Exosome, mesenchymal stem cell and application of exosome and mesenchymal stem cell in treatment of type 2 diabetes mellitus

Abstract

The invention provides an exosome, a mesenchymal stem cell and application of the exosome and the mesenchymal stem cell in treatment of type 2 diabetes mellitus, and belongs to the technical field of biological medicine. The exosome secreted by the umbilical cord mesenchymal stem cells of the composite pretreatment group is used for treating the type 2 diabetes mellitus, and the obvious advantages are shown. Compared with a model control group, the exosome has the advantages that the glucose tolerance of diabetic rats treated by the exosome is obviously improved, the blood glucose value is reduced by about 47.5% within 120 minutes, the area under a blood glucose curve (AUC) is reduced by about 50.5%, the serum insulin is increased by about 147.7%, insulin secretion is effectively promoted, the glycosylated hemoglobin level is obviously reduced, and the long-term blood glucose control effect is good. By integrating various indexes, the exosome obtained by the composite pretreatment scheme provided by the invention has a remarkable effect of improving the illness state of rats with type 2 diabetes mellitus, provides a new effective strategy for treatment of type 2 diabetes mellitus, and shows a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to exosomes, mesenchymal stem cells and applications thereof in treating type 2 diabetes. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a common metabolic disease, and the number of patients worldwide continues to rise, which seriously threatens human health. Although traditional treatments can control the disease to a certain extent, it is difficult to achieve a complete cure and has many limitations. It is urgent to find new and effective treatments. As an important medium for intercellular communication, exosomes have shown great potential in the treatment of T2DM in recent years and have become a research hotspot. T2DM is mainly caused by insulin resistance and pancreatic β-cell dysfunction, with hyperglycemia as a typical feature. Long-term illness can cause a series of serious complications such as cardiovascular disease, nephropathy, and retinopathy. At present, the treatment of T2DM includes lifestyle intervention, drug therapy (such as metformin, insulin secretagogues, etc.), insulin injection, etc. However, drug therapy is often accompanied by adverse reactions such as hypoglycemia, weight gain, and gastrointestinal reactions. Long-term use may also cause drug tolerance, affecting the treatment effect; although insulin injection can effectively control blood sugar, frequent injections bring inconvenience to patients and may cause injection site infection, fat atrophy and other problems.

[0003] Exosomes are extracellular vesicles with a diameter of 30-150nm secreted by cells and widely present in various body fluids. They are rich in bioactive substances such as proteins, nucleic acids (mRNA, miRNA, etc.), lipids, etc., which give exosomes unique biological functions. Exosomes have low immunogenicity and can avoid being quickly cleared by the immune system, and can be used as natural drug carriers. At the same time, it can transmit biological information between cells, regulate the physiological functions of receptor cells, and participate in a variety of physiological and pathological processes. Studies have found that exosomes derived from mesenchymal stem cells have a positive effect on the treatment of T2DM. Mesenchymal stem cell exosomes can regulate the insulin signaling pathway, enhance insulin sensitivity, and promote glucose uptake and utilization by delivering specific miRNAs, thereby lowering blood sugar levels.

[0004] The activity of exosomes secreted by mesenchymal stem cells is within a certain range and is affected by cell culture conditions, exosome purification methods, etc. For example, the prior art records that in the process of mesenchymal stem cell culture, providing a physical environment conducive to the growth of chondrocytes, or adding cartilage protection small molecules, chondrogenic induction factors, inflammatory factors and other stimulating cells can achieve the improvement of the regulatory function of exosomes secreted by mesenchymal stem cells. For example, curcumin, as a polyphenol compound with antioxidant properties, plays an important role in the prevention and treatment of osteoarthritis. After mesenchymal stem cells are treated with curcumin, the exosomes they secrete show unique efficacy. These exosomes can promote the proliferation of chondrocytes and inhibit their apoptosis, which has a significant effect on alleviating osteoarthritis lesions. Further mechanism studies revealed that the exosomes secreted by mesenchymal stem cells treated with curcumin showed high expression of miR-124 and miR-143. miR-124 and miR-143 inhibit cartilage degradation by targeting and inhibiting the nuclear factor-activated B cell kappa-light chain enhancement and ROCK1 / TLR9 signaling pathways, respectively, thereby playing a positive regulatory role in the development of osteoarthritis (QIU B, XU X, YI P, et al. Curcumin reinforces MSC-derived exosomes in attenuating osteoarthritis via modulatingthe miR-124 / NF-kB and miR-143 / ROCK1 / TLR9 signalling pathways. J Cell Mol Med.2020; 24(18):10855-10865).

[0005] So far, there has been no research on enhancing the activity of exosomes in the treatment of type 2 diabetes. Summary of the invention

[0006] The purpose of the present invention is to provide an exosome, a mesenchymal stem cell and use thereof in the treatment of type 2 diabetes, which has excellent therapeutic activity for type 2 diabetes.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for preparing exosomes derived from mesenchymal stem cells, characterized in that it comprises the following steps: The mesenchymal stem cells are cultured in a culture medium containing 3-8 μmol / L resveratrol and 5-20 μmol / L metformin to obtain pretreated mesenchymal stem cells; The pretreated mesenchymal stem cells are used to extract secretory bodies to obtain the exosomes derived from the mesenchymal stem cells.

[0008] Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0009] Preferably, the amount of the culture medium is 10 4 ~10 6 cells / mL.

[0010] Preferably, the culture medium is α-MEM culture medium.

[0011] Preferably, the culture time is 24 to 72 hours.

[0012] The present invention also provides exosomes derived from mesenchymal stem cells obtained by the above preparation method.

[0013] The present invention also provides the use of the exosomes derived from mesenchymal stem cells in the preparation of a drug for treating type 2 diabetes.

[0014] The present invention also provides an application of a mesenchymal stem cell culture medium additive in enhancing the activity of mesenchymal stem cell exosomes, wherein the mesenchymal stem cell culture medium additive contains resveratrol and metformin; The concentration of resveratrol in the mesenchymal stem cell culture medium additive is 3-8 μmol / L, and the concentration of metformin is 5-20 μmol / L.

[0015] Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0016] The present invention also provides a mesenchymal stem cell, which can secrete the exosomes derived from the mesenchymal stem cell.

[0017] Beneficial effects of the present invention: The present invention utilizes exosomes secreted by umbilical cord mesenchymal stem cells in the composite pretreatment group to treat type 2 diabetes, showing significant advantages. Compared with the model control group, the glucose tolerance of diabetic rats treated with exosomes obtained by the composite pretreatment scheme was significantly improved, the 120-min blood glucose value decreased by about 47.5%, the area under the blood glucose curve (AUC) decreased by about 50.5%, and the serum insulin increased by about 147.7%, which effectively promoted insulin secretion and significantly decreased the level of glycosylated hemoglobin, indicating that the long-term blood glucose control effect was good. Comprehensively considering various indicators, the exosomes obtained by the composite pretreatment scheme provided by the present invention have a significant effect on improving the condition of type 2 diabetic rats, providing a new and effective strategy for the treatment of type 2 diabetes, and showing good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The graphs are the blood glucose measurement results of each treatment group; Figure 2 The area under the blood glucose curve of each group is shown in the figure; Figure 3 The results of serum insulin level test in each group are shown in Figure 2; Figure 4 The graphs show the glycated hemoglobin test results for each group. DETAILED DESCRIPTION

[0019] The present invention provides a method for preparing exosomes derived from mesenchymal stem cells, characterized in that it comprises the following steps: culturing mesenchymal stem cells with a culture medium containing 3-8 μmol / L resveratrol and 5-20 μmol / L metformin to obtain pretreated mesenchymal stem cells; and extracting secretions from the pretreated mesenchymal stem cells to obtain the exosomes derived from the mesenchymal stem cells.

[0020] In the present invention, preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells. Preferably, the amount of the culture medium is 10 4 ~10 6 cells / mL. Preferably, the culture medium is α-MEM culture medium. Preferably, the culture time is 24 to 72 hours.

[0021] The present invention also provides exosomes derived from mesenchymal stem cells obtained by the above preparation method.

[0022] The present invention also provides the use of the exosomes derived from mesenchymal stem cells in the preparation of a drug for treating type 2 diabetes. Preferably, the application of the exosomes includes but is not limited to the following: 1. Through tail vein injection, exosomes can quickly enter the blood circulation and be distributed to various tissues and organs throughout the body, especially the pancreatic islets, liver, muscles and other organs closely related to blood sugar regulation, thereby directly improving insulin resistance and promoting insulin secretion. This method of administration can ensure the bioavailability of exosomes, quickly reach an effective therapeutic concentration in the body, and is relatively simple to operate, which has high feasibility in animal experiments and future clinical applications.

[0023] 2. Use the method of local injection of pancreatic islets to make exosomes act precisely on damaged pancreatic islet tissue. The pancreatic islet is a key organ for regulating blood sugar. The function of pancreatic β cells in patients with type 2 diabetes is impaired. Local delivery of exosomes can increase its concentration in the pancreatic islet, enhance the repair and protection of pancreatic islet cells, promote pancreatic β cell regeneration and normal insulin secretion, and improve the core pathological mechanism of diabetes in a more targeted manner.

[0024] 3. Combine exosomes with degradable biological scaffold materials to prepare an implantable drug sustained-release system. This method allows exosomes to be released slowly and continuously in the body, prolonging their duration of action, reducing the number of dosings, and reducing the treatment burden on patients. For example, exosomes are wrapped in a hydrogel scaffold and implanted subcutaneously or intraperitoneally. The hydrogel can provide protection for the exosomes to prevent them from being quickly cleared, while gradually releasing the exosomes to maintain a stable blood drug concentration and achieve long-term therapeutic effects.

[0025] 4. Use nanotechnology to encapsulate or modify exosomes into nanoparticles to improve the stability and targeting of exosomes. Nanoparticles can be surface modified to connect ligands that specifically recognize surface markers of diabetes-related lesions, so that exosomes can be accurately enriched in lesion tissues, improve the therapeutic effect while reducing the impact on peripheral normal tissues, reduce potential adverse reactions, and provide a safer and more efficient route of administration for the clinical application of exosomes.

[0026] The present invention also provides an application of a mesenchymal stem cell culture medium additive in enhancing the activity of mesenchymal stem cell exosomes, wherein the mesenchymal stem cell culture medium additive contains resveratrol and metformin; the concentration of resveratrol in the mesenchymal stem cell culture medium additive is 3-8 μmol / L, and the concentration of metformin is 5-20 μmol / L. Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Example 1

[0029] α-MEM culture medium (containing 10% FBS) containing 5 μmol / L resveratrol and 10 μmol / L metformin was prepared for the culture of umbilical cord mesenchymal stem cells.

[0030] Experimental example

[0031] 1. Experimental Materials Neonatal umbilical cord, taken from healthy full-term newborns.

[0032] Experimental animals: Clean-grade male Wistar rats, 7-9 weeks old, weighing 200-240 g, were used to construct the rat model of type 2 diabetes mellitus (T2DM).

[0033] Reagents: α-MEM culture medium, high-quality fetal bovine serum (FBS), 0.25% trypsin-EDTA digestion solution, resveratrol, metformin, PBS buffer, exosome extraction reagent, blood glucose meter test strips, insulin ELISA test kit, glycated hemoglobin test kit, streptozotocin (STZ), citric acid-sodium citrate buffer (pH 4.5), and high-sugar and high-fat feed.

[0034] Instruments: sterile operation clean bench, CO2 constant temperature incubator, low-temperature centrifuge, phase contrast microscope, transmission electron microscope (TEM), Nanosight particle size analyzer, blood glucose meter, and microplate reader.

[0035] 2. Experimental Procedure 2.1 Isolation of neonatal umbilical cord mesenchymal stem cells Following medical ethics standards and obtaining informed consent from the mother, the umbilical cord of the newborn was collected aseptically in the delivery room, quickly placed in a sterile container containing α-MEM medium containing double antibiotics (penicillin 100 U / mL, streptomycin 100 μg / mL), and brought back to the laboratory. In the clean bench, the umbilical cord was gently rinsed several times with preheated PBS buffer until the residual blood on the surface was washed away.

[0036] Transfer the cleaned umbilical cord to a sterile dish, remove the surface blood vessels and connective tissue, and cut the remaining part into about 1-2 mm 3 Transfer the tissue pieces into a 50 mL centrifuge tube, add an appropriate amount of 0.25% trypsin-EDTA digestion solution, ensure that the tissue pieces are completely immersed, and digest in a 37°C shaker for 35 - 40 min, gently shaking the centrifuge tube every 10 min.

[0037] After digestion, add an equal volume of α-MEM medium containing 10% FBS to terminate the reaction, and gently pipette to disperse the tissue blocks into a single cell suspension. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Resuspend the cells in α-MEM medium containing 10% FBS and adjust the cell concentration to 1×10 5 The cells were inoculated into T25 culture flasks and cultured in a 37°C, 5% CO2 incubator.

[0038] The medium was changed for the first time 24 hours after inoculation to remove non-adherent cells and tissue fragments, and then the medium was changed every 3-4 days. The cell growth status was observed under a phase contrast microscope every day, and the cells were passaged when the cell confluence reached 80%-90%.

[0039] 2.2 Pretreatment and culture of umbilical cord mesenchymal stem cells In this study, we selected well-growing 3rd-5th generation umbilical cord mesenchymal stem cells, discarded the culture medium in the culture flask, and gently rinsed the cells 2-3 times with PBS buffer. Add an appropriate amount of 0.25% trypsin-EDTA digestion solution, digest at 37℃ for 2-3min, observe under a phase contrast microscope, and when the cells become round and begin to detach from the flask wall, add α-MEM culture medium containing 10% FBS to terminate digestion. Gently blow the cells to form a uniform suspension, and inoculate them into a new culture flask at a ratio of 1:3-1:4 to continue culturing.

[0040] When the cell confluence reached 70%-80%, the cells were divided into six groups (the amount of culture medium was about 1×10 5 cells / mL): Composite pretreatment group: α-MEM medium (containing 10% FBS) containing 5 μmol / L resveratrol and 10 μmol / L metformin was added.

[0041] Resveratrol pretreatment group: α-MEM medium (containing 10% FBS) containing 5 μmol / L resveratrol was added.

[0042] Metformin pretreatment group: α-MEM medium (containing 10% FBS) containing 10 μmol / L metformin was added.

[0043] Resveratrol combined with high-concentration metformin treatment group: α-MEM culture medium (containing 10% FBS) containing 5 μmol / L resveratrol and 20 μmol / L metformin was added.

[0044] Resveratrol combined with low-concentration metformin treatment group: α-MEM culture medium (containing 10% FBS) containing 5 μmol / L resveratrol and 5 μmol / L metformin was added.

[0045] Control group: α-MEM medium (containing 10% FBS) without resveratrol and metformin was added.

[0046] The six groups of cells were placed in a 37°C, 5% CO2 incubator and cultured for 48 h.

[0047] 2.3. Secretome extraction After 48 hours of pretreatment, the supernatants from the six groups of cell culture flasks were collected into centrifuge tubes and centrifuged at 4°C and 300×g for 10 minutes to remove cell debris. The supernatant was transferred to a new centrifuge tube and centrifuged at 4°C and 2000×g for 20 minutes to further remove larger cell debris. The supernatant after centrifugation was transferred to an ultrafiltration tube and centrifuged at 4°C and 10000×g for 40 minutes to concentrate the supernatant to about 1-2 mL. Use the exosome extraction reagent to extract exosomes according to the instructions, resuspend with an appropriate amount of PBS buffer, and store at -80°C for later use.

[0048] 2.4. Exosome verification: Take an appropriate amount of exosome resuspension and drop it on the copper mesh. Let it stand at room temperature for 5 min to allow the exosomes to adsorb on the copper mesh. Gently absorb the excess liquid with filter paper, add 2% phosphotungstic acid solution for negative staining, and stain at room temperature for 3-5 min. Use filter paper to absorb the excess dye again, wait for the copper mesh to dry naturally, and observe the morphological characteristics of exosomes under a transmission electron microscope. The results show that the obtained exosomes show typical exosome characteristics of flat, disc-shaped, and biconcave surfaces.

[0049] The exosome resuspension was appropriately diluted with PBS buffer to make its concentration within the detection range of the Nanosight particle size analyzer, and then injected into the instrument sample pool for particle size analysis. The exosome particle size distribution was recorded. The results showed that the particle size range was 60-100nm, indicating that the obtained exosomes were of high purity and could be used for subsequent experiments.

[0050] 2.5 Construction of type 2 diabetes rat model Wistar rats were purchased and kept in an animal room with an ambient temperature of (23 ± 2) °C, humidity of (55 ± 10)%, and a 12-h light / dark cycle. After 1 week of free access to food and water, all rats except the normal control group were fed a high-sugar and high-fat diet for 5 weeks to induce insulin resistance.

[0051] After 5 weeks of high-sugar and high-fat diet feeding, rats were fasted for 12 h and intraperitoneally injected with 35 mg / kg streptozotocin (STZ, prepared with 0.1 mol / L citric acid-sodium citrate buffer, pH 4.5). 72 h after STZ injection, blood was collected from the tail vein and fasting blood glucose was measured with a blood glucose meter. Rats with fasting blood glucose ≥16.7 mmol / L were considered to have successfully established a type 2 diabetes model.

[0052] 2.6 Animal Experiment Grouping and Dosing The successfully constructed type 2 diabetic rats were randomly divided into six groups, with 10 rats in each group: Composite pretreatment group: exosomes extracted from the composite pretreatment group were injected into the tail vein (the dose was 100 μg / kg, diluted to an appropriate volume with PBS buffer).

[0053] Resveratrol pretreatment group: Exosomes extracted from the resveratrol pretreatment group were injected into the tail vein (the dose was 100 μg / kg, diluted to an appropriate volume with PBS buffer).

[0054] Metformin pretreatment group: exosomes extracted from the metformin pretreatment group were injected into the tail vein (dose of 100 μg / kg, diluted to an appropriate volume with PBS buffer).

[0055] Resveratrol combined with high concentration of metformin: Exosomes extracted from the resveratrol combined with high concentration of metformin treatment group were injected into the tail vein (dose of 100 μg / kg, diluted to an appropriate volume with PBS buffer).

[0056] Resveratrol combined with low concentration of metformin: Exosomes extracted from the resveratrol combined with low concentration of metformin treatment group were injected into the tail vein (dose of 100 μg / kg, diluted to an appropriate volume with PBS buffer).

[0057] Model control group: an equal volume of PBS buffer was injected into the tail vein.

[0058] Another group of normal rats was set up as the normal control group without any treatment.

[0059] The drug was administered for 4 consecutive weeks, 3 times a week.

[0060] 3. Indicator detection and results 3.1 Glucose tolerance test: (intraperitoneal glucose tolerance test, IPGTT) After 4 weeks of administration, all rats were fasted for 12 h and intraperitoneally injected with glucose (2 g / kg). Blood was collected from the tail vein at 0, 30, 60, and 120 min after glucose injection, and blood glucose levels were measured with a blood glucose meter. The glucose tolerance curve was drawn and the area under the blood glucose curve (AUC) was calculated. One-way ANOVA was used to analyze the differences between the groups. If there were significant differences, Tukey's multiple comparison test was further used for pairwise comparison. The results are shown in Tables 1 and 2. Figure 1~2 As shown: Table 1 Glucose tolerance test results Group 0min blood glucose (mmol / L) 30-minute blood sugar (mmol / L) 60-minute blood glucose (mmol / L) 120min blood glucose (mmol / L) Blood glucose area under the curve (AUC) Normal control group 5.37±0.31 7.93±0.52 6.64±0.41 5.82±0.37 230.33±12.85 Model control group 18.86±1.33 26.90±1.73 24.63±1.52 20.90±1.63 617.05±27.22 Pretreatment compound reagent 11.21±0.94*** 14.82±1.13*** 12.28±1.03*** 9.22±0.83*** 282.47±16.24*** Resveratrol + high concentration metformin pretreatment 13.72±1.10** 18.23±1.34** 15.18±1.22** 12.15±0.98** 352.68±20.51** Resveratrol + low concentration metformin pretreatment 14.08±1.12** 18.52±0.32** 15.42±1.23** 12.45±1.02** 359.36±21.25** Resveratrol pretreatment group 16.62±1.22* 21.73±1.43* 16.40±1.32* 13.58±1.51* 385.17±22.38* Metformin pretreatment group 15.47±1.18* 19.51±1.37* 16.27±1.28* 13.42±1.08* 381.64±21.93* Among them, *, P < 0.05, the difference was significant compared with the model control group, **, P < 0.01, the difference was extremely significant compared with the model control group, ***, P < 0.001, the difference was extremely significant compared with the model control group; The results showed that the normal control group had normal blood sugar metabolism, while the model control group showed significant hyperglycemia and severe glucose metabolism disorders. The pretreatment composite reagent group provided by the present invention had the best effect in lowering blood sugar and improving blood sugar regulation ability, and the blood sugar values ​​at each time point were significantly lower than those of the model control group. The secretory bodies pretreated with the combined reagent of resveratrol and metformin also had obvious effects. The blood sugar and the area under the blood sugar curve (AUC) at each time point were significantly different from those of the model control group. The secretory bodies obtained by pretreatment with resveratrol or metformin alone could also improve blood sugar. The blood sugar and AUC at each time point were significantly different from those of the model control group, but the effect was relatively weak. After the umbilical cord mesenchymal stem cells were pretreated with the two drugs, the content of bioactive substances related to enhancing insulin sensitivity in the secreted exosomes may increase. After entering the body of diabetic rats, they act on target cells, repair and optimize the insulin signal transduction pathway, make cells more sensitive to insulin, and thus effectively lower blood sugar levels. The mechanism needs to be further explored in subsequent studies.

[0061] 3.2 Serum insulin level detection: After the glucose tolerance test, the rats were fasted for 12 h. The next day, blood was collected from the heart and centrifuged at 3000×g for 15 min to separate the serum. The serum insulin level was detected using the insulin ELISA test kit according to the instructions. The same one-way analysis of variance and Tukey's multiple comparison test were used for statistical analysis. The results are shown in Table 2 and Figure 3 As shown: Table 2 Serum insulin level test results Group Serum insulin level (mIU / L) Normal control group 15.87±1.31 Model control group 5.35±0.81 Composite pretreatment group 12.63±1.01** Resveratrol + high concentration metformin 9.12±0.90* Resveratrol + low concentration metformin 8.75±0.87* Resveratrol pretreatment group 8.02±0.84* Metformin pretreatment group 7.85±0.82* Among them, *, P < 0.05, there is a significant difference compared with the model control group, **, P < 0.01, there is an extremely significant difference compared with the model control group; The serum insulin level in the normal control group was within the normal range, reflecting that the rats in this group had normal insulin secretion function. The serum insulin level in the model control group was significantly lower than that in the normal control group, indicating that the pancreatic β-cell function of type 2 diabetic rats was impaired, insulin secretion was insufficient, and the model was successfully established.

[0062] The serum insulin level of the pretreatment composite reagent group provided by the present invention (12.63 ± 1.01) mIU / L was significantly different from that of the model control group (P < 0.01), close to the level of the normal control group. The exosomes in this group achieved effective improvement in insulin secretion, which may have a repairing effect on damaged pancreatic β cells or enhance the synthesis and secretion of insulin. The serum insulin levels of resveratrol + high concentration metformin, resveratrol + low concentration metformin, resveratrol pretreatment group and metformin pretreatment group were higher than those of the model control group (P < 0.05), but lower than those of the pretreatment composite reagent group provided in the embodiment of the present invention, indicating that the exosomes in each group can improve insulin secretion to a certain extent, but the pretreatment composite reagent group provided by the present invention has the best effect;.

[0063] 3.3 Glycated hemoglobin detection: After the glucose tolerance test, the rats fasted for 12 hours. The next day, blood was collected from the heart and centrifuged at 3000×g for 15 minutes to separate the serum. The glycosylated hemoglobin level was detected using the glycosylated hemoglobin detection kit according to the instructions. The statistical analysis method was the same as before. The results are shown in Table 3 and Figure 4 As shown: Table 3 Glycated hemoglobin level test results Group Glycated hemoglobin (%) Normal control group 4.43±0.30 Model control group 10.15±0.60 Composite pretreatment group 5.72±0.34** Resveratrol + High Concentration Metformin 7.52±0.46* Resveratrol + low concentration metformin 7.80±0.48* Resveratrol pretreatment group 8.35±0.54* Metformin pretreatment group 8.22±0.52* Among them: *, P < 0.05, significant difference compared with the model control group; **, P < 0.01, extremely significant difference compared with the model control group; The glycated hemoglobin in the normal control group was within the normal range, reflecting good long-term blood sugar control. The glycated hemoglobin in the model control group was significantly higher than that in the normal control group, indicating that the long-term hyperglycemia state caused glycation modification of hemoglobin.

[0064] The glycated hemoglobin (5.72 ± 0.34)% of the pretreatment composite reagent group provided by the present invention is significantly different from that of the model control group (P < 0.01), which is close to the normal level, indicating that the treatment scheme can not only improve short-term blood sugar levels, but also has a significant effect on long-term blood sugar control, by stabilizing blood sugar levels and reducing the glycation reaction of glucose and hemoglobin. The glycated hemoglobin levels of resveratrol + high concentration metformin, resveratrol + low concentration metformin, resveratrol pretreatment group and metformin pretreatment group were lower than the model control group (P < 0.05), but higher than the pretreatment composite reagent group provided by the present invention, indicating that each treatment scheme has an effect on long-term blood sugar control, but the exosomes using the culture medium of Example 1 of the present invention have the best effect.

[0065] Based on the above index detection and analysis results, the exosomes secreted by umbilical cord mesenchymal stem cells pretreated with different concentrations of combined reagents have different degrees of influence on the glucose tolerance, serum insulin level and glycosylated hemoglobin level of type 2 diabetic rats, but the specific ratio of the composite pretreatment group provided in this study has the most significant effect and has the best application prospect; this may be because resveratrol and metformin may have a synergistic effect in improving the activity of exosomes, but this synergistic effect depends on the appropriate concentration ratio. When the medium concentration is combined (resveratrol 5μmol / L, metformin 10μmol / L), the two can just achieve the exosome activity stimulation effect.

[0066] 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 preparing exosomes derived from mesenchymal stem cells, characterized in that: The following steps are involved: The mesenchymal stem cells are cultured in a culture medium containing 3-8 μmol / L resveratrol and 5-20 μmol / L metformin to obtain pretreated mesenchymal stem cells; The pretreated mesenchymal stem cells are used to extract secretory bodies to obtain the exosomes derived from the mesenchymal stem cells.

2. The method for preparing exosomes derived from mesenchymal stem cells according to claim 1, characterized in that: The mesenchymal stem cells are umbilical cord mesenchymal stem cells.

3. The method for preparing exosomes derived from mesenchymal stem cells according to claim 2, characterized in that: The amount of the culture medium is 10 4 ~10 6 cells / mL.

4. The method for preparing exosomes derived from mesenchymal stem cells according to claim 1, characterized in that: The culture medium is α-MEM culture medium.

5. The method for preparing exosomes derived from mesenchymal stem cells according to claim 1, characterized in that: The culture time is 24 to 72 hours.

6. Exosomes derived from mesenchymal stem cells obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the exosomes derived from mesenchymal stem cells according to claim 6 in the preparation of a drug for treating type 2 diabetes.

8. Use of a mesenchymal stem cell culture medium additive in enhancing the activity of mesenchymal stem cell exosomes, characterized in that: The mesenchymal stem cell culture medium additive contains resveratrol and metformin; The concentration of resveratrol in the mesenchymal stem cell culture medium additive is 3-8 μmol / L, and the concentration of metformin is 5-20 μmol / L.

9. The use according to claim 8, characterized in that: The mesenchymal stem cells are umbilical cord mesenchymal stem cells.

10. A mesenchymal stem cell, characterized in that: It is capable of secreting the exosomes derived from mesenchymal stem cells as described in claim 6.

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