Method for repairing damaged pancreas islet beta cells, product and application

Abstract

The invention relates to the technical field of cells, and particularly discloses a method for repairing damaged pancreatic beta cells, a product and application. The research finds that the sEV and the liquiritigenin are combined to improve the repairing effect of the injured pancreatic beta cells, the invention further provides a method for repairing the injured pancreatic beta cells with the non-disease diagnosis or treatment purpose, and the sEV and the liquiritigenin are combined to repair the injured pancreatic beta cells. According to the repair method provided by the invention, only a small amount of liquiritigenin and sEV can generate a synergistic effect for repair, and the repair method can be used as a new mode for treating diabetes mellitus by combining sEV and medicines. The repairing method provided by the invention is high in repairing efficiency, and the method for repairing the damaged pancreatic beta cells is expected to be applied clinically.

Description

Technical Field

[0001] The present invention relates to the field of cell technology, and in particular to a method, product and application for repairing damaged pancreatic beta cells. Background Art

[0002] Diabetes is a complex metabolic disease whose core feature is hyperglycemia. This hyperglycemic state is caused by defects in insulin secretion or impaired biological functions. Long-term hyperglycemia can cause chronic damage to multiple organs and tissues, leading to functional disorders. Insulin is secreted by pancreatic β cells. Both type 1 diabetes and type 2 diabetes have the problem of pancreatic β cell failure. The difference between the two is that type 1 diabetes requires exogenous insulin to maintain life in the early stages of the disease, while β cell failure in type 2 diabetes usually occurs in the late stages of the disease.

[0003] Therefore, repairing damaged pancreatic β-cell function has always been the goal pursued by the medical community and diabetic patients. People are currently pinning their hopes on extracellular vesicles to achieve this goal, and alternative strategies for diabetes treatment represented by vesicle therapy have been widely studied. Among them, mesenchymal stem cell extracellular vesicle therapy is one of the key areas of research, and currently it is mostly focused on using extracellular vesicles of a certain type of mesenchymal stem cell to repair pancreatic β-cell damage.

[0004] Extracellular vesicles (EVs) are a type of vesicle released by prokaryotes and higher eukaryotic cells, and are conserved throughout the evolution of organisms. Extracellular vesicles are divided into three types according to their production mechanism, namely exosomes (EXO), microvesicles (MV), and apoptotic bodies, and there are certain differences in the diameter of the vesicles. Because their sizes overlap, there are certain challenges in distinguishing exosomes from other extracellular vesicles. For the convenience of research and description, "sEV" (small extracellular vesicles) and "lEV" (large extracellular vesicles) are usually used to refer to extracellular vesicles of different sizes.

[0005] Mesenchymal stem cells (MSC) are a powerful multipotent stem cell with multiple advantages. They are widely available and abundant in number, making them relatively easy to obtain. In addition, the separation, culture, expansion and purification process of MSC is relatively simple, which greatly improves its feasibility in clinical application. More importantly, the use of MSC does not involve ethical disputes and does not cause immune rejection, which greatly facilitates its application in the medical field. The immunoregulatory function of MSC itself enables it to show unique therapeutic effects in the treatment of various diseases. At present, clinical studies have confirmed that MSC can effectively treat complex diseases such as cardiovascular and cerebrovascular diseases, diabetes, liver and kidney damage, and brain and spinal cord nerve damage. The powerful repair ability of this cell gives it broad application prospects in the fields of tissue engineering and regenerative medicine.

[0006] Liquiritigenin (LQ) is a flavonoid isolated from licorice, which has cytoprotective, anti-inflammatory and anti-tumor properties. In addition, LQ is a plant-derived estrogen receptor β agonist, which has been reported to be an antidiabetic drug with hypoglycemic effects in streptozotocin (STZ)-induced diabetic mice.

[0007] During the long-term treatment of diabetes, a large amount of medication is required. Excessive doses can easily have adverse effects on the body, and many of these drugs are toxic, and their effects on human use are unknown. The use of mesenchymal stem cell extracellular vesicles to treat diabetes is generally cumbersome, the in vitro culture and collection cycle is long, and the yield is low, making it unsuitable for widespread clinical use. Currently, the efficiency of extracellular vesicles (EVs) in repairing damage is low, and a large amount of drugs are still used for treatment. The use of EVs for repair is in the exploratory stage and cannot meet clinical needs. Summary of the invention

[0008] One of the purposes of the present invention is to provide a new and efficient method for repairing pancreatic β cells.

[0009] The present invention provides a method for repairing damaged pancreatic β cells for non-disease diagnosis or treatment purposes, which uses sEV and glycyrrhizin in combination to repair damaged pancreatic β cells.

[0010] The present invention found that sEV and glycyrrhizin have a synergistic effect in repairing damaged pancreatic β cells. The use of glycyrrhizin alone has limited repair effects on pancreatic β cells, but when used together with sEV, the original repair efficiency of sEV can be further improved, achieving sEV-mediated efficient repair of intercellular communication damage. Compared with traditional extracellular vesicle therapy, the present invention only uses a non-toxic and low-cost small chemical molecule (glycyrrhizin has been proven to have many benefits to the human body and is a medicinal product) in combination with sEV, which improves the repair efficiency and level of pancreatic β cells, greatly reduces the cost of treatment, and is safer and more efficient.

[0011] The method of the present invention can be applied to non-disease diagnosis or treatment purposes such as research on the mechanism of pancreatic β cell damage, development of pancreatic β cell damage treatment products, and drug efficacy evaluation.

[0012] In the method of the present invention, licorice is added for treatment while the pancreatic β cells are damaged, and then sEV is added for treatment after the pancreatic β cells are damaged.

[0013] In the method of the present invention, the working concentration of liquiritigenin is 5-10 μM, and the working concentration of sEV is 25-50 μg / mL.

[0014] In the method of the present invention, preferably, the working concentration of glycyrrhizin is 5 μM, and the working concentration of sEV is 25 μg / mL.

[0015] In the method of the present invention, the licorice radical treatment time is 24 hours, and the sEV treatment time is 48 hours.

[0016] In the method of the present invention, the sEV is a small extracellular vesicle produced by stem cells with repair function; the particle size range of sEV is 75-250nm.

[0017] Preferably, the stem cells are mesenchymal stem cells, more preferably human placental mesenchymal stem cells.

[0018] In the method of the present invention, when the stem cells are used to produce small extracellular vesicles, the serum in the culture system is serum without extracellular vesicles.

[0019] As a specific embodiment, the method for repairing damaged pancreatic beta cells provided by the present invention may include: single repair and combined repair; the working concentration of sEV in the single repair is 1.56-50 μg / mL, and the working time is 24-72h; in the combined repair, the working concentration of sEV is 25 μg / mL, the working time is 48h, and the working concentration of glycyrrhizin is 1-10 μM.

[0020] The specific repair process may include: after plating INS-1 cells, when the cells are 30%-50% confluent, adding a damage system with a palmitic acid working concentration of 0.5mM and a protection system with a glycyrrhizin working concentration of 5μM, culturing for 24 hours, and then adding a culture system with a sEV working concentration of 25μg / mL, replacing the sEV culture system every 24 hours, and culturing for a total of 48 hours.

[0021] In the method provided by the present invention, in order to ensure good repair efficiency, EV-free serum is used in the INS-1 cell culture system.

[0022] In the method provided by the present invention, the sEV is derived from human placental mesenchymal stem cells (hPMSC), and EV-free serum is used in the human placental mesenchymal stem cell (hPMSC) culture system; the particle size range of the isolated sEV is 75nm-250nm. The PMSC used in the present invention is currently recognized as a cell for studying extracellular vesicles to repair diseases.

[0023] In the method provided by the present invention, when obtaining sEV, the method used is specifically as follows: culturing human placental mesenchymal stem cells (hPMSC), collecting the culture medium every 48 hours, centrifuging at 300g and 2000g to remove dead cells and cell debris, centrifuging at 12000g to remove large extracellular vesicle precipitates, ultracentrifuging at 120000g to remove the supernatant to obtain the precipitate, adding PBS to resuspend the precipitate and then ultracentrifuging at 120000g once, removing the supernatant and resuspending the precipitate with PBS, and storing at 4°C for future use.

[0024] The present invention also provides a product for repairing damaged pancreatic beta cells, which comprises small extracellular vesicles produced by cells with repairing function and glycyrrhizin.

[0025] The present invention also provides the use of the above product in improving the repair efficiency of damaged pancreatic islet β cells.

[0026] The beneficial effects of the present invention are at least: The method provided by the present invention can repair damaged pancreatic beta cells more efficiently and safely, and can be used for the treatment of late-stage diabetes, so as to avoid or solve the problems of poor effect and high price caused by the use of EV or glycyrrhizin alone. In addition, it overcomes the problems of cumbersome steps, long repair time, toxicity of chemical small molecules, etc. in traditional stem cell therapy, as well as the problem that the efficiency of EV in repairing damage is too low.

[0027] The method provided by the present invention can be used to culture PMSC in vitro and collect the sEVs secreted by them, and then inject sEVs and glycyrrhizin into the patient's body in combination, without relying on a large amount of sEVs and glycyrrhizin for treatment alone. This method greatly reduces the cost of treatment and eliminates immune rejection reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The flowchart and results of the pancreatic β cell injury model of the present invention; wherein A: schematic diagram of the preparation of palmitic acid solution and the injury mode of INS-1 cells; B: the effect of palmitic acid concentration and injury time on INS-1 survival; C: the effect of palmitic acid concentration on INS-1 apoptosis; D: statistical graph of the effect of palmitic acid concentration on INS-1 apoptosis.

[0029] Figure 2 Schematic diagram of the process of separating sEV and lEV and the identification results of the present invention; wherein A: schematic diagram of the process of ultracentrifugation separation of sEV and lEV by PMSC; B: results of Western Blot detection of sEV and lEV specific markers; C: results of DLS detection of sEV particle size distribution; D: results of DLS detection of lEV particle size distribution; E: sEV morphology observed by transmission electron microscopy; F: lEV morphology observed by transmission electron microscopy.

[0030] Figure 3These are the results of the mesenchymal stem cells sEV and lEV repairing damaged INS-1 of the present invention; wherein A: the effect of sEV and lEV on the survival of INS-1; B: the effect of sEV and lEV on the survival of damaged pancreatic β cells; C: the effect of different sEV repair times on the survival of damaged pancreatic β cells; D: the effect of different sEV concentrations on the survival of damaged pancreatic β cells; E: the effect of sEV and lEV after trypsin inactivation on the survival of damaged pancreatic β cells.

[0031] Figure 4 Schematic diagram and results of the study on the mode of action of mesenchymal stem cell sEV entering INS-1 of the present invention; wherein A: schematic diagram of the preparation process of PKH67-labeled sEV; BE: fluorescence results of sEV carrying PKH67 label added to INS-1 cells supplemented with DMSO or EIPA and incubated with the cells for 3 h, 14 h, 24 h, and 48 h; F: average fluorescence intensity results of each treatment group at different incubation times; G: effect of sEV on the survival of damaged pancreatic β cells after EIPA pretreatment.

[0032] Figure 5 It is the effect of the mesenchymal stem cell sEV of the present invention on the apoptosis of damaged INS-1 cells; wherein A: the effect of sEV on the apoptosis of damaged pancreatic β cells; B: the statistical results of the effect of sEV on the apoptosis of damaged pancreatic β cells; C: Western Blot detection results of apoptosis protein markers in different repair groups; D: WB band grayscale analysis results of apoptosis protein markers in different repair groups.

[0033] Figure 6 The figures are as follows: A: a schematic diagram of the combined treatment process of sEV and LQ for damaged INS-1 cells; B: the results of the first group of sEV and LQ used alone or in combination on the survival of damaged INS-1 cells; C: the results of the second group of sEV and LQ used alone or in combination on the survival of damaged INS-1 cells; D: the results of the single or combined use of sEV and BA for prevention on the survival of damaged INS-1 cells; E: the statistical results of the single or combined use of sEV and LQ on the apoptosis of damaged INS-1 cells; F: the results of the single or combined use of sEV and LQ on the apoptosis of damaged INS-1 cells.

[0034] In each figure (if any), ns means no significant difference. represents P < 0.05, represents P < 0.01, Represents P < 0.001. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0036] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0037] Example 1 Construction of pancreatic β cell injury model

[0038] This example provides a method for constructing a pancreatic β cell injury model. The details are as follows: The palmitic acid solution was prepared by weighing palmitic acid and dissolving it in sodium hydroxide solution to prepare sodium palmitic acid solution, then weighing BSA (fatty acid-free) and adding it to PBS and mixing. Sodium palmitic acid solution was added to BSA fatty acid-free solution to prepare palmitic acid solution. The solution was thoroughly mixed, cooled to room temperature, filtered through a 0.22 μm filter membrane, and stored in aliquots at -20 °C.

[0039] The experimental method of the effect of palmitic acid on INS-1 survival in a concentration- and time-dependent manner was as follows: when INS-1 cells grew to 70-80%, the cells were digested and counted, and the cells were plated in 96-well plates and divided into three groups at 37 °C and 5% CO 2 Cultivate; dilute the palmitic acid solution prepared above to different concentrations with INS-1 complete culture medium, add it to the corresponding group and continue culturing for different time periods; observe cell survival using CCK8 method.

[0040] The schematic diagram of palmitic acid solution preparation and INS-1 cell injury model is shown in Figure 1 A in the figure; the effects of palmitic acid concentration and injury time on INS-1 survival are shown in Figure 1 B in which PA represents the group treated with the prepared palmitic acid solution, and BSA represents the control group treated with only BSA.

[0041] The experimental method of the concentration-dependent effect of palmitic acid on INS-1 apoptosis is as follows: when INS-1 cells grow to 70-80%, the cells are digested and counted, and the cells are plated in a 6-well plate at 37°C and 5% CO 2Cultivate; dilute the palmitic acid solution prepared above to different concentrations with INS-1 complete medium, add to the corresponding group and continue culturing for 48 hours; digest the cells, add PBS to resuspend the cells, and discard the supernatant; add Binding buffer, gently blow to obtain a single cell suspension, add Annexin V-FITC and PI Staining Solution, gently blow to obtain a single cell suspension, incubate at room temperature away from light. Add Binding buffer and gently mix; flow cytometry to detect cell apoptosis.

[0042] The results of the effect of palmitic acid concentration on INS-1 apoptosis are shown in Figure 1 C. The statistical graph of the effect of palmitic acid concentration on INS-1 apoptosis is shown in Figure 1 D, where NC is the normal INS-1 cell control group.

[0043] With the increase of palmitic acid damage concentration and the extension of time, the degree of palmitic acid damage to INS-1 increased, indicating that palmitic acid damages INS-1 in a time- and concentration-dependent manner.

[0044] Example 2 Isolation and identification of sEV

[0045] This example provides a method for isolating and identifying sEVs. The details are as follows: The FBS in human placental mesenchymal stem cell (hPMSC) culture medium was prepared using a Beckman Coulter ultracentrifuge and a Type 70 Ti rotor at 35,000 RPM for 18 h to remove EVs.

[0046] The hPMSC culture medium system is DMEM / F12+10% FBS+10ng / mL bFGF+1% penicillin-streptomycin. hPMSCs are inoculated into 15 cm dishes, 4 dishes are inoculated, and 25 mL of culture medium is added to each dish. The culture medium is incubated at 37°C and 5% CO 2 , cultured at saturated humidity, and the culture medium was collected every 48 h.

[0047] Normally culture PMSCs, collect the culture supernatant of PMSCs, centrifuge at 300×g, 4℃ for 5 min to remove dead cells, and then centrifuge the collected supernatant at 2000×g, 4℃ for 15 min to remove cell debris. Centrifuge at 12000×g, 4℃ for 60 min to collect the precipitate and resuspend it in PBS into an EP tube to obtain lEV. Transfer the supernatant just after ultracentrifugation to a new ultracentrifuge tube, ultracentrifuge at 120000×g, 4℃ for 120 min to collect the precipitate and resuspend it in PBS into an EP tube to obtain sEV. Add 20 mL of pre-cooled PBS to sEV and lEV respectively, wash once according to their respective ultracentrifugation conditions, and retain the precipitate. The final pellet is suspended in an appropriate amount of PBS and temporarily stored at 4℃, or stored at -80℃ for a long time.

[0048] Schematic diagram of the ultracentrifugation separation process of PMSC sEV and lEV is shown in Figure 2 A in.

[0049] Western Blot was used to identify the expression of Marker Alix, CD63, CD81, TSG101, AnnexinV, and CD 40 in sEV and lEV. The results are as follows: Figure 2 As shown in B, EV is a mixture of sEV and lEV; DLS was used to identify the particle size range of sEV and lEV, and the results are shown in Figure 2 As shown in Figures C and D, the average value of sEV is 162.6nm, the peak value is 146.1nm; the sEV particle size range is 75-250nm. The morphology of sEV and lEV was observed using transmission electron microscopy. The results are as follows Figure 2 As shown in E and F, the scale bar is 100 nm, sEVs have a clear double membrane structure.

[0050] Example 3 Combination of mesenchymal stem cell sEV and licorice root extract to repair pancreatic β cell damage

[0051] In this example, the recipient cells to be repaired are the INS-1 damaged cell model constructed in Example 1 (the concentration of PA is 0.5 mM, and the treatment time is 48 h). The sEV and lEV used in this example are isolated in Example 2. The complete culture medium for culturing INS-1 during the repair of damage is composed of FBS without EV.

[0052] The specific process is as follows: (1) Effects of sEV and lEV on the survival of normal INS-1 cells. INS-1 cells were plated in advance, sEV and lEV were diluted to different protein concentrations with INS-1 complete culture medium, and added to the cells at 37 °C and 5% CO 2 After culturing for 24 h, cell survival was detected by CCK8 method; The results of the effects of sEV and lEV on INS-1 survival are shown in Figure 3 A in the figure, where NC is the control group.

[0053] (2) Effects of sEV and lEV on the survival of damaged cells. After the pancreatic β-cell injury model was established, sEV and lEV were diluted with INS-1 complete medium to a protein concentration of 25 μg / mL and added to the cells at 37°C and 5% CO. 2 After 48 h of culture (adding once every 24 h), cell survival was detected by CCK8 method; The results of the effects of sEV and lEV on the survival of damaged pancreatic β cells are shown in Figure 3 In B, NC is the normal INS-1 cell control group.

[0054] (3) Effects of different sEV repair times on the survival of damaged cells. After the pancreatic β-cell injury model was established, sEVs were diluted with INS-1 complete medium to a protein concentration of 25 μg / mL and added to the cells at 37 °C and 5% CO. 2 After culturing for 24 h, 48 h, and 72 h (adding cells every 24 h), cell survival was detected by CCK8 method; The results of the effects of different sEV repair time on the survival of damaged pancreatic β cells are shown in Figure 3 C, where NC is the normal INS-1 cell control group.

[0055] (4) Effects of different sEV concentrations on the survival of damaged cells. After the pancreatic β-cell injury model was established, sEV was diluted to 6 different protein concentrations using INS-1 complete medium and added to the cells at 37 °C and 5% CO. 2 After 48 h of culture, cell survival was detected by CCK8 assay; The results of the effects of different sEV concentrations on the survival of damaged pancreatic β cells are shown in Figure 3 D in.

[0056] (5) Effect of trypsin inactivation of sEV and lEV on the survival of damaged cells. Sterile PBS was used to prepare trypsin at a concentration of 50 ng / mL, and PMSCs were cultured and the culture medium was collected to extract sEV and lEV. Two equal portions of sEV and lEV protein were quantified, and 5% trypsin was added to one portion of sEV and lEV to a final concentration of at least 1%; the sEV and lEV to which trypsin had been added were incubated in a 37°C water bath for 30 min, and then transferred to ultracentrifuge tubes. 20 mL PBS was added to each tube and ultracentrifuged and washed twice according to the centrifugation collection method for each particle; the collected precipitate was retained and the volume of PBS was adjusted to the same for both; after the pancreatic β-cell injury model was established, the inactivated sEV and lEV were added, and the CCK8 method was used to observe the effect on the survival of damaged pancreatic β-cells.

[0057] The results of the effects of trypsin inactivation of sEV and lEV on the survival of damaged pancreatic β cells are shown in Figure 3 E in.

[0058] (6) PKH67 staining to observe sEV entering INS-1. PMSCs were cultured normally and the culture medium was collected to extract sEVs and control their protein content to about 200 μg; 4 μL of 1 mM PKH67 was added and supplemented to 1 mL with Diluent C, with a final concentration of 4 μM. Then 200 μg of sEVs were diluted to 1 mL with Diluent C and mixed with the diluted PKH67, with a final concentration of PKH67 of 2 μM. The mixture was flicked and centrifuged briefly, and incubated at room temperature for 10 min; the reaction was terminated by incubation at 37 °C for 1 min with an equal amount of EV-free fetal bovine serum; the sEV-PKH67 complex was washed twice by ultracentrifugation with PMSC complete culture medium without EV to ensure that there was no unattached dye on the sEVs, and then 20 mL of pre-cooled PBS was added and ultracentrifuged once to remove the culture medium and retain the precipitate; the precipitate was resuspended with an appropriate amount of pre-cooled PBS. These precipitates are PKH67-labeled sEVs, and the operation should be protected from light.

[0059] Schematic diagram of the preparation process of PKH67-labeled sEVs is shown in Figure 4 A in.

[0060] INS-1 was seeded into 24-well plates in advance, and the experiment was carried out when the cell density reached 40-50%. INS-1 was first treated with 100 μM EIPA at 37 °C for 30 min, and then PKH67-labeled sEV was added to obtain the EIPA+sEV+PKH67 labeling group.

[0061] In addition, the sEV+PKH67 labeling group (NC) was set up without EIPA treatment and the sEV labeled with PKH67 was directly added. The DMSO+sEV+PKH67 labeling group was constructed in a similar way to the EIPA+sEV+PKH67 labeling group (DMSO was used instead of EIPA treatment). Each group was incubated with INS-1 for 3 h, 14 h, 24 h, and 48 h, respectively. The fluorescence distribution of INS-1 was observed with an inverted fluorescence microscope, and the fluorescence intensity was analyzed using Image J software.

[0062] PKH67-tagged sEVs were added to INS-1 cells supplemented with DMSO or EIPA and incubated with the cells for 3 h, 14 h, 24 h, and 48 h. The fluorescence results are shown in Figure 2. Figure 4 BE in the . Scale bar is 100 μm. The average fluorescence intensity of each treatment group at different incubation times is shown in Figure 4 F in.

[0063] (7) Effect of EIPA pretreatment of INS-1 on sEV repair function. After the pancreatic β-cell injury model was established, EIPA was diluted with INS-1 complete medium to a final concentration of 100 μM. The control group was diluted with an equal amount of DMSO and added to a 24-well plate at 37 °C and 5% CO. 2 The sEVs that had been tested by Bradford were diluted to a protein concentration of 25 μg / mL with INS-1 complete medium and added to a 24-well plate at 37°C and 5% CO. 2 After 48 h of culture (once added every 24 h), the effect on the survival of damaged pancreatic β cells was observed using the CCK8 method.

[0064] The results of the effects of sEV on the survival of damaged pancreatic β cells after EIPA pretreatment are shown in Figure 4 The G in.

[0065] (8) Effect of sEV repair on the apoptosis rate of damaged cells and the expression of apoptosis-related proteins. When the INS-1 cells in a 10 cm dish grew to 70-80%, the cells were digested and counted, and the cells were plated in a 6-well plate and incubated at 37 °C with 5% CO 2 After the pancreatic β-cell injury model was established, the culture medium was discarded and the sEVs that had been tested by Bradford were diluted to a protein concentration of 25 μg / mL with INS-1 complete culture medium at 37 °C and 5% CO. 2 After 48 hours of culture (add once every 24 hours), a portion of the cells were collected and the expression of apoptosis-related proteins was detected by WB. The cells of the other portion were digested and resuspended in PBS, and the supernatant was discarded. Binding buffer was added and gently blown to obtain a single cell suspension, and Annexin V-FITC and PI Staining Solution were added and gently blown to obtain a single cell suspension. Incubate at room temperature in the dark. Binding buffer was added and gently mixed; apoptosis was detected by flow cytometry.

[0066] The results of the effects of sEV on apoptosis of damaged pancreatic β cells are shown in Figure 5 A in Figure 2. The statistical results of the effect of sEV on the apoptosis of damaged pancreatic β cells are shown in Figure 5 B. Western Blot detection of apoptosis protein markers in different repair groups is shown in Figure 5 C in the figure; D in the figure: The results of WB band grayscale analysis of apoptotic protein markers in different repair groups are shown in Figure 5 D in.

[0067] (9) Effect of combined use of sEV and glycyrrhizin on the survival of damaged pancreatic β cells. INS-1 cells were plated in advance. In the first group, 0.5 mM palmitic acid was added to construct an injury model and 1-10 μM glycyrrhizin was added. The cells were incubated together for 24 h. Then, the sEV that had been tested by Bradford was diluted with INS-1 complete culture medium to a protein concentration of 25 μg / mL at 37 °C and 5% CO. 2 After 48 h of culture (once every 24 h), the effect on the survival of damaged pancreatic β cells was observed by CCK8 method. The second group was added with 0.5 mM palmitic acid and incubated for 24 h to construct an injury model. After that, the sEVs that had been tested by Bradford were diluted with INS-1 complete medium to a protein concentration of 25 μg / mL, mixed with 5 μM licorice, and incubated at 37 °C with 5% CO 2 After 48 hours of culture (adding once every 24 hours), the CCK8 method was used to observe the effect on the survival of damaged pancreatic β cells. The two groups were treated with sEV and licorice as controls.

[0068] The schematic diagram of the process of the combined treatment of INS-1 with sEV and LQ in the present invention is shown in Figure 6 A in the first group. The results of the effects of sEV and LQ alone or in combination on the survival of damaged INS-1 cells are shown in Figure 6 B in the figure; the second group of sEV and LQ used alone or in combination on the survival of damaged INS-1 cells results are shown in Figure 6 C in the figure. NC is the blank control group, None is the injury model group without intervention, and sEV is the group that is directly treated with sEV without prior treatment with licorice.

[0069] (10) Comparative experiment: Effect of combined use of sEV and Baicalin on the survival of damaged pancreatic β cells. INS-1 cells were plated in advance and pretreated with 50 μM Baicalin (BA). Then, 0.5 mM palmitic acid was added and incubated for 24 h to construct an injury model. The sEV that had been tested by Bradford was diluted with INS-1 complete culture medium to a protein concentration of 25 μg / mL and incubated at 37 °C and 5% CO 2 After 48 hours of culture (adding once every 24 hours), the CCK8 method was used to observe the effect on the survival of damaged pancreatic β cells. Groups treated with sEV and Baicalin alone were set up as controls.

[0070] The results of the effects of sEV and BA alone or in combination on the survival of damaged INS-1 cells are shown in Figure 6 D in the figure; NC is the blank control group, None is the injury model group without intervention, and sEV is the group that is directly treated with sEV without prior treatment with Baicalin.

[0071] (11) Effect of combined use of sEV and glycyrrhizin on apoptosis of damaged pancreatic β cells. INS-1 cells were plated in advance, and 5 μM glycyrrhizin was added to construct an injury model while adding 0.5 mM palmitic acid. The cells were incubated together for 24 h. Then, sEV that had been tested by Bradford was diluted with INS-1 complete culture medium to a protein concentration of 25 μg / mL. The cells were incubated at 37 °C and 5% CO 2 After 48 hours of culture (adding once every 24 hours), flow cytometry was used to detect the effect on apoptosis of damaged pancreatic β cells. Groups treated with sEV and licorice were set up as controls.

[0072] The statistical results of the effects of sEV and LQ alone or in combination on apoptosis of damaged INS-1 cells are shown in Figure 6 The results of the effects of sEV and LQ alone or in combination on the apoptosis of damaged INS-1 cells are shown in Figure 6 F in the figure. NC is the blank control group, and PA is the injury model group without intervention.

[0073] The test results are: (1) sEV can improve the survival of damaged pancreatic β cells. The four different concentrations of sEV and lEV in the treatment group had no effect on the survival of normal cells. sEV had a significant repair effect on damaged pancreatic β cells, while the addition of lEV had no obvious repair effect. Adding sEV to repair for 48 hours can significantly improve cell survival, and the effect is most significant when the repair concentration is 25 μg / mL. The repair effect of trypsin-inactivated sEV on damaged INS-1 disappeared.

[0074] (2) sEV repair reduces the apoptosis level of damaged pancreatic β cells. After sEV repair, the apoptosis rate of damaged INS-1 cells was significantly reduced, the protein level of apoptosis-related protein BCL-2 increased, and the protein level of BAX decreased.

[0075] (3) sEVs enter INS-1 through macropinocytosis to exert their effects. sEVs carrying the PKH67 label were added to INS-1 cells and incubated with them. Observation under an inverted fluorescence microscope revealed a faint green fluorescence after 3 h, which increased in intensity after 14 h, spread throughout the cell population after 24 h, and significantly weakened after 48 h. When the macropinocytosis inhibitor EIPA was added to INS-1 cells, it was found that sEV endocytosis was inhibited. After EIPA pretreatment, the degree of repair of damaged pancreatic β cells by sEVs was significantly reduced.

[0076] (4) sEV combined with glycyrrhizin can further repair pancreatic β-cell damage. Before adding sEV to repair damaged INS-1 cells, they were pretreated with glycyrrhizin. It was found that the combined use of the two had a more significant repair effect than using them alone. The cell survival rate increased and the apoptosis rate decreased, indicating that the combined use of sEV and glycyrrhizin had a better effect.

[0077] In summary, the present invention uses placental mesenchymal stem cells sEV alone to repair pancreatic β cell damage, which is not achievable with the prior art, indicating that the repair efficiency of sEV in the present invention is greatly improved. Different technical means have been used to verify that the sEV+liquiritigenin treatment group has a higher repair efficiency, that is, sEV plays a leading role in repairing pancreatic β cell damage. The repair method provided by the present invention makes EV-mediated intercellular communication truly a new way to repair damage. Moreover, the repair efficiency of the repair method is very high and is expected to be applied clinically.

[0078] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for repairing damaged pancreatic β cells for non-disease diagnosis or treatment purposes, characterized in that: The combination of sEV and licorice root was used to repair damaged pancreatic β cells.

2. The method according to claim 1, characterized in that Licoricein was added for treatment at the same time as pancreatic β-cell damage, and then sEV was added for treatment after pancreatic β-cell damage.

3. The method according to claim 1 or 2, characterized in that: The working concentration of the licorice root is 5-10 μM, and the working concentration of sEV is 25-50 μg / mL.

4. The method according to claim 3, characterized in that The working concentration of the licorice root is 5 μM, and the working concentration of sEV is 25 μg / mL.

5. The method according to any one of claims 2 to 4, characterized in that: The licorice radical treatment time was 24 hours, and the sEV treatment time was 48 hours.

6. The method according to any one of claims 1 to 5, characterized in that: The sEVs are small extracellular vesicles produced by stem cells with repair functions; the particle size range of sEVs is 75-250nm.

7. The method according to claim 6, characterized in that The stem cells are mesenchymal stem cells, preferably human placental mesenchymal stem cells.

8. The method according to claim 6 or 7, characterized in that: When the stem cells are used to produce small extracellular vesicles, the serum in the culture system is serum without extracellular vesicles.

9. A product for repairing damaged pancreatic β cells, characterized in that: Includes small extracellular vesicles and glycyrrhizin produced by cells with repair functions.

10. Use of the product according to claim 9 in improving the repair efficiency of damaged pancreatic β cells.

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