
5) changing the culture medium once every 24 hours by using a DMEM culture medium containing 10% FBS and high glucose, and adopting a half-liquid changing method, namely sucking out half of the original culture medium and adding half of fresh culture medium;
6) until the cells are successfully differentiated.
Example 10
The method for inducing the differentiation of the mouse preadipocyte 3T3-L1 comprises the following steps:
1)3T3-L1 cells were recovered conventionally, and placed in 10% FBS-containing high-glucose DMEM medium at 37 deg.C with 5% CO2Culturing under saturated humidity condition.
2) At 5X 104Inoculating cells into a 12-well plate at a density of one milliliter (mL), wherein each well is 1mL, and starting differentiation when the cells grow to 85%;
3) culturing with DMEM culture solution containing 0.55 mmol/L3-isobutyl-1-methylxanthine, 5.5 μ g/mL insulin, 0.9 μmol/L dexamethasone, 0.9 μmol/L rosiglitazone, 10% FBS, and high glucose for 48 hr;
4) then culturing the cells in a 10% FBS high-glucose DMEM culture solution containing 4.5 mu g/mL of insulin for 48 hours;
5) changing the culture medium once every 24 hours by using a DMEM culture medium containing 10% FBS and high glucose, and adopting a half-liquid changing method, namely sucking out half of the original culture medium and adding half of fresh culture medium;
6) until the cells are successfully differentiated.
Example 11
The method for inducing the differentiation of the mouse preadipocyte 3T3-L1 comprises the following steps:
1)3T3-L1 cells were recovered conventionally, and placed in 10% FBS-containing high-glucose DMEM medium at 37 deg.C with 5% CO2Culturing under saturated humidity condition.
2) At 6X 104Inoculating cells into a 12-well plate at a density of one milliliter (mL), wherein each well is 1mL, and starting differentiation when the cells grow to 85%;
3) culturing with DMEM culture solution containing 0.45 mmol/L3-isobutyl-1-methylxanthine, 4.5 μ g/mL insulin, 1.1 μmol/L dexamethasone, 1.1 μmol/L rosiglitazone, 10% FBS, and high glucose for 48 hr;
4) then culturing the cells in a 10% FBS high-glucose DMEM culture solution containing 4.5 mu g/mL of insulin for 48 hours;
5) changing the culture medium once every 24 hours by using a DMEM culture medium containing 10% FBS and high glucose, and adopting a half-liquid changing method, namely sucking out half of the original culture medium and adding half of fresh culture medium;
6) until the cells are successfully differentiated.
Example 12
The method for inducing the differentiation of the mouse preadipocyte 3T3-L1 comprises the following steps:
1)3T3-L1 cells were recovered conventionally, and placed in 10% FBS-containing high-glucose DMEM medium at 37 deg.C with 5% CO2Culturing under saturated humidity condition.
2) At 4X 104Inoculating cells into a 12-well plate at a density of one milliliter (mL), wherein each well is 1mL, and starting differentiation when the cells grow to 85%;
3) culturing with DMEM culture solution containing 0.55 mmol/L3-isobutyl-1-methylxanthine, 5.5 μ g/mL insulin, 0.9 μmol/L dexamethasone, 1.1 μmol/L rosiglitazone, 10% FBS, and high glucose for 48 hr;
4) then culturing the cells in a 10% FBS high-glucose DMEM culture solution containing 4.5 mu g/mL of insulin for 48 hours;
5) changing the culture medium once every 24 hours by using a DMEM culture medium containing 10% FBS and high glucose, and adopting a half-liquid changing method, namely sucking out half of the original culture medium and adding half of fresh culture medium;
6) until the cells are successfully differentiated.
During the research process, the inventor also sets a plurality of experimental groups, wherein the experimental groups comprise the following groups:
comparative example 1
The method for inducing the differentiation of the mouse preadipocyte 3T3-L1 comprises the following steps:
1)3T3-L1 cells were recovered conventionally, and placed in 10% FBS-containing high-glucose DMEM medium at 37 deg.C with 5% CO2Culturing under saturated humidity condition.
2) At 5X 104Inoculating cells into a 12-well plate at a density of one milliliter (mL), wherein each well is 1mL, and starting differentiation when the cells grow to 85%;
3) culturing with DMEM culture solution containing 0.5 mmol/L3-isobutyl-1-methylxanthine, 5 μ g/mL insulin, 1 μmol/L dexamethasone, 10% FBS, and high sugar for 2 days;
4) then culturing for 2 days by using a 10% FBS high-glucose DMEM culture solution containing 5 mu g/mL of insulin;
5) changing the culture medium with 10% FBS (fetal bovine serum) in DMEM (DMEM) culture medium once a day by adopting a half-liquid changing method, namely sucking out half of the original culture medium and adding half of fresh culture medium;
6) until the cells are successfully differentiated.
Comparative example 2
The method for inducing the differentiation of the mouse preadipocyte 3T3-L1 comprises the following steps:
1)3T3-L1 cells were recovered conventionally, and placed in 10% FBS-containing high-glucose DMEM medium at 37 deg.C with 5% CO2Culturing under saturated humidity condition.
2) At 4X 104Inoculating cells into a 12-well plate at a density of one milliliter (mL), wherein each well is 1mL, and starting differentiation when the cells grow to 85%;
3) culturing with DMEM culture solution containing 0.45 mmol/L3-isobutyl-1-methylxanthine, 5.5 μ g/mL insulin, 0.9 μmol/L dexamethasone, 1.2 μmol/L rosiglitazone, 10% FBS, and high glucose for 60 hr;
4) then culturing the cells in a 10% FBS high-glucose DMEM culture solution containing 5.5 mu g/mL of insulin for 36 hours;
5) changing the culture medium once every 24 hours by using a DMEM culture medium containing 10% FBS and high glucose, and adopting a half-liquid changing method, namely sucking out half of the original culture medium and adding half of fresh culture medium;
6) until the cells are successfully differentiated.
Comparative example 3
The method for inducing the differentiation of the mouse preadipocyte 3T3-L1 comprises the following steps:
1)3T3-L1 cells were recovered conventionally, and placed in 10% FBS-containing high-glucose DMEM medium at 37 deg.C with 5% CO2Culturing under saturated humidity condition.
2) At 6X 104Density of/mLInoculating cells into a 12-hole plate, wherein each hole is 1mL, and starting differentiation when the cells grow to 85%;
3) culturing with DMEM culture solution containing 0.55 mmol/L3-isobutyl-1-methylxanthine, 4 μ g/mL insulin, 1.1 μmol/L dexamethasone, 0.9 μmol/L rosiglitazone, 10% FBS, and high glucose for 36 hr;
4) then culturing the cells in a 10% FBS high-glucose DMEM culture solution containing 4.5 mu g/mL of insulin for 60 hours;
5) changing the culture medium once every 24 hours by using a DMEM culture medium containing 10% FBS and high glucose, and adopting a half-liquid changing method, namely sucking out half of the original culture medium and adding half of fresh culture medium;
6) until the cells are successfully differentiated.
EXAMPLE 13 Effect of inducing differentiation
Each of examples 7-12 and comparative examples 1-3 above was repeated 6 times. In the case of the cells of examples 7 to 12 and comparative examples 1 to 3, which were observed to differentiate during the induction of differentiation, it was found that the cells of examples 7 to 12 were successfully differentiated on day 3 from the start of the induction of step 3), exhibited an adipocyte phenotype in which lipid droplets were observed, and were successfully differentiated substantially in total on day 6, whereas the cells of comparative examples 1 to 3 generally exhibited lipid droplets on days 4 to 5. The method of the invention has the advantage of short differentiation period.
Performing oil red staining on each group of cells on the 7 th day from the induction of the step 3), detecting the amount of lipid drops of the cells, further judging the differentiation degree of the cells, and calculating the preadipocyte conversion rate. The specific method for dyeing the oil red comprises the following steps: cells were washed twice with phosphate buffered saline PBS, fixed with 0.4% paraformaldehyde for 30 minutes, oil red stock was mixed with water at a ratio of 3:2, and then stained for 1 hour, and washed twice with phosphate buffered saline PBS. The cells were differentiated in 60% isopropanol for 1 minute and photographed twice with Phosphate Buffered Saline (PBS). And judging the differentiation uniformity of each group of cells through visual observation, and scoring, wherein the total score is 10, and the higher the score is, the better the differentiation uniformity is. Data are all provided with
Figure BDA0001504529120000081
The statistical treatment is shown by the t-test.
The statistics of the transformation rate and differentiation uniformity of preadipocytes in each group are shown in Table 1. Comparison between groups showed that the conversion rates of examples 7-12 were all significantly higher than those of comparative examples 1-3, the differences were statistically significant (P <0.05), and the conversion rates of example 7 were significantly higher than those of examples 8-12, the differences were also statistically significant (P < 0.05); the differentiation uniformity scores of examples 7-12 were all significantly higher than those of comparative examples 1-3, with the differences being statistically significant (P <0.05), and the differentiation uniformity score of example 7 was also significantly higher than those of examples 8-12, with the differences being statistically significant (P < 0.05). The result shows that the method selects proper inducer and culture medium, the induced differentiation steps and method are reasonable, the conversion rate is obviously improved, and the mature fat cells with consistent differentiation degree are obtained.
TABLE 1 preadipocyte conversion and differentiation uniformity for each group
Group of Conversion (%) Differentiation uniformity (minute)
Example 7 98.3±8.5 9.3±0.6
Example 8 85.0±7.4 8.6±0.9
Example 9 92.9±7.8 8.4±0.7
Example 10 87.9±10.3 8.8±1.2
Example 11 90.3±9.1 8.9±0.9
Example 12 96.6±3.4 9.0±0.8
Comparative example 1 69.2±4.3 7.3±0.5
Comparative example 2 68.9±6.7 6.9±0.8
Comparative example 3 73.5±5.1 6.8±0.7
In conclusion, the method has the advantages of short differentiation period, high transformation rate and consistent differentiation.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (5)

1. An induction differentiation agent of mouse preadipocyte 3T3-L1 is characterized by comprising the following components: 0.5mmol/L of 3-isobutyl-1-methylxanthine, 5. mu.g/mL of insulin, 1. mu. mol/L of dexamethasone, 1. mu. mol/L of rosiglitazone, 100mL/L of FBS.
2. The use of the induction differentiation agent as claimed in claim 1 for inducing mouse preadipocytes 3T3-L1 to differentiate into mature adipocytes.
3. An induced differentiation medium of mouse preadipocytes 3T3-L1, wherein the induced differentiation medium comprises the induced differentiation agent of claim 1.
4. A method for inducing differentiation of mouse preadipocytes 3T3-L1, wherein the method comprises the following steps:
1)3T3-L1 cells were recovered conventionally, and placed in 10% FBS-containing high-glucose DMEM medium at 37 deg.C with 5% CO2Culturing under saturated humidity condition;
2) by (4-6). times.104Inoculating cells to a cell culture plate at a density of/mL, and starting differentiation when the cells grow to 85%;
3) culturing with DMEM culture solution containing 0.5 mmol/L3-isobutyl-1-methylxanthine, 5 μ g/mL insulin, 1 μmol/L dexamethasone, 1 μmol/L rosiglitazone, 10% FBS, and high sugar for 36-60 hr;
4) then culturing the mixture for 36 to 60 hours by using a 10 percent FBS high-glucose DMEM culture solution containing 5 mu g/mL of insulin;
5) then culturing with DMEM culture solution containing 10% FBS high sugar, changing the culture solution once every day, and adopting a half-liquid changing method;
6) until the cell differentiation was successful.
5. The method for inducing differentiation according to claim 4, wherein the time required until the cell differentiation is successful in step 6) is 6 to 7 days from step 3).
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1657529A (en) * 2004-02-20 2005-08-24 中国科学院上海药物研究所 2-substituted imine thia zlidine derivative its preparation technology and medicinal composition

Family Cites Families (5)

* Cited by examiner, † Cited by third party
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CN1548529A (en) * 2003-05-09 2004-11-24 中国人民解放军军事医学科学院基础医 Separation method of buffering stem cell in human placenta
US8809384B2 (en) * 2011-03-25 2014-08-19 Hoffmann-La Roche Inc. Azaspirodecanone compounds
US10632165B2 (en) * 2016-01-18 2020-04-28 Academia Sinica Beneficial effect of Bidens pilosa on fat decrease and muscle increase
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Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1657529A (en) * 2004-02-20 2005-08-24 中国科学院上海药物研究所 2-substituted imine thia zlidine derivative its preparation technology and medicinal composition

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
小檗碱对3T3-L1 前体脂肪细胞增殖与分化的影响;李东伟等;《阜阳师范学院学报（自然科学版）》;20160630;第33卷(第2期);第51页左栏第1.2.1节 *
李东伟等.小檗碱对3T3-L1 前体脂肪细胞增殖与分化的影响.《阜阳师范学院学报（自然科学版）》.2016,第33卷(第2期), *
组胺对分化的3T3L1细胞UCP2表达的影响;曾慧等;《中南药学》;20080229;第6卷(第1期);第2页右栏第1.2.1节 *

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