Method for inducing umbilical cord mesenchymal stem cells to differentiate insulin-like cells and application thereof

Abstract

The invention discloses a method for inducing umbilical cord mesenchymal stem cells to differentiate insulin-like cells and application of the method, purified umbilical cord mesenchymal stem cells are reasonably cultured by adopting culture media with different substances, and the umbilical cord mesenchymal stem cells can be efficiently and stably induced to be differentiated into insulin-like cells. The percentage of insulin positive cells can reach 80%, and the cell preparation prepared after the umbilical cord mesenchymal stem cells are induced can be used for treating clinical diabetics and is high in curative effect.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, and in particular to a method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells and an application thereof. Background Art

[0002] Umbilical cord mesenchymal stem cells (MSCs) refer to a type of multifunctional stem cells found in the umbilical cord tissue of newborns. They can differentiate into many types of tissue cells and have broad prospects for clinical application. Umbilical cord mesenchymal stem cells (MSCs) have high differentiation potential and can differentiate into bone cells, nerve cells, insulin-like cells, etc. under certain conditions. It has broad prospects for clinical application in tissue engineering such as bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium and myocardium. Diabetes is the third most serious chronic non-communicable disease threatening human health after tumors and vascular diseases. It has the characteristics of high mortality, high disability and high medical expenses. Insulin is a hormone secreted by pancreatic β cells in the human pancreas and is essential for regulating blood sugar. For example, patients with type 1 diabetes (T1D) lose pancreatic β cells due to autoimmune reactions and are unable to secrete insulin autonomously. They then need to rely on exogenous insulin injections to maintain blood sugar balance. Stem cell differentiation into insulin-secreting cells can provide new pathways and directions for diabetes treatment. Although the existing technology, such as the Chinese invention with application number 2016109796896 "Method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-secreting-like cells", relies on a single culture medium in the process of inducing umbilical cord mesenchymal stem cells to differentiate into insulin-secreting-like cells, the induction differentiation efficiency is low, and the positive expression rate of insulin cells (based on markers) is less than 25%. Summary of the invention

[0003] Therefore, based on the above background, the present invention provides a method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells and its application, which has a high insulin cell positive rate and can provide a new approach and direction for the treatment of diabetes.

[0004] The technical solution of the present invention is:

[0005] A method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells, comprising the following steps:

[0006] S1: Preparation of umbilical cord mesenchymal stem cells from umbilical cord;

[0007] S2: Purified umbilical cord mesenchymal stem cells were taken and added to α-MEM culture medium containing 10 ng / ml bFGF, 10 ng / ml EGF and 5 wt% human platelet lysate to induce the umbilical cord mesenchymal stem cells to differentiate into nestin-positive progenitor cells NIP;

[0008] S3: After the cells fill the culture flask, the cells are digested and α-MEM culture medium containing glucose and 5 wt% human platelet lysate is added for 2-4 days;

[0009] S4: centrifugation, discarding the culture supernatant, taking the cells, washing them, adding them to α-MEM culture medium containing ITS-A and 5wt% human platelet lysate, and culturing them for 3-5 days;

[0010] S5: centrifugation, discarding the culture supernatant, taking out the cells, adding them to α-MEM culture medium containing 5wt% human platelet lysate with 1wt% ITS-A and niacinamide, and culturing for 2-4 days without changing the medium;

[0011] S6: Take the cells, discard the culture supernatant, add them to α-MEM culture medium containing 5wt% human platelet lysate with 30ng / ml β-cell regulatory factor, 10ng / ml IGF, 1wt% ITS-A and niacinamide, and culture for 2-5 days without changing the medium.

[0012] Furthermore, the concentration of glucose in the α-MEM culture medium in step S3 is 25 mM.

[0013] Furthermore, the concentration of ITS-A in the α-MEM culture medium in steps S4 to S6 is 1 wt %.

[0014] Furthermore, the concentration of niacinamide in the α-MEM culture medium in steps S5 and S6 is 10 mM.

[0015] Furthermore, the cell culture conditions in steps S3 to S6 are: temperature: 37°C-38°C, 5% CO 2 .

[0016] Furthermore, the preparation of umbilical cord mesenchymal stem cells in step S1 comprises the following steps:

[0017] S1.1 Take the umbilical cord, remove the artery and vein, cut it into small pieces, stick it in a culture dish, and drip the umbilical cord mesenchymal stem cell culture medium onto the small pieces of umbilical cord;

[0018] S1.2 After 2-6 hours of incubation, add umbilical cord mesenchymal stem cell-specific culture medium;

[0019] S1.3 After 5-7 days of continuous culture, the cells were digested and subcultured with trypsin and supplemented with a special culture medium for umbilical cord mesenchymal stem cells during the culture process;

[0020] S1.4 When the cell density around the tissue block reaches about 90%, trypsinize the cells, count them, transfer them to culture, and mark them as P1 cells;

[0021] S1.5 The cells are subcultured until the third generation, at which time the cells are digested and collected;

[0022] S1.6 Purify and expand the third generation cells collected in step S1.5 in α-MEM medium containing 5wt% human platelet lysate.

[0023] Furthermore, the special culture medium for umbilical cord mesenchymal stem cells in steps S1.1, S1.2 and S1.3 is an α-MEM culture medium containing 5 wt% human platelet lysate, 100 U / ml penicillin and 100 U / ml streptomycin.

[0024] Based on the same inventive concept, the present invention also provides a cell preparation, which is prepared from cells induced by the method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells.

[0025] Based on the same inventive concept, the present invention also provides a method for preparing a cell preparation, characterized in that it comprises the following steps: 1) taking cells induced by the method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells;

[0026] 2) The induced cells were washed with PBS, trypsinized, centrifuged, and washed again with PBS;

[0027] 3) Discard the supernatant, resuspend the cells with physiological saline, and then wash them twice by centrifugation.

[0028] 4) Discard the supernatant, resuspend the cells with physiological saline, count the cell number and activity, and prepare a cell suspension.

[0029] Based on the same inventive concept, the present invention also applies the cells induced by the above-mentioned method of inducing umbilical cord mesenchymal stem cells to prepare preparations for treating or assisting in treating diabetes.

[0030] The beneficial effects achieved by the technical solution of the present invention are:

[0031] The present invention reasonably cultures the purified umbilical cord mesenchymal stem cells using culture media with different substances, provides suitable environmental conditions for different stages of differentiation of the umbilical cord mesenchymal stem cells, and can efficiently and stably induce them to differentiate into insulin-like cells, with the percentage of insulin-positive cells reaching 80%.

[0032] The cell preparation prepared by inducing umbilical cord mesenchymal stem cells can be used to treat clinical diabetics with high efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Attached Figure 1This is a diagram showing the growth of P2 human umbilical cord mesenchymal stem cells cultured in a φ90 mm culture dish in Example 1.

[0034] Attached Figure 2 This is a graph showing the growth of P4 human umbilical cord mesenchymal stem cells in a T25 culture flask in Example 1.

[0035] Attached Figure 3 This is the morphology of stem cells after 6 days of induction in Example 1.

[0036] Attached Figure 4 This is the morphology of stem cells after 12 days of induction in Example 1.

[0037] Attached Figure 5 This is the stem cell flow cytometry detection diagram of Example 1.

[0038] Attached Figure 6 This is a staining for the multilineage differentiation of stem cells in the example. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] The technical solution of the present invention is a method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells, comprising the following steps:

[0041] S1: Preparation of umbilical cord mesenchymal stem cells from umbilical cord;

[0042] The preparation of umbilical cord mesenchymal stem cells in this step includes the following steps:

[0043] S1.1 Take the umbilical cord, remove the artery and vein, cut it into small pieces, stick it in a culture dish, and drip the umbilical cord mesenchymal stem cell culture medium onto the small pieces of umbilical cord;

[0044] S1.2 After 2-6 hours of incubation, add umbilical cord mesenchymal stem cell-specific culture medium;

[0045] The special culture medium for umbilical cord mesenchymal stem cells of S1.1 and S1.2 is α-MEM culture medium containing 5wt% human platelet lysate, 100U / ml penicillin, and 100U / ml streptomycin.

[0046] S1.3 After 5-7 days of continuous culture, the cells were digested and subcultured with trypsin and supplemented with a special culture medium for umbilical cord mesenchymal stem cells during the culture process;

[0047] S1.4 When the cell density around the tissue block reaches about 90%, trypsinize the cells, count them, transfer them to culture, and mark them as P1 cells;

[0048] S1.5 The cells are subcultured until the third generation, at which time the cells are digested and collected;

[0049] S1.6 Purify and expand the third generation cells collected in step S1.5 in α-MEM medium containing 5wt% human platelet lysate.

[0050] S2: Purified umbilical cord mesenchymal stem cells were taken and added to α-MEM culture medium containing 10 ng / ml bFGF, 10 ng / ml EGF and 5 wt% human platelet lysate to induce the differentiation of umbilical cord mesenchymal stem cells into nestin-positive progenitor cells NIP; after the cells filled the culture flask, the P4 cells were digested and α-MEM culture medium containing 25 mM glucose and 5 wt% human platelet lysate was added, and the culture was continued at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator for 3 days, and the cell changes were dynamically observed;

[0051] In this step, purified mesenchymal stem cells of the 3rd to 6th generations are used for induction of differentiation. Since the genomes of the 1st and 2nd generations of mesenchymal stem cells have unstable factors and the number of cell amplification is insufficient, they are not suitable for clinical application. After the P7 generation, the proliferation ability and genetic stability of mesenchymal stem cells decrease sharply, and the number of cell apoptosis also increases, and they are not suitable for efficient induction of differentiation into insulin cells.

[0052] S3: After the cells fill the culture flask, digest the cells and add α-MEM culture medium containing 25 mM glucose and 5 wt% human platelet lysate. Temperature: 37°C-38°C, 5% CO 2 Cultivate for 2-4 days;

[0053] S4: Centrifuge, discard the culture supernatant, take the cells, wash the cells, and add them to α-MEM culture medium containing 5wt% human platelet lysate with 1wt% ITS-A, temperature: 37℃-38℃, 5% CO 2 Cultivate for 3-5 days;

[0054] Adding ITS-A to the culture medium in this step and the following steps can better promote cell proliferation, promote cell growth and differentiation in vitro, and improve cell activity and differentiation efficiency.

[0055] S5: Take the cells, discard the culture supernatant, and add them to α-MEM culture medium containing 5 wt% human platelet lysate with 1 wt% ITS-A and 10 mM nicotinamide, temperature: 37°C-38°C, 5% CO 2 , culture for 2-4 days without changing the medium;

[0056] Adding niacinamide to the culture medium in this step and the next step helps the mesenchymal stem cells to differentiate and proliferate into insulin-like cells, and helps to increase the differentiation rate and positive rate of insulin cells.

[0057] S6: Take the cells, discard the culture supernatant, add them to α-MEM culture medium containing 5wt% human platelet lysate with 30ng / ml β-cell regulatory factor, 10ng / ml IGF, 1wt% ITS-A and 10mM niacinamide, and culture for 2-5 days without changing the medium.

[0058] Example 1: This example further describes the implementation of the present invention in detail.

[0059] (1) Preparation of umbilical cord mesenchymal stem cells, comprising the following steps:

[0060] 1) Take fresh umbilical cord (healthy newborns born naturally or by cesarean section), wash the residual blood on the surface of the umbilical cord with PBS in a GMP standardized laboratory, and cut it into pieces of about 2 cm;

[0061] After washing again with PBS, remove the two arteries in the umbilical cord with ophthalmic forceps. Cut the umbilical cord along the umbilical vein and scrape the vein with ophthalmic forceps. Wash the umbilical cord segment with the artery and vein removed and cut it into 1 mm pieces with ophthalmic scissors. 3 The tissue blocks are evenly attached to a φ90mm culture dish with a spacing of about 5mm. After 5-10 minutes, α-MEM culture medium containing 10% autologous cord blood inactivated plasma, 100U / ml penicillin, and 100U / ml streptomycin is dripped onto each tissue block (just enough to infiltrate the tissue block).

[0062] Umbilical cord donors should at least be free of diseases caused by infection with pathogenic microorganisms such as hepatitis B virus, hepatitis C virus, HIV, Treponema pallidum, Epstein-Barr virus, and cytomegalovirus.

[0063] 2) Place at 37°C, 5% CO 2 After incubation in the incubator for 4 hours, take out the cells and add 2-3 ml of umbilical cord mesenchymal stem cell-specific culture medium.

[0064] Replace the culture medium (special culture medium for umbilical cord mesenchymal stem cells) semi-quantitatively every 2-3 days; be gentle throughout the process to avoid tissue chunks from falling off.

[0065] 3) After about 5 days of umbilical cord tissue culture, some cells can be seen crawling out from around the tissue mass, with a tiny spindle-shaped morphology;

[0066] After 7 days, the cells began to proliferate rapidly and formed cell colonies of varying sizes. After the cells were fully grown, they were digested and passaged with 0.25% trypsin.

[0067] On days 12-15, a large number of spindle-shaped cells can be seen crawling out of the tissue mass in the culture dish.

[0068] When the cell density around the tissue block reached about 90%, the cells were digested with trypsin and counted according to 1×10 3 / cm 2 Transfer cells to 25 cm 2 The cells were cultured in flasks and labeled as P1 cells.

[0069] After the cells fill the culture flask (about 3 days), normal passage was performed at a ratio of 1:3. The cell growth was observed at any time.

[0070] When the cells reached the third generation, trypsin was digested and a certain amount of cells were collected for cell counting, viability and phenotype determination, and cryopreservation.

[0071] The adherent cells all expressed CD44 and CD29, low expression of CD106, no expression of hematopoietic cell phenotype CD14, CD34, CD45 and endothelial cell phenotype CD31, and no expression of HLA-DR: the cell doubling time was 30h, and cell cycle analysis showed that the proportions of mesenchymal stem cells in the G0-G1 phase and +G2+M phase after culture were 78.84% and 11.16%, respectively.

[0072] 4) The third generation cells were purified and amplified using α-MEM medium containing 5 wt% human platelet lysate, 100 U / ml penicillin, and 100 U / ml streptomycin;

[0073] During the process of culturing mesenchymal stem cells, the cells will be gradually purified as the number of generations increases. At the P3 generation, the cells grow in a long spindle shape and adhere to the wall, with a typical "fish-like" or "whirlpool-like" distribution.

[0074] (2) Inducing differentiation of insulin-like cells, comprising the following steps:

[0075] 1) Purified P3 cells were taken and induced to differentiate into nestin-positive progenitor cells (NIP) using factors such as bFGF and EGF;

[0076] 2) On the 4th day of culture, the cell culture supernatant was discarded, the cells were washed once with PBS, and α-MEM culture medium containing 5 wt% human platelet lysate of ITS-A (insulin-transferrin-seleno-sodium pyruvate) was added: 37°C, 5% CO 2 The cells were cultured in a cell culture incubator for 4 days without changing the medium, and the cell status was observed every day.

[0077] 3) On day 8, the cell culture supernatant was discarded, and α-MEM culture medium containing 5 wt% human platelet lysate containing 1 wt% ITS-A and 10 mM nicotinamide was added and cultured at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator for 3 days without changing the medium, and the cell changes were dynamically observed.

[0078] 4) On day 11, the cell culture supernatant was discarded and α-MEM medium containing 5% human platelet lysate containing 30 ng / ml β-cell regulator, 10 ng / ml IGF, 1 wt% ITS-A and 10 mM nicotinamide was added and cultured at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator for 4 days without changing the medium. The cell changes were dynamically observed and then identified from multiple aspects such as morphology.

[0079] During the process of inducing differentiation of insulin-like cells, the cells gradually changed from spindle-shaped to polygonal and round, and the refractive index increased until most of the cells became round.

[0080] Flow cytometry identification of human umbilical cord mesenchymal stem cells after induced differentiation:

[0081] Take the induced cells, wash with PBS and then digest with trypsin (the digestion time should not be too long, generally 20 seconds is enough), add culture medium to neutralize the trypsin and put it into a 15ml centrifuge tube, then centrifuge it in a centrifuge (1000rpm / 5min), and wash it again with PBS.

[0082] After cell counting, cells (5×10 5 ) were dispensed into 15 ml centrifuge tubes, and after centrifugation, the volume was fixed to 400 μl with PBS containing 0.1 wt% FBS. Subsequently, the relative dose of antibody was added according to the dose recommended in the antibody manual, and the mixture was incubated at 4°C in the dark for 30 minutes.

[0083] After staining, the cells were washed with PBS and then fixed to 600 μl. The cells were filtered into a flow cytometer using a 200-mesh nylon gauze.

[0084] The test was performed on a FACSCalibur to exclude non-specific signal interference and ensure the accuracy of the experiment. The antibody tube was used as a negative reference. After adjustment, the expression of the corresponding antibody in each tube was tested. The results are shown in the table. Figure 5 The percentage of insulin-positive cells can reach 80%.

[0085] Example 2: The cell preparation used in this example is prepared by removing the supernatant from the cells cultured in step 4) of Example 1.

[0086] Take the induced cells, wash with PBS and then digest with trypsin (the digestion time should not be too long, generally 20 seconds is enough): add culture medium (α-MEM) to neutralize the trypsin and put it into a 15ml centrifuge tube, then centrifuge it in a centrifuge (1000rpm / 5min), and wash it again with PBS.

[0087] The supernatant was discarded, the cells were resuspended in physiological saline, and then centrifuged and washed twice.

[0088] The supernatant was discarded, the cells were resuspended with physiological saline, the cell number and activity were counted, and a cell suspension was prepared.

[0089] The subsequent clinical application will be reviewed by the ethics committee.

[0090] A total of 325 patients diagnosed with diabetes were recruited (203 males, 122 females, aged 10-70 years old, 129 patients with type 1 diabetes, 196 patients with type 2 diabetes). All patients were evaluated by the endocrinology department. All patients controlled their diet and performed appropriate physical exercise during the treatment process.

[0091] Before the cell preparation treatment, insulin and oral hypoglycemic drugs were adjusted to maintain the patient's blood sugar at a stable level (blood sugar level at 7.0-10.0mmol / L, glycosylated hemoglobin level at 6.0-8.0%). Cell preparations were intravenously infused 1 week later, and then once every 2 weeks for a total of 2-3 times. The total number of cells infused for each patient was (1-3)×10 6 / kg, and the average total number of cells transfused per patient was 1.8×10 6 / kg.

[0092] During the cell preparation treatment, try not to take any hypoglycemic drugs or inject insulin unless the patient has obvious hypoglycemia or hyperglycemia and requires emergency treatment.

[0093] Efficacy evaluation criteria

[0094] (1) Significant effect: Blood sugar is reduced by more than 20% without oral hypoglycemic drugs or insulin injections.

[0095] (2) Effective: When blood sugar is controlled at the pre-treatment level, the dosage of hypoglycemic drugs or insulin is reduced by 1 / 3;

[0096] All patients underwent detailed medical history, physical examination, and relevant laboratory tests:

[0097] (1) Medical history: age, gender, height, weight, duration of diabetes, dosage of insulin and oral hypoglycemic drugs, blood sugar control, history of drug and food allergies, etc.;

[0098] (2) Physical examination: presence of diabetic complications;

[0099] (3) Inspection:

[0100] ① Fasting blood sugar and 2 hours after meals;

[0101] ② C-peptide release experiment: Each patient had fasting venous blood drawn for testing of blood sugar and C-peptide, and then took 100g of steamed bread or bread orally. The blood sugar and C-peptide were drawn 1, 2, and 3 hours after the start of eating.

[0102] ③HbA1c;

[0103] ④T cell subsets;

[0104] ⑤Regulate T lymphocytes

[0105] ⑥ Check whether there are any obvious adverse reactions. If any adverse reactions occur, they should be recorded in time and active symptomatic treatment should be given.

[0106] Efficacy evaluation:

[0107] 325 patients were treated with the cell preparation containing insulin-like cells prepared by the present invention. The results showed that 293 patients had their blood sugar levels reduced by 20% and their glycosylated hemoglobin returned to normal without oral hypoglycemic drugs or insulin injections. The blood sugar levels were stable and no complications occurred.

[0108] In addition, 98 patients with type 2 diabetes did not need to take hypoglycemic drugs or reduced the dosage, and no renal function and peripheral nerve complications occurred. Blood sugar and glycosylated hemoglobin fluctuated slightly. Ultimately, the occurrence and development of diabetic complications were controlled clinically, achieving the goal of clinical cure.

[0109] In addition, during the tissue repair process, the cell preparation of the present invention can also protect the residual β cells through paracrine effects, improve the insulin sensitivity of peripheral target tissues, inhibit oxidative stress, stimulate capillary angiogenesis, promote cell proliferation, and reduce cell apoptosis. Mesenchymal stem cells can promote the regeneration of pancreatic β cells: they have the potential to differentiate into pancreatic β cells, and can supplement the loss of pancreatic β cells caused by diabetes, thereby restoring or improving the ability to secrete insulin. 2. Immunomodulatory effect: In type 1 diabetes, autoimmune reaction is the main cause of pancreatic β cell destruction. Mesenchymal stem cells can reduce autoimmune reactions and protect the remaining pancreatic β cells by inhibiting T cell proliferation and reducing the secretion of inflammatory factors. 3. Improve microvascular lesions: Diabetic patients are often accompanied by microvascular lesions, which affect organ function. Mesenchymal stem cells can promote the formation of new blood vessels, improve blood circulation, and help alleviate complications caused by diabetes. 4. Anti-inflammatory effect: Mesenchymal stem cells have significant anti-inflammatory effects, can reduce the production of inflammatory mediators, improve insulin resistance, and have positive significance for the treatment of type 2 diabetes.

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells, characterized in that: The steps include: S1: Preparation of umbilical cord mesenchymal stem cells from umbilical cord; S2: Purified umbilical cord mesenchymal stem cells are taken and added into α-MEM culture medium containing bFGF, EGF and 5wt% human platelet lysate to induce the umbilical cord mesenchymal stem cells to differentiate into nestin-positive progenitor cells NIP; S3: After the cells fill the culture flask, the cells are digested and α-MEM culture medium containing glucose and 5 wt% human platelet lysate is added for 2-4 days; S4: Take the cells, discard the culture supernatant, wash them, add them to α-MEM culture medium containing ITS-A and 5wt% human platelet lysate, and culture them for 3-5 days; S5: Take the cells, discard the culture supernatant, add them to α-MEM culture medium containing ITS-A and niacinamide and 5 wt% human platelet lysate, and culture for 2-4 days without changing the medium; S6: Take the cells, discard the culture supernatant, add them to α-MEM culture medium containing 5 wt% human platelet lysate with β-cell regulin, IGF, ITS-A and niacinamide, and culture for 2-5 days without changing the medium.

2. The method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells according to claim 1, characterized in that: The concentration of glucose in the α-MEM culture medium in step S3 is 25 mM.

3. The method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells according to claim 1, characterized in that: The concentration of ITS-A in the α-MEM culture medium in steps S4 to S6 is 1 wt %.

4. The method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells according to claim 1, characterized in that: The concentration of nicotinamide in the α-MEM culture medium in steps S5 and S6 is 10 mM.

5. The method of inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells according to claim 1, characterized in that: The conditions for cell culture in steps S3 to S6 are: temperature: 37° C.-38° C., 5% CO 2 .

6. The method of inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells according to claim 1, characterized in that: The preparation of umbilical cord mesenchymal stem cells in step S1 comprises the following steps: S1.1 Take the umbilical cord, remove the artery and vein, cut it into small pieces, stick it in a culture dish, and drip the umbilical cord mesenchymal stem cell culture medium onto the small pieces of umbilical cord; S1.2 After 2-6 hours of incubation, add umbilical cord mesenchymal stem cell-specific culture medium; S1.3 After continuing to culture for 5-7 days, the cells were digested and subcultured with trypsin, and the umbilical cord mesenchymal stem cell-specific culture medium used in step S1.2 was added during the culture process; S1.4 When the cell density around the tissue block reaches about 90%, trypsinize the cells, count them, transfer them to culture, and mark them as P1 cells; S1.5 The cells are subcultured until the third generation, at which time the cells are digested and collected; S1.6 Purify and expand the third generation cells collected in step S1.5 in α-MEM medium containing 5wt% human platelet lysate.

7. The method of inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells according to claim 6, characterized in that: The special culture medium for umbilical cord mesenchymal stem cells in step S1.2 is α-MEM culture medium containing 5wt% human platelet lysate, 100U / ml penicillin, and 100U / ml streptomycin.

8. A cell preparation, characterized in that: The method is prepared from cells induced by the method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells as described in any one of claims 1 to 7.

9. A method for preparing a cell preparation, characterized in that: It includes the following steps: 1) Cells induced by the method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells according to any one of claims 1 to 7; 2) The induced cells were washed with PBS, trypsinized, centrifuged, and washed again with PBS; 3) Discard the supernatant, resuspend the cells with physiological saline, and then wash them twice by centrifugation. 4) Discard the supernatant, resuspend the cells with physiological saline, count the cell number and activity, and prepare a cell suspension.

10. Use of the method for inducing umbilical cord mesenchymal stem cells to differentiate into insulin-like cells according to any one of claims 1 to 7 in preparing a preparation for treating or assisting in treating diabetes.

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