CN113151161B - Application of insulin-like growth factor 2 in promoting differentiation of cancer cells into adipocytes - Google Patents

Application of insulin-like growth factor 2 in promoting differentiation of cancer cells into adipocytes Download PDF

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CN113151161B
CN113151161B CN202110441475.4A CN202110441475A CN113151161B CN 113151161 B CN113151161 B CN 113151161B CN 202110441475 A CN202110441475 A CN 202110441475A CN 113151161 B CN113151161 B CN 113151161B
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张传茂
王向阳
辛广伟
迟王菲
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Abstract

The invention provides an application of insulin-like growth factor 2 in promoting differentiation of cancer cells into adipocytes, which is characterized in that before cancer cells are subjected to in vitro induced differentiation, the cells are pretreated by insulin-like growth factor 2 (IGF 2) with the final concentration of 50-400ng/mL, and then the cancer cells are cultured in an induced differentiation culture medium (1) for 4-8 days; then the induced differentiation culture medium (1) is changed into an induced differentiation culture medium (2) to be cultured for 1-4 days; then changing to a basic culture medium for culturing for 6-12 days. The mode of firstly carrying out IGF2 pretreatment and then carrying out induced differentiation can successfully induce and differentiate cancer cells into fat cells with high efficiency, the number of the obtained fat cells is obviously more than that of a control group which is not subjected to IGF2 pretreatment, and the expression of fat cell marker genes is increased to 11-17 times of that of the control group. The method can efficiently induce and differentiate the cancer cells into the fat cells, effectively inhibit the proliferation and the metastasis of the cancer cells and has good clinical application prospect.

Description

Application of insulin-like growth factor 2 in promoting differentiation of cancer cells into adipocytes
Technical Field
The invention relates to the technical field of biology, in particular to application of insulin-like growth factor 2 in promoting differentiation of cancer cells into adipocytes.
Background
During the progression of cancer conditions, there is a stage called "epithelial-mesenchymal transition" (EMT) in which cancer cells have traits similar to those of "stem cells" with the potential to transform into a variety of cell types. Epithelial cells can transform to mesenchymal cells, so that the polarity of the cells is lost, the adhesion capability is reduced, the cytoskeleton is changed, the migration and movement capabilities of the cells are enhanced, and the cancer cells are separated from an initial tumor and metastasize to a far place by utilizing EMT. Studies have demonstrated that EMT is a key event implicated in the invasion and metastasis of cancer. After the tumor cells are spread through blood circulation, the tumor cells undergo mesenchymal-epithelial transformation (MET) to form secondary tumor metastases. MET is the reverse process of EMT, and cancer cells change from mesenchymal cell morphology to epithelial cell morphology, and have enhanced proliferation ability and faster growth.
Scientists use rosiglitazone in combination with MEK inhibitor tramitinib in mice to differentiate breast cancer cells into adipocytes, reducing tumor invasiveness and inhibiting tumor metastasis, mainly using the method: the breast cancer cells were seeded into a culture dish, cultured overnight, and then treated with 200ng/ml of human recombinant BMP2 protein for 3 days, followed by treatment with 200ng/ml of BMP2 and 2uM of Rosiglitazone (Rosiglitazone) for 4 days, and then treated with 2. Mu.M of Rosiglitazone for 3 days. The culture medium adopted by the method is a DMEM culture medium containing 10% fetal bovine serum. The results show that after the induction of rosiglitazone and bone morphogenetic protein-2 (BMP-2), the breast cancer cells after EMT are transformed into fat cells, express various fat cell markers, and have the same response to isoproterenol and insulin as the fat cells; and after inducing differentiation for 9 days, researchers remove the medium for inducing differentiation, replace the medium with a common medium, observe and find that the differentiated adipocytes maintain the characteristics of the adipocytes, and do not have the condition of recovering to mesenchymal-like cancer cells, confirming that the induced differentiation is irreversible.
However, this method of inducing differentiation of cancer cells has significant drawbacks. The method can only induce and differentiate cancer cells after EMT into fat cells, namely can only induce and differentiate cancer cells in a mesenchymal state, but cannot induce and differentiate cancer cells in an epithelial state. The BMP2 is used in this method to promote EMT transformation in cancer cells. Therefore, the method has harsh conditions for inducing differentiation of cancer cells, has great limitation and no universality.
In previous studies, the inventors found that insulin-like growth factor 2 has an excellent effect of promoting transformation in promoting the differentiation of human dermal fibroblasts into adipocytes (see chinese patent CN 111518752A), but normal human dermal fibroblasts and cancer cells have obvious differences in growth and development, physiological mechanism and the like, and are mainly expressed in that: first, human skin fibroblasts have a limited number of passages, do not have the ability to proliferate indefinitely, metastasize, and do not undergo EMT or MET transformation. Cancer cells, which are immortalized and capable of malignant metastasis, undergo EMT or MET transformation. Second, human dermal fibroblasts are generally in a quiescent state, and usually proliferate in large amounts during wound repair, and secrete collagen fibrils and extracellular matrix, which are involved in wound healing. Cancer cells are in a state of proliferation or metastasis at the moment, and hardly secrete collagen fibrils and extracellular matrix. Third, human skin fibroblasts are differentiated from mesenchymal cells at an embryonic stage, and generally exist in connective tissues of the skin in a mesenchymal state. Cancer cells are various in types, wide in sources, in an epithelial state in some cases, and in a mesenchymal state in some cases. Therefore, it is difficult and complicated to induce differentiation of cancer cells. The method is an effective method which is easy to research and has strong universality and is used for inducing the differentiation of cancer cells into fat cells.
Disclosure of Invention
The invention aims to provide application of insulin-like growth factor 2 (IGF 2) in promoting differentiation of cancer cells into fat cells.
In a first aspect, the invention provides the use of insulin-like growth factor 2 to promote differentiation of cancer cells into adipocytes.
The invention discovers that pretreatment by using insulin-like growth factor 2 can effectively improve the efficiency of cancer cells to differentiate into fat cells before inducing the cancer cells to differentiate into fat cells.
The application is characterized in that the cancer cells are pretreated by insulin-like growth factor 2 with the final concentration of 50-400ng/mL before the cancer cells are subjected to in vitro induced differentiation.
Preferably, cancer cells are pretreated with insulin-like growth factor 2 at a final concentration of 100-300 ng/mL.
In a second aspect, the present invention provides a method for inducing differentiation of cancer cells into adipocytes in vitro, comprising the steps of:
(1) Culturing cancer cells in a basal culture medium, adding insulin-like growth factor 2 with the final concentration of 50-400ng/mL when the confluence degree of the cells reaches 50-70%, and pretreating the cancer cells;
(2) Replacing the basic culture medium in the step (1) with an induced differentiation culture medium (1), and culturing for 4-8 days;
(3) Changing the induced differentiation culture medium (1) into an induced differentiation culture medium (2), and culturing for 1-4 days;
(4) Changing into a basic culture medium, and culturing for 6-12 days;
wherein the basic culture medium is a high-glucose DMEM culture medium containing fetal calf serum and antibiotics;
the induced differentiation culture medium (1) is a basal culture medium containing dexamethasone, insulin, rosiglitazone and 1-methyl-3-isobutylxanthine;
the induced differentiation culture medium (2) is a basal culture medium containing insulin.
In step (1) of the above method, IGF2 is added to a final concentration of 100-300 ng/mL. Preferably 200ng/mL IGF2.
In step (1), the cancer cells are pretreated for 12 to 48 hours, preferably 20 to 30 hours.
In the method, the basic culture medium is a high-glucose DMEM culture medium containing 5-20% of fetal calf serum, 100U/mL penicillin and 100 mu g/mL streptomycin;
the induced differentiation culture medium (1) is a basic culture medium of 0.5-1 mu M dexamethasone, 5-20 mu g/mL insulin, 1-2 mu M rosiglitazone and 0.2-0.5mM 1-methyl-3-isobutyl xanthine;
the induced differentiation culture medium (2) is a basal culture medium containing 5-20 mug/mL of insulin.
When the corresponding induced differentiation culture medium and the basic culture medium are adopted for culture, the culture medium is changed once every two days, or the culture medium of the cancer cells is changed according to the actual situation and the conventional operation requirements in the field.
Preferably, the differentiation induction medium (1) is a basal medium of 1. Mu.M dexamethasone, 10. Mu.g/mL insulin, 2. Mu.M rosiglitazone, 0.5mM 1-methyl-3-isobutylxanthine;
the induced differentiation culture medium (2) is a basal culture medium containing 10 mu g/mL of insulin.
The environmental conditions for induced differentiation of the method of the present invention are 35 ℃ to 37 ℃ and 5% of CO 2 。
The whole process of inducing and differentiating cancer cells mainly comprises two parts of pretreatment and induced differentiation. Insulin-like growth factor 2 pretreatment was performed for 12-48 hours when the confluency of cancer cells reached 50% -70%. Then, induction differentiation is carried out according to a differentiation mode of '4 +2+ 10', namely a differentiation medium (1) is induced for 4 days, a differentiation medium (2) is induced for 2 days, and a basic medium is continuously cultured for 10 days. The mode of firstly carrying out IGF2 pretreatment and then inducing differentiation greatly improves the efficiency of cancer cells to differentiate into fat cells.
The present invention provides a specific procedure for inducing differentiation of cancer cells into adipocytes, which is used to explain the present invention, but not to limit the present invention:
taking out the cover glass soaked in 75% alcohol, burning and sterilizing on an alcohol lamp, and then paving in 35mm culture dishes, wherein 4 cover glass are paved on each culture dish; add 4mL of basal medium to the culture dish and spread the overlapping coverslips with forceps for later passage of the cells. After the cancer cells in the 10cm culture dish are full, sucking the culture medium in the culture dish, and washing for 2 times with Phosphate Buffer Solution (PBS), wherein each time is 2mL; then sucking off PBS, adding 1mL of pancreatin with the concentration of 0.25%, and repeatedly shaking the culture dish by two hands to enable the pancreatin to uniformly infiltrate the bottom of the culture dish; finally placing in a container containing 5% of CO 2 Digesting for 1-2min at 37 ℃ in the cell culture box; the digested cells were removed and 0.5mL basal medium was added to the culture dish to stop the digestion; then repeatedly blowing and sucking by using a pipettor, uniformly blowing and transferring the cells into a 1.5mL centrifuge tube, and centrifuging for 4 minutes at 1000 rpm/min; sucking off the supernatant, adding 1mL of a basic culture medium, and gently and repeatedly blowing and sucking to uniformly mix the cells; then, according to the following steps of 1:6, evenly dividing the cell suspension into 6 35mm culture dishes paved with cover glass, repeatedly shaking the culture dishes by hands,mixing the cells evenly, and then putting the mixture into a cell culture box for culture;
after the confluence degree of the cells reaches 50-70%, IGF2 with the final concentration of 50-400ng/mL is added, and the cells are pretreated for 12-48 hours;
sucking off the basic culture medium, adding 3mL of induced differentiation culture medium (1), putting the culture medium back into the cell culture box for continuous induced culture for 4-8 days, and replacing the fresh culture medium every two days; when the culture medium is replaced, the culture medium is gently added along the wall of the culture dish;
sucking off the induced differentiation culture medium (1), adding 3mL of induced differentiation culture medium (2), and carrying out induced culture for 1-4 days; as the induced differentiation proceeded, a shiny circular fat drop was found in the cells under a 10-fold microscope;
sucking off the induced differentiation culture medium (2), adding 3mL of a basic culture medium, continuously culturing for 6-12 days, and replacing the fresh culture medium every two days; under a microscope, cells containing fat drops are more and more, and fat drops in the cells are larger and larger;
as the induced differentiation proceeded, a fat drop with a circular blinking appeared gradually in the cancer cell under 10-fold microscope and became gradually larger. After the induction of differentiation is complete, fat droplets accumulated in mature adipocytes can be detected by oil red staining. In addition, the expression of marker genes adipinectin, fabp4 and Leptin of mature adipocytes can be detected by fluorescent quantitative PCR to further verify the induced differentiation of cancer cells. In the whole induction differentiation process, the culture medium is required to be attached to the wall of the culture dish when being replaced, so that cells are prevented from being blown up.
After the induction differentiation is finished, taking out the cover glass in the culture dish for oil red staining, and detecting the induction differentiation effect; in addition, RNA of the residual cells of the culture dish is extracted, fluorescent quantitative PCR is carried out, the expression of marker genes Adiponectin, fabp4 and Leptin of mature adipocytes is detected, and the induced differentiation result is further verified;
(1) Oil red dyeing
Oil red O is a strong fat solvent and dye agent, capable of binding triglycerides and is therefore frequently used for fat dyeing. The invention carries out oil red staining on the cells after induced differentiation to detect fat drops in mature fat cells. The specific operation is as follows:
(1) the coverslip was washed 3 times with PBS and then fixed with 4% Paraformaldehyde (PFA) for 20 minutes;
(2) after 3 washes with PBS, equilibrate with 60% isopropanol for 5 minutes;
(3) hermetically dyeing with oil red O (dissolved in 60% isopropanol) at a concentration of 3g/L for 10 minutes;
(4) washing with 60% isopropanol for 3 times;
(5) staining with hematoxylin for 2 minutes;
(6) washing with PBS for 3 times, air drying, and sealing to form image;
(7) counting the number of cells which are positive in oil red staining;
(2) Mature adipocyte marker gene detection: adipoectin, fabp4 and Leptin are marker genes for mature adipocytes, and are often used to measure the effect of adipocyte differentiation induction. The invention uses Trizol to extract total RNA from induced and differentiated cells, carries out fluorescent quantitative PCR after reverse transcription, and detects the expression levels of three genes, namely Adiponectin, fabp4 and Leptin.
In a third aspect, the invention also provides the application of the adipose cells obtained by inducing differentiation by the method of the invention and the application of the insulin-like growth factor 2 in inhibiting cancer cell proliferation and metastasis by promoting the differentiation of cancer cells into adipose cells.
In a fourth aspect, the invention provides an application of insulin-like growth factor 2 in preparing a medicament for improving the efficiency of cancer cell induced differentiation into adipocytes, and an application of insulin-like growth factor 2 in preparing a medicament for inhibiting cancer cell proliferation and metastasis by promoting cancer cell differentiation into adipocytes.
The cancer cells to which the method of the present invention is applicable are not required to be transformed by EMT, and are not affected by the state thereof, and may be cancer cells in an epithelial state or cancer cells in a mesenchymal state. Therefore, the method has generality. Suitable cancer cells include, but are not limited to, heLa (human cervical cancer cells), U2OS (human osteosarcoma cells), SW620 (human colon cancer cells), SW480 (human colon cancer cells), HCT116 (human colon cancer cells), HT29 (human colon cancer cells), A549 (human non-small cell lung cancer cells), MDA-MB-231 (human breast cancer cells), hepG2 (human liver cancer cells), T47D (human breast cancer cells) cell lines, and isolated primary cancer cells.
According to the method, various cancer cells are successfully induced and differentiated into the fat cells, so that the efficiency of differentiating the cancer cells into the fat cells is greatly improved, and compared with the method of pretreating the cancer cells without IGF2, the method disclosed by the invention can improve the efficiency of differentiating the cancer cells into the fat cells by 30% -150%. Taking HeLa cells, U2OS cells, A549 cells and SW620 cells as examples, cancer cells are successfully induced to differentiate into adipocytes by adopting an induction differentiation mode of "4+2+10 days" after cancer cells are pretreated by IGF2. The oil red staining result shows that the number of the fat cells obtained by the method is obviously more than that of the control group, the expression of the fat cell marker gene is improved to 11-17 times of that of the control group, and the efficiency of differentiating cancer cells into fat cells is improved by about 30-150%.
Cancer cells have the ability to proliferate and metastasize maliciously, while mature adipocytes belong to terminally differentiated cells and do not have the ability to proliferate. It is believed that in the near future, cancer cells can be efficiently induced and differentiated into adipocytes in a mode of "IGF 2 pretreatment followed by differentiation induction", and the proliferation and metastasis of cancer cells are effectively inhibited, thereby providing a new concept and method for clinical cancer treatment.
Drawings
FIG. 1 is a graph showing the results of oil red staining of cells after the induction of differentiation in example 1.
FIG. 2 is a graph showing the results of detecting mature adipocyte marker genes of the adipocytes obtained by induced differentiation in example 1.
FIG. 3 is a graph showing the results of oil red staining of cells after the completion of the induced differentiation in example 2.
FIG. 4 is a graph showing the results of detecting a marker gene of mature adipocytes derived from the induced differentiation of example 2.
FIG. 5 is a graph showing the results of oil red staining of cells after the differentiation induction in example 3.
FIG. 6 is a graph showing the results of oil red staining of cells after the completion of the induced differentiation in example 4.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples follow conventional experimental conditions and, unless otherwise specified, the reagents used in the present invention are commercially available. In the examples of the invention, P represents P <0.001, P <0.0001, and the differences between the different treatment groups were statistically significant.
In the embodiment of the invention, the primers for quantitatively detecting the marker genes of the three mature adipocytes such as adipiectin, fabp4 and Leptin by PCR are respectively as follows:
Adiponectin:5’-GATGGCAGAGATGGCAC-3’
5’-GCTGAGCGGTATACATAGG-3’
Fabp4:5’-ACGAGAGGATGATAAACTGGTGG-3’
5’-GCGAACTTCAGTCCAGGTCAAC-3’
Leptin:5’-CACCAAAACCCTCATCAAGACA-3’
5’-CTTTCTGTTTGGAGGAGACTGACT-3’
example 1
This example illustrates the results of HeLa cells in an epithelial state induced differentiation, using 200ng/mL IGF2 pretreatment for 24 hours.
1. The media involved in the induction process were as follows:
basic culture medium: high-glucose DMEM +10% fetal bovine serum +100U/mL penicillin + 100. Mu.g/mL streptomycin;
differentiation induction medium (1): dexamethasone, insulin, rosiglitazone and 1-methyl-3-isobutylxanthine are added into a basal medium, and the final concentrations of the dexamethasone, the insulin, the rosiglitazone and the 1-methyl-3-isobutylxanthine in the basal medium are respectively 1 mu M,10 mu g/mL,2 mu M and 0.5mM;
differentiation induction medium (2): to the basal medium, insulin was added to a final concentration of 10. Mu.g/mL.
2. The induction process comprises the following steps:
(1) The HeLa cells were passaged into a 35mm petri dish plated with a cover glass, 3mL of a basal medium was added, and the medium was placed in a chamber containing 5% CO 2 Culturing at 37 ℃ in the cell culture box;
(2) After the cell density reaches 50-70%, IGF2 with the final concentration of 200ng/mL is added, and the cells are pretreated for 24 hours;
(3) Sucking off the basic culture medium, adding the induced differentiation culture medium (1), continuously carrying out induced culture for 4 days, and replacing the fresh culture medium every two days;
(4) Absorbing the induced differentiation culture medium (1), adding the induced differentiation culture medium (2), and carrying out induced culture for 2 days;
(5) Sucking off the induced differentiation culture medium (2), adding a basic culture medium, continuously culturing for 10 days, and replacing a fresh culture medium every two days;
3. after induction differentiation is finished, the result of induction differentiation is detected through oil red staining and mature fat cell marker gene detection. The results are as follows:
(1) Oil red staining results: as shown in fig. 1, the stained part was stained fat (red after oil red staining), and the uninduced HeLa cells had no fat accumulation, while the HeLa cells induced to differentiate for 16 days had significant fat accumulation. And compared with a control group (without IGF2 pretreatment), 200ng/mL of IGF2 can obviously promote the differentiation of the fat cells after being subjected to the induction after 24 hours of pretreatment.
The number of cells positive for oil red staining in uninduced HeLa cells was 0;
in the control group in which differentiation was induced for 16 days, the efficiency of differentiation of HeLa cells into adipocytes was about 75%,
the efficiency of differentiation into adipocytes of HeLa cells of this example group (24 hours pretreatment with 200ng/mL IGF 2) induced to differentiate for 16 days was about 98%;
(2) Mature adipocyte marker gene detection
As shown in FIG. 2, by quantitative PCR (quantitative PCR), marker genes of three mature adipocytes, i.e., adiponectin, fabp4, and Leptin, were detected. The expression of the three marker genes in the 200nM IGF2-pretreated group was 17.5-fold, 11-fold and 15.5-fold higher than that in the control group (not pretreated with IGF2).
Example 2
This example illustrates the results of induced differentiation of U2OS cells in the mesenchymal state, using IGF2 pretreatment at 100ng/mL for 24 hours.
The culture medium and the specific experimental steps involved in this embodiment are the same as those in embodiment 1, and the differentiation induction mode of "4+2+10" is also adopted. Except that the cancer cells were U2OS cells and the IGF2 treatment concentration was 100ng/mL.
The control group was a group without IGF2 pretreatment.
After induction differentiation is finished, the result of induction differentiation is detected through oil red staining and mature fat cell marker gene detection. The results were as follows:
(1) Oil red staining results: as shown in fig. 3, the stained portion was stained fat (red after oil red staining), and the non-induced U2OS cells had no fat accumulation, while the U2OS cells induced to differentiate for 16 days had significant fat accumulation. Compared with a control group, the induction of 100ng/mL IGF2 after pretreatment for 24 hours can obviously promote the differentiation of fat cells.
The number of cells positive for staining with oil red in uninduced U2OS cells is 0;
in the control group induced to differentiate for 16 days, the efficiency of differentiation of U2OS cells into adipocytes was about 20%,
the efficiency of differentiation into adipocytes of U2OS cells of this example group (100 ng/mL IGF2 pretreatment for 24 hours) induced for 16 days was about 45%.
(2) Mature adipocyte marker gene detection
As shown in FIG. 4, by quantitative PCR, marker genes of two mature adipocytes, adiponectin and Leptin, were detected. The expression of two marker genes in 100ng/mL IGF2 pretreatment group is 2 times and 1.2 times that in control group (without IGF2 pretreatment).
Example 3
This example illustrates the results of SW620 cells in an epithelial state inducing differentiation, using 200ng/mL IGF2 pretreatment for 24 hours.
The culture medium and the specific experimental steps involved in this embodiment are the same as example 1, and the differentiation induction mode of "4+2+10" is also adopted. Except that the cancer cells were SW620 cells.
The control group was a group without IGF2 pretreatment.
After the induction differentiation is finished, the result of the induction differentiation is detected by oil red staining. The results were as follows: as shown in fig. 5, the stained part was stained fat (red after oil red staining), and there was no fat accumulation in the non-induced SW620 cells, whereas there was significant fat accumulation in the SW620 cells induced to differentiate for 16 days. Compared with a control group, the induction of 200ng/mL IGF2 after 24 hours can obviously promote the differentiation of fat cells.
The number of cells staining positive for oil red in uninduced SW620 cells was 0;
in the control group in which differentiation was induced for 16 days, the efficiency of differentiation of SW620 cells into adipocytes was about 30%,
the efficiency of the SW620 cells differentiating into adipocytes in this example group (200 ng/mL IGF2 pretreatment for 24 hours) induced to differentiate for 16 days was about 50%.
Example 4
This example illustrates the results of inducing differentiation of A549 cells in an epithelial state, as exemplified by 200ng/mL IGF2 pretreatment for 24 hours.
The culture medium and the specific experimental steps involved in this embodiment are the same as example 1, and the differentiation induction mode of "4+2+10" is also adopted. Except that the cancer cells were a549 cells.
The control group was a group without IGF2 pretreatment.
After the induction differentiation was completed, the results were as follows by oil red staining: as shown in fig. 6, the stained portion was stained fat (red color after oil red staining), and the non-induced a549 cells had no fat accumulation, whereas the a549 cells induced to differentiate for 16 days had significant fat accumulation. Compared with a control group, the induction of 200ng/mL IGF2 after 24 hours of pretreatment can obviously promote the differentiation of the fat cells.
The number of cells positive for oil red staining in the uninduced A549 cells is 0;
in the control group induced to differentiate for 16 days, the efficiency of differentiation of a549 cells into adipocytes was about 35%,
the efficiency of differentiation of A549 cells into adipocytes in this example group (200 ng/mL IGF2 pretreatment for 24 hours) induced to differentiate for 16 days was about 85%.
Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (3)

1. A method for inducing differentiation of cancer cells into adipocytes in vitro, comprising the steps of:
(1) Culturing cancer cells in a basal culture medium, adding insulin-like growth factor 2 with the final concentration of 100-300ng/mL when the confluence degree of the cells reaches 50-70%, and pretreating the cancer cells for 20-30 hours; the cancer cell is a cancer cell in an epithelial state and/or a cancer cell in a mesenchymal state;
(2) Changing the basic culture medium in the step (1) into an induced differentiation culture medium (1), and culturing for 4 days;
(3) Changing the induced differentiation medium (1) into an induced differentiation medium (2), and culturing for 2 days;
(4) Changing to a basic culture medium, and culturing for 10 days;
wherein the basic culture medium is a high-glucose DMEM culture medium only containing 5% -20% fetal calf serum, 100U/mL penicillin and 100 mu g/mL streptomycin;
the induced differentiation culture medium (1) is a basic culture medium only containing 0.5-1 mu M dexamethasone, 5-20 mu g/mL insulin, 1-2 mu M rosiglitazone and 0.2-0.5mM 1-methyl-3-isobutyl xanthine;
the induced differentiation culture medium (2) is a basal culture medium only containing 5-20 mu g/mL of insulin.
2. The method according to claim 1, wherein the differentiation-inducing medium (1) is the basal medium containing only 1 μ M dexamethasone, 10 μ g/mL insulin, 2 μ M rosiglitazone, 0.5mM 1-methyl-3-isobutylxanthine;
the induced differentiation medium (2) is the basal medium containing only 10 mug/mL of insulin.
3. The method of any one of claims 1-2, wherein the differentiation is induced under conditions of 35 ℃ to 37 ℃ and 5% CO 2 。
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