A stem cell therapy for type 2 diabetes

Abstract

The application relates to the technical field of stem cell engineering and regenerative medicine, and specifically discloses a stem cell treatment method for treating type 2 diabetes. The method comprises the following steps: S1, extracting placenta-derived mesenchymal stem cells; S2, performing multi-gene modification on the mesenchymal stem cells to obtain gene-modified mesenchymal stem cells; S3, pretreating the gene-modified mesenchymal stem cells with a culture medium; S4, extracting microvesicles from the mesenchymal stem cells; S5, mixing the microvesicles, baicalin, L-arginine and vitamin D in a buffer to form a composite preparation; S6, introducing the composite preparation into a patient's body through abdominal cavity intervention; and S7, performing synergistic treatment by combining oral administration of an SGLT2 inhibitor after infusion, and regularly monitoring islet function indexes. The stem cell treatment method can be used for treating type 2 diabetes, and has the advantages of being targeted to improve the islet microenvironment, promoting the recovery of beta cell function and being durable in action.

Description

Technical Field

[0001] This application relates to the fields of stem cell engineering and regenerative medicine, and more specifically, to a stem cell therapy for treating type 2 diabetes. Background Technology

[0002] The application of stem cell engineering and regenerative medicine in the field of diabetes revolves around the repair of pancreatic islet function and the reconstruction of blood glucose homeostasis. Utilizing seed cells such as mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells, islet-like cells with insulin-secreting function are obtained through directed differentiation techniques, or functional stem cells are directly infused into patients. Combined with technologies such as biomaterial scaffold transplantation, gene editing modification, and microencapsulation with immunoisolation, these technologies are widely applied to islet cell replacement therapy for type 1 diabetes, repair of pancreatic β-cell function and relief of insulin resistance in type 2 diabetes, as well as tissue repair of diabetic complications, and basic research and clinical translation in areas such as the construction of diabetes disease models and the development and screening of novel hypoglycemic drugs. Its advantages lie in its more fundamental treatment mechanism, breaking through the limitations of traditional drug-based hypoglycemic and insulin injection-based symptomatic treatments. It repairs or replaces damaged islet tissue at the cellular level, potentially achieving autonomous insulin secretion and long-term blood glucose stability.

[0003] Related stem cell engineering and regenerative medicine use mesenchymal stem cells and employ techniques such as directed differentiation, gene editing, biomaterial scaffolds, and microencapsulation to achieve pancreatic islet cell replacement, functional repair, and treatment of complications. However, current technologies still face multiple challenges, including the risk of immune rejection, insufficient cell survival and functional stability after transplantation, difficulty in regulating the directed differentiation and long-term integration of cells in vivo, low targeted delivery efficiency, and potential tumorigenic risks. As a result, the therapeutic effects are often limited to short-term or local treatments, and functional recovery is incomplete. Summary of the Invention

[0004] In order to address the problem that the therapeutic effects of mesenchymal stem cells used in related stem cell engineering and regenerative medicine are limited to short-term or local treatments and that functional recovery is incomplete, this application provides a stem cell therapy method for treating type 2 diabetes.

[0005] This application provides a stem cell therapy method for treating type 2 diabetes, employing the following technical solution:

[0006] A stem cell therapy for treating type 2 diabetes includes the following steps:

[0007] S1. Placental mesenchymal stem cells were extracted, and primary stem cell populations were obtained by protease digestion and gradient centrifugation. High-purity mesenchymal stem cells were then obtained by amplification culture in serum-free medium.

[0008] S2. The mesenchymal stem cells obtained in S1 are subjected to multi-gene modification, and the GIP and FGF1 dual-factor expression sequences are introduced into them through a non-viral vector to obtain gene-modified mesenchymal stem cells.

[0009] S3. The gene-modified mesenchymal stem cells obtained in step S2 were pretreated with a culture medium containing epigallocatechin gallate and sitagliptin.

[0010] S4. Microvesicles were extracted from the mesenchymal stem cells pretreated in S3 to obtain active microvesicles;

[0011] S5. The microvesicles extracted in S4 are mixed with baicalin, L-arginine and vitamin D in a buffer solution to form a microvesicle-drug complex formulation.

[0012] S6. The compound preparation prepared in S5 is introduced into the body of a type 2 diabetic patient via an intraperitoneal intervention.

[0013] S7. After infusion, synergistic treatment with oral SGLT2 inhibitors is administered, and pancreatic function indicators are monitored regularly.

[0014] By employing the above-mentioned technical solution, placental mesenchymal stem cells are used as a therapeutic carrier. These cells possess low immunogenicity and multi-lineage differentiation potential. Through multi-gene modification, they stably express glucose-dependent insulinotropic peptides and fibroblast growth factor 1 (GF-1). These two factors synergistically act on pancreatic β-cells, promoting their proliferation and improving insulin secretion function. Subsequently, the cells are pretreated with epigallocatechin gallate and sitagliptin. Epigallocatechin gallate enhances the cells' antioxidant capacity, while sitagliptin prolongs the endogenous incretin effect by inhibiting dipeptidyl peptidase-4. The combined effect of these two factors... This method enhances stem cell survival and paracrine activity; then, it extracts the released microvesicles, integrates the modified stem cell secretory group with baicalin, L-arginine, and vitamin D. Baicalin assists in anti-inflammation, L-arginine promotes nitric oxide synthesis to improve microcirculation, and vitamin D regulates immune and calcium signaling, forming a multifunctional compound preparation. Finally, it achieves local high-concentration delivery through intraperitoneal intervention, and combines it with SGLT2 inhibitors to synergistically lower blood sugar and protect pancreatic islet function through multiple pathways. Therefore, it achieves a therapeutic effect that can specifically improve the pancreatic islet microenvironment, promote β-cell function recovery, and has a long-lasting effect.

[0015] Preferably, the procedure before step S1 includes pretreatment of the tissue: the obtained placental tissue is rinsed with Hank's balanced salt solution until no residue remains, cut into tissue blocks with a particle size of 0.5-2 mm³, and then 0.15% proteinase K is added and the tissue is shaken and digested at 35°C for 25-40 min.

[0016] By employing the above-mentioned technical solution, Hank's balanced salt solution is used for rinsing to remove residual blood and potential contaminants from the placental tissue, reducing the risk of microbial contamination during subsequent culture. Shredding the tissue to a specified particle size range increases the digestion contact area, ensuring uniform digestion. Using a specific concentration of proteinase K and gentle agitation digestion at 35°C dissociates the extracellular matrix and connective proteins while avoiding over-digestion that could damage cell membrane proteins and cell viability. This lays the foundation for subsequent gradient centrifugation to separate highly active primary stem cell populations. Therefore, a high cell yield and tissue pretreatment with high initial viability are achieved.

[0017] Preferably, in step S1, the amplification culture conditions are as follows: cultured in a 36.5°C, 4.5% CO2 incubator using DMEM high glucose medium containing 8% human serum substitute for 6 to 8 days, with the medium being replaced every 36 hours until the cell confluence reaches 85% to 95%.

[0018] By employing the above-mentioned technical approach, culturing at 36.5°C can slightly reduce the cell metabolic rate, which is beneficial for maintaining genomic stability and reducing culture stress. A CO2 concentration of 4.5% maintains the pH balance of the culture medium within a range suitable for mesenchymal stem cell growth. Simultaneously, using DMEM high-glucose medium containing 8% human serum substitutes avoids the risks associated with animal-derived components while providing sufficient nutrients and growth factors to support cell expansion. The subsequent high-glucose environment aligns with the energy metabolism characteristics of mesenchymal stem cells. Changing the culture medium every 36 hours promptly removes metabolic waste and replenishes fresh nutrients, preventing cell aging. Harvesting at 85% to 95% confluence ensures that the cells are in the logarithmic growth phase without contact inhibition. Therefore, a population of mesenchymal stem cells with high expansion efficiency, uniform cell state, and complete biological function is obtained.

[0019] Preferably, the mesenchymal stem cells isolated in step S1 need to be identified by surface markers, requiring a positive rate of ≥95% for CD73, CD90, and CD105, and a positive rate of ≤2% for CD34 and CD45.

[0020] By employing the above-mentioned technical solutions, the isolated cell population is identified using specific surface markers. The positive expression rate of typical mesenchymal stem cell markers CD73, CD90, and CD105 is required to reach over 95%, ensuring that the cell population possesses clear mesenchymal stem cell characteristics and purity. Simultaneously, the positive rates of hematopoietic stem cell markers CD34 and leukocyte common antigen CD45 are required to be below 2%, excluding contamination from hematopoietic cell lines and other impurities, and ensuring the consistency, predictability, and safety of the cell matrix for subsequent gene modification and therapeutic applications.

[0021] Preferably, in step S2, the non-viral vector is an electroporation transfection system with a transfection voltage of 120–150 V and a transfection time of 15–25 ms; the gene-modified cells need to be tested for the expression efficiency of GIP and FGF1 by ELISA, requiring a dual-factor secretion level of not less than 50 ng / 10 6 cells / 24h.

[0022] By employing the above-mentioned technical solution, electroporation is used as a non-viral vector transfection method. A brief, high-intensity electric field creates reversible micropores on the cell membrane, allowing plasmid DNA encoding GIP and FGF1 to efficiently enter the cell. Controlling the voltage to 120-150V and setting the pulse duration within a window of 15-25ms ensures high transfection efficiency while maintaining cell viability and functional integrity. After transfection, secreted proteins are quantified using ELISA, requiring the secretion levels of both factors to reach specified standards. This ensures that the modified stem cells can continuously provide sufficient therapeutic factors, providing a source of support for subsequent microvesicles to carry these factors and exert long-term regulatory effects. Therefore, gene-modified mesenchymal stem cells that stably and efficiently express the target therapeutic factors and are in good cellular condition are obtained.

[0023] Preferably, in step S3, the pretreatment medium is RPMI-1640 medium, wherein the concentration of epigallocatechin gallate is 5–10 μmol / L, the concentration of sitagliptin is 8–15 μmol / L, the pretreatment time is 18–36 h, and the cell density is 5 × 10⁻⁶ cells / year. 4 ~5×10 5 cells / mL.

[0024] By employing the above-mentioned technical approach, RPMI-1640 medium was selected for pretreatment. This medium has a balanced nutrient composition and is suitable for short-term cell maintenance during drug treatment. Epigallocatechin gallate was added at a specific concentration range to enhance the cells' ability to resist oxidative damage during subsequent in vivo transplantation by utilizing its antioxidant capacity and ability to activate endogenous protective pathways. Sitagliptin was also added, which, by inhibiting DPP-4 activity, not only simulates the protective effect of incretin in the culture medium environment but also acts on stem cells to enhance their survival and paracrine function. The pretreatment time was set at 18 to 36 hours to ensure that the drug has time to exert its cellular biological effects without causing toxicity. Controlling the appropriate cell density avoids overcrowding or nutrient competition. Therefore, mesenchymal stem cells with enhanced function, improved stress resistance, and better suitability for subsequent microvesicle induction were obtained.

[0025] Preferably, in step S4, the microvesicle extraction is performed using size exclusion chromatography: first, cell debris is removed by centrifugation at 1500×g for 15 min, and then microvesicle components with a particle size of 50-200 nm are collected by column chromatography; the obtained microvesicles must meet the requirements of positive expression of marker proteins CD9 and CD81, and the concentration must not be less than 2×10⁻⁶. 10 particles / mL.

[0026] By employing the above technical solution, large particulate impurities such as cell debris are first removed by low-speed centrifugation, providing conditions for subsequent fine separation. Then, size exclusion chromatography is used to collect target microvesicle populations with particle sizes of 50-200 nm at high resolution. Microvesicles in this particle size range are rich in bioactive proteins, nucleic acids, and lipids, and are easily taken up by target cells. The obtained product is tested for transmembrane proteins CD9 and CD81, and the positive results confirm that the extracted product is indeed an exosome-like microvesicle. A specific lower limit of particle concentration is required to ensure that sufficient active carriers are obtained for each treatment. Therefore, a high-purity, high-concentration therapeutic microvesicle product with clearly defined biological markers is obtained.

[0027] Preferably, in step S5, the amount of baicalein added is 1-3 g / L, L-arginine is 0.3-0.6 g / L, and vitamin D is 0.1-0.3 g / L; the order of addition is to first dissolve baicalein in the buffer solution, then add L-arginine and vitamin D, and finally mix with microvesicles.

[0028] By adopting the above technical solution, baicalin, which has low solubility in water, is first dissolved in a buffer solution to ensure its dispersion and avoid precipitation when mixed with other components. L-arginine, as a nitric oxide precursor, and vitamin D, as a lipid-soluble hormone, are added sequentially to ensure their stability in the liquid phase. Finally, the mixed drug solution is co-incubated with microvesicles. The lipid bilayer structure of the microvesicles can adsorb or load some drug components and form complexes through surface interactions. The selected concentration range of baicalin exerts anti-inflammatory and vascular endothelial protective effects, the concentration of L-arginine helps improve local blood flow and pancreatic microcirculation, and the concentration of vitamin D participates in immune regulation and β-cell function protection. The three components work synergistically with the microvesicles in the buffer system to jointly construct a multi-target drug delivery system. Therefore, a microvesicle-drug complex formulation with stable physicochemical properties and complementary functions of each component is obtained.

[0029] Preferably, in step S6, the dosage of the intraperitoneal interventional microvesicles is 5 × 10⁻⁶. 10 ~8×10 10 The dosage is 0.5-1.5 mL / min, and the compound preparation should be brought to room temperature before infusion.

[0030] By adopting the above technical solution, the dosage of microvesicles is controlled according to the patient's weight, ensuring the accuracy of individualized drug delivery and the appropriateness of treatment intensity. This dosage range is based on preclinical studies and can achieve effective concentrations in target tissues without causing adverse reactions. Controlling the infusion rate at 0.5-1.5 mL / min is considered slow infusion, which is conducive to the uniform distribution and absorption of the compound preparation in the peritoneal cavity and reduces local discomfort caused by excessively rapid infusion. The preparation is equilibrated to room temperature before infusion to avoid spasms or pain caused by cold liquid entering the body cavity, thereby improving patient tolerance and treatment safety. Therefore, a safe, controllable, and well-tolerated intraperitoneal interventional drug delivery effect is achieved.

[0031] Preferably, in step S7, the oral dose of the SGLT2 inhibitor is 10-25 mg / day, and administration begins within 12 hours after infusion; efficacy monitoring is based on a decrease in glycated hemoglobin to below 6.0% and an increase in fasting C-peptide level by more than 2 times as the effective criteria.

[0032] By adopting the above technical solution, the combined oral administration of SGLT2 inhibitors within 12 hours after the infusion of stem cell-derived preparations can promptly exert the drug's osmotic diuretic and weight-loss effects, helping to reduce glycemic load and providing a favorable metabolic basis for the reparative microenvironment created by stem cells and microvesicles. SGLT2 inhibitors can also synergistically enhance the reparative effects of stem cell therapy by improving insulin resistance and reducing β-cell glucosinolates. Lowering HbA1c to below 6.0% is used as the standard for long-term glycemic control, while increasing fasting C-peptide levels serves as evidence of improved β-cell function. The combination of these two indicators can comprehensively and objectively evaluate the therapeutic effect on the pathological aspects of type 2 diabetes.

[0033] In summary, this application has the following beneficial effects:

[0034] 1. This application uses placental mesenchymal stem cells as a therapeutic carrier and modifies them through multiple genes to stably express GIP and FGF1 dual factors. Then, it pre-treats them with epigallocatechin gallate and sitagliptin to enhance cell activity. Microvesicles are then extracted and combined with baicalin, L-arginine, and vitamin D to form a compound preparation. Finally, the preparation is delivered via intraperitoneal intervention and combined with SGLT2 inhibitors for synergistic treatment. This forms a multi-linked approach from cell preparation and functional enhancement to targeted delivery, achieving a targeted improvement of the pancreatic islet microenvironment, promoting the recovery of β-cell function, and providing a long-lasting therapeutic effect.

[0035] 2. In this application, placental tissue is preferably digested with proteinase K and amplified under specific conditions. Because the tissue pretreatment and amplification process are closely coordinated, it not only ensures the high yield and activity of primary stem cells, but also provides a uniform and functionally complete cell population for subsequent gene modification. Therefore, a basic material with pure cell matrix and stable biological characteristics is obtained to support the effective implementation of subsequent treatment steps.

[0036] 3. The method of this application involves sequentially mixing microvesicles with baicalin, L-arginine, and vitamin D to form a composite preparation, and controlling the intraperitoneal intervention dosage and rate. The microvesicles act as carriers and synergistically integrate with multiple active ingredients in a buffer system, taking into account multiple effects such as anti-inflammatory, microcirculation improvement, and immune regulation. Furthermore, the administration method is matched with the characteristics of the preparation, thus achieving a multi-target synergistic, locally high-concentration delivery and well-tolerated therapeutic effect. Attached Figure Description

[0037] Figure 1 This is a flowchart of a stem cell therapy method for treating type 2 diabetes proposed in this application. Detailed Implementation

[0038] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0039] Example 1: This example provides a stem cell therapy method for treating type 2 diabetes, comprising the following steps:

[0040] S1: Placental mesenchymal stem cells were extracted, and primary stem cell populations were obtained by protease digestion and gradient centrifugation. High-purity mesenchymal stem cells were then obtained by amplification culture in serum-free medium.

[0041] The process included pretreatment of the tissue before extraction: the obtained placental tissue was rinsed with Hank's balanced salt solution until no residue remained, and then minced to a particle size of 0.5 mm. 3 The tissue blocks were then digested with 0.15% proteinase K at 35°C for 25 minutes with shaking. The amplification culture conditions were as follows: cultured in DMEM high glucose medium containing 8% human serum substitute in a 36.5°C, 4.5% CO2 incubator for 6 days, changing the medium every 36 hours until the cell confluence reached 85%. Mesenchymal stem cells were also required to undergo surface marker identification, with CD73, CD90, and CD105 positivity rates ≥95% and CD34 and CD45 positivity rates ≤2%.

[0042] S2: The mesenchymal stem cells obtained in S1 are modified with multiple genes and introduced into the GIP and FGF1 dual-factor expression sequences through a non-viral vector to obtain gene-modified mesenchymal stem cells.

[0043] The non-viral vector was transfected via electroporation at a voltage of 120V for 15ms. Genetically modified cells were then analyzed using ELISA to determine the expression efficiency of GIP and FGF1, requiring a dual-factor secretion level of at least 50 ng / 10⁻⁶. 6 cells / 24h.

[0044] S3: The genetically modified mesenchymal stem cells obtained in step S2 were pretreated with a culture medium containing epigallocatechin gallate and sitagliptin.

[0045] The pretreatment medium was RPMI-1640 medium, with epigallocatechin gallate concentration of 5 μmol / L and sitagliptin concentration of 8 μmol / L. The pretreatment time was 18 h, and the cell density was 5 × 10⁶ cells / year. 4 cells / mL.

[0046] S4: Microvesicles were extracted from mesenchymal stem cells pretreated with S3 to obtain active microvesicles.

[0047] The microvesicle extraction method employed size exclusion chromatography: cell debris was first removed by centrifugation at 1500×g for 15 min, followed by column chromatography to separate and collect microvesicle components with a particle size of 50-200 nm. The obtained microvesicles were required to be positive for the marker proteins CD9 and CD81, with a concentration of not less than 2×10⁻⁶. 10 particles / mL.

[0048] S5: The microvesicles extracted in S4 are mixed with baicalin, L-arginine, and vitamin D in a buffer solution to form a microvesicle-drug complex.

[0049] The amount of baicalein added was 1 g / L, L-arginine was 0.3 g / L, and vitamin D was 0.1 g / L. The order of addition was to first dissolve baicalein in the buffer solution, then add L-arginine and vitamin D, and finally mix with microvesicles.

[0050] S6: The compound preparation prepared in S5 is introduced into the body of a type 2 diabetic patient via an intraperitoneal intervention.

[0051] The dosage of microvesicles used in intraperitoneal intervention was 5×10⁻⁶. 10 The dosage is 0.5 mL / min, and the compound preparation should be brought to room temperature before infusion.

[0052] S7: After infusion, combine with oral SGLT2 inhibitors for synergistic treatment, and monitor pancreatic function indicators regularly.

[0053] The oral dose of SGLT2 inhibitor is 10 mg / day, and administration should begin within 1 hour after infusion. The efficacy monitoring criteria are a decrease in HbA1c to below 6.0% and an increase in fasting C-peptide level by more than 2 times.

[0054] Example 2: This example provides a stem cell therapy method for treating type 2 diabetes, comprising the following steps:

[0055] S1: Placental mesenchymal stem cells were extracted, and primary stem cell populations were obtained by protease digestion and gradient centrifugation. High-purity mesenchymal stem cells were then obtained by amplification culture in serum-free medium.

[0056] The process included pretreatment of the tissue before extraction: the obtained placental tissue was rinsed with Hank's balanced salt solution until no residue remained, and then minced to a particle size of 1.25 mm. 3 The tissue blocks were then digested with 0.15% proteinase K at 35°C for 32.5 min with shaking. The amplification culture conditions were as follows: cultured in DMEM high glucose medium containing 8% human serum substitute in a 36.5°C, 4.5% CO2 incubator for 7 days, changing the medium every 36 hours until the cell confluence reached 90%. Mesenchymal stem cells were also required to undergo surface marker identification, with CD73, CD90, and CD105 positivity rates ≥95% and CD34 and CD45 positivity rates ≤2%.

[0057] S2: The mesenchymal stem cells obtained in S1 are modified with multiple genes and introduced into the GIP and FGF1 dual-factor expression sequences through a non-viral vector to obtain gene-modified mesenchymal stem cells.

[0058] The non-viral vector was transfected via electroporation at a voltage of 135V for 20ms. Genetically modified cells were then analyzed using ELISA to determine the expression efficiency of GIP and FGF1, requiring a dual-factor secretion level of at least 50 ng / 10⁻⁶. 6 cells / 24h.

[0059] S3: The genetically modified mesenchymal stem cells obtained in step S2 were pretreated with a culture medium containing epigallocatechin gallate and sitagliptin.

[0060] The pretreatment medium was RPMI-1640 medium, with epigallocatechin gallate concentration of 7.5 μmol / L and sitagliptin concentration of 11.5 μmol / L. The pretreatment time was 27 h, and the cell density was 2.75 × 10⁻⁶ cells / year. 5 cells / mL.

[0061] S4: Microvesicles were extracted from mesenchymal stem cells pretreated with S3 to obtain active microvesicles.

[0062] The microvesicle extraction method employed size exclusion chromatography: cell debris was first removed by centrifugation at 1500×g for 15 min, followed by column chromatography to separate and collect microvesicle components with a particle size of 50-200 nm. The obtained microvesicles were required to be positive for the marker proteins CD9 and CD81, with a concentration of not less than 2×10⁻⁶. 10 particles / mL.

[0063] S5: The microvesicles extracted in S4 are mixed with baicalin, L-arginine, and vitamin D in a buffer solution to form a microvesicle-drug complex.

[0064] The amount of baicalein added was 2 g / L, L-arginine was 0.45 g / L, and vitamin D was 0.2 g / L. The order of addition was to first dissolve baicalein in the buffer solution, then add L-arginine and vitamin D, and finally mix with microvesicles.

[0065] S6: The compound preparation prepared in S5 is introduced into the body of a type 2 diabetic patient via an intraperitoneal intervention.

[0066] The dosage of microvesicles used in the intraperitoneal intervention was 6.5 × 10⁻⁶. 10 The dosage is 100 particles / kg body weight, and the infusion rate is 1.0 mL / min. The compound preparation needs to be equilibrated to room temperature before infusion.

[0067] S7: After infusion, combine with oral SGLT2 inhibitors for synergistic treatment, and monitor pancreatic function indicators regularly.

[0068] The oral dose of the SGLT2 inhibitor is 17.5 mg / day, and administration should begin within 6 hours after infusion. The efficacy monitoring criteria are a decrease in HbA1c to below 6.0% and an increase in fasting C-peptide level by more than 2 times.

[0069] Example 3: This example provides a stem cell therapy method for treating type 2 diabetes, comprising the following steps:

[0070] S1: Placental mesenchymal stem cells were extracted, and primary stem cell populations were obtained by protease digestion and gradient centrifugation. High-purity mesenchymal stem cells were then obtained by amplification culture in serum-free medium.

[0071] The tissue was pretreated before extraction: the obtained placental tissue was rinsed with Hank's balanced salt solution until no residue remained, and then minced to a particle size of 2 mm. 3The tissue blocks were then digested with 0.15% proteinase K at 35°C for 40 minutes with shaking. The amplification culture conditions were as follows: cultured in DMEM high glucose medium containing 8% human serum substitute in a 36.5°C, 4.5% CO2 incubator for 8 days, changing the medium every 36 hours until the cell confluence reached 95%. Mesenchymal stem cells were also required to undergo surface marker identification, with CD73, CD90, and CD105 positivity rates ≥95% and CD34 and CD45 positivity rates ≤2%.

[0072] S2: The mesenchymal stem cells obtained in S1 are modified with multiple genes and introduced into the GIP and FGF1 dual-factor expression sequences through a non-viral vector to obtain gene-modified mesenchymal stem cells.

[0073] The non-viral vector was transfected via electroporation at a voltage of 150V for 25ms. Genetically modified cells were then analyzed using ELISA to determine the expression efficiency of GIP and FGF1, requiring a dual-factor secretion level of at least 50 ng / 10⁻⁶. 6 cells / 24h.

[0074] S3: The genetically modified mesenchymal stem cells obtained in step S2 were pretreated with a culture medium containing epigallocatechin gallate and sitagliptin.

[0075] The pretreatment medium was RPMI-1640 medium, with epigallocatechin gallate at a concentration of 10 μmol / L and sitagliptin at a concentration of 15 μmol / L. The pretreatment time was 36 h, and the cell density was 5 × 10⁶ cells / year. 5 cells / mL.

[0076] S4: Microvesicles were extracted from mesenchymal stem cells pretreated with S3 to obtain active microvesicles.

[0077] The microvesicle extraction method employed size exclusion chromatography: cell debris was first removed by centrifugation at 1500×g for 15 min, followed by column chromatography to separate and collect microvesicle components with a particle size of 50-200 nm. The obtained microvesicles were required to be positive for the marker proteins CD9 and CD81, with a concentration of not less than 2×10⁻⁶. 10 particles / mL.

[0078] S5: The microvesicles extracted in S4 are mixed with baicalin, L-arginine, and vitamin D in a buffer solution to form a microvesicle-drug complex.

[0079] The amount of baicalein added was 3 g / L, L-arginine was 0.6 g / L, and vitamin D was 0.3 g / L. The order of addition was to first dissolve baicalein in the buffer solution, then add L-arginine and vitamin D, and finally mix with microvesicles.

[0080] S6: The compound preparation prepared in S5 is introduced into the body of a type 2 diabetic patient via an intraperitoneal intervention.

[0081] The dosage of microvesicles used in intraperitoneal intervention was 8×10⁻⁶. 10 The dosage is 1.5 mL / min, and the compound preparation should be brought to room temperature before infusion.

[0082] S7: After infusion, combine with oral SGLT2 inhibitors for synergistic treatment, and monitor pancreatic function indicators regularly.

[0083] The oral dose of SGLT2 inhibitor is 25 mg / day, and administration should begin within 12 hours after infusion. The efficacy monitoring criteria are a decrease in HbA1c to below 6.0% and an increase in fasting C-peptide level by more than 2 times.

[0084] Comparative Example 1: This comparative example refers to Example 1, except that in step S1, during the pretreatment of the placental tissue, the tissue blocks were minced to a particle size of 0.35 mm. 3 The rest of the content is the same as in Example 1.

[0085] Comparative Example 2: This comparative example is based on the content of Example 1, except that the proteinase K digestion time of placental tissue in step S1 is 17.5 min, and the rest is the same as Example 1.

[0086] Comparative Example 3: This comparative example refers to the content of Example 1, except that the voltage of electroporation transfection in step S2 is 180V, and the rest is the same as Example 1.

[0087] Comparative Example 4: This comparative example is the same as that in Example 1, except that the concentration of epigallocatechin gallate in the pretreatment culture medium in step S3 is 3.5 μmol / L. The rest of the contents are the same as those in Example 1.

[0088] Comparative Example 5: This comparative example is based on the content of Example 1, except that the amount of baicalin added in step S5 is 1.5 g / L, and the rest of the content is the same as in Example 1.

[0089] Comparative Example 6: This comparative example refers to Example 1, except that the dose of microvesicles used in the peritoneal intervention in step S6 is 3.5 × 10⁻⁶. 10 The particle / kg body weight ratio is the same as in Example 1.

[0090] Performance testing

[0091] Sample preparation: Genetically modified mesenchymal stem cells, microvesicles, and microvesicle-drug composite preparations were prepared according to the methods described in Examples 1-3; at the same time, corresponding comparative samples were prepared according to the methods described in Comparative Examples 1-6; all samples were prepared according to the steps in their respective examples or comparative examples.

[0092] Mesenchymal stem cell yield and activity detection: Primary stem cell populations obtained in step S1 of Examples 1-3 and Comparative Examples 1-6 were used to count cells using a hemocytometer to obtain stem cell yield, which was expressed as the number of cells obtained per gram of placental tissue. At the same time, cell activity was detected by trypan blue staining. The percentage of live cells was calculated by microscopic observation. After trypan blue staining, dead cells appeared blue, while live cells refused to be stained. The percentage of live cells was counted.

[0093] Detection of GIP and FGF1 expression efficiency after gene modification: Gene-modified mesenchymal stem cells obtained in step S2 of Examples 1-3 and Comparative Examples 1-6 were cultured under standard culture conditions for 24 hours. The supernatant was collected, and the concentrations of GIP and FGF1 were detected using an ELISA kit according to the instructions. The expression efficiency was expressed as per 10-1. 6 The amount of factors secreted by the cells within 24 hours is represented by the average value of three replicates for each sample.

[0094] Microvesicle concentration: Microvesicles extracted in step S4 of Examples 1-3 and Comparative Examples 1-6 were used to detect the microvesicle concentration using a nanoparticle tracking analyzer. The samples were appropriately diluted before being injected for analysis.

[0095] Anti-inflammatory effect test of compound preparation: The microvesicle-drug compound preparations prepared in step S5 of Examples 1-3 and Comparative Examples 1-6 were co-cultured with macrophage cell lines. Lipopolysaccharide was added to induce an inflammatory response. After 24 hours of culture, the supernatant was collected, and the concentrations of tumor necrosis factor α and interleukin 6 were detected by ELISA. The untreated group was used as a control to obtain the inhibition rate of inflammatory factors.

[0096] Detection of pancreatic function improvement in animal models: Type 2 diabetic mice were randomly divided into groups and injected with the compound preparations of Examples 1-3 and Comparative Examples 1-6, respectively. A blank control group and a model control group were set up. The blood glucose level, glycated hemoglobin and fasting C-peptide level of the mice were measured regularly. The treatment period was 4 weeks. After the animals were sacrificed, pancreatic tissue was taken for pathological analysis.

[0097] Table 1: Comparison of Detection Parameter Data between Examples and Comparative Examples

[0098]

[0099] Table 2: Comparison of Detection Parameters for Anti-inflammatory Effects and Improvement of Pancreatic Function

[0100]

[0101] Example Conclusion:

[0102] Based on Examples 1-3 and Comparative Example 1, and referring to Tables 1 and 2, it can be seen that improving the particle size of the tissue blocks can maintain a high yield and activity of stem cells; the tissue was minced to 0.5-2 mm. 3 The optimal particle size achieves a balance between physical digestion and enzymatic digestion efficiency. Too small a particle size can excessively disrupt tissue microstructure and intercellular connections, leading to damage to a large number of cells in the early stages of digestion and a sharp decline in cell activity. Simultaneously, excessively small fragments expose too large a surface area, causing proteinase K to act excessively and rapidly on the cells themselves, damaging cell membrane proteins and further reducing the viable cell ratio. The optimized particle size in this embodiment ensures that the enzyme solution fully penetrates the tissue to release stem cells while protecting the original vitality and integrity of the cells, thus providing high-quality seed cells for subsequent steps.

[0103] As can be seen from Examples 1-3 and Comparative Example 2, and in conjunction with Tables 1 and 2, controlling the proteinase K digestion time is a crucial factor in ensuring efficient stem cell extraction. Sufficient digestion time is necessary to ensure effective degradation of the extracellular matrix, thereby completely dissociating mesenchymal stem cells from the placental tissue. Insufficient digestion time results in a large number of stem cells remaining trapped in incompletely digested tissue blocks, which cannot be effectively harvested during centrifugation, directly leading to a low stem cell yield. These insufficiently released cells also cannot enter the subsequent expansion process, resulting in a waste of cell resources and insufficient final therapeutic product yield.

[0104] As can be seen from Examples 1-3 and Comparative Example 3, and in conjunction with Tables 1 and 2, the improvement of electroporation voltage has an impact on the success of gene modification. Electroporation allows exogenous genes to enter cells by forming temporary hydrophilic channels on the cell membrane. If the voltage is too low, the cell membrane cannot be effectively broken down, resulting in low transfection efficiency. If the voltage is too high, irreversible membrane damage will occur, leading to apoptosis or necrosis of a large number of cells and a decrease in the survival rate. Even if some cells survive, their cellular function will be impaired due to electrical stress, resulting in low expression efficiency of therapeutic factors. The voltage range of 120-150V used in the examples achieves a balance between forming effective channels and maintaining cell viability, ensuring high activity and high secretion capacity of transgenic cells.

[0105] Based on Examples 1-3 and Comparative Example 4, and in conjunction with Tables 1 and 2, it can be seen that the appropriate selection of the concentration of epigallocatechin gallate has an impact on the pretreatment effect of stem cells. EGCG, as a potent polyphenol, can activate intracellular antioxidant stress pathways and may enhance cellular metabolic adaptability and anti-apoptotic capacity by regulating mitochondrial function. If the pretreatment concentration is too low, it is insufficient to fully activate these protective signaling pathways, resulting in insufficient tolerance and functional activity of cells during the subsequent microvesicle extraction stress process and when facing an inflammatory environment after reinfusion into the body. This leads to a decrease in both the quantity and quality of microvesicles secreted by cells, thereby weakening the anti-inflammatory and repair efficacy of the final compound preparation.

[0106] Based on Examples 1-3 and Comparative Example 5, and in conjunction with Tables 1 and 2, it can be seen that optimizing the amount of baicalin added can affect the anti-inflammatory properties of the microvesicle-drug composite formulation. Baicalin has anti-inflammatory and antioxidant properties. After being mixed with microvesicles, it is loaded onto the surface or interior of the microvesicles through hydrophobic interactions or membrane fusion. The amount added needs to match the drug loading capacity of the microvesicles. Inappropriate addition can lead to excessive aggregation of drug molecules, affecting the stability or dispersibility of microvesicles, thereby reducing the bioavailability and targeted delivery efficiency of the drug. The addition amount in the examples ensures that the drug can be effectively loaded and synergistically interact with the microvesicles, jointly exerting a stronger anti-inflammatory effect at the target tissue site, thereby providing a microenvironment for the recovery of pancreatic islet function.

[0107] As can be seen from Examples 1-3 and Comparative Example 6, and in conjunction with Tables 1 and 2, the microvesicle dosage setting has an impact on the therapeutic effect. Therapeutic microvesicles need to reach a certain effective concentration threshold in vivo in order to accumulate locally in the pancreas and exert a sufficient paracrine effect through systematic distribution and targeted homing. Insufficient dosage means that this therapeutic threshold cannot be crossed, resulting in an insufficient total amount of infused microvesicles to form an effective signaling network around the islets, thus making it difficult to fully regulate the immune response, promote angiogenesis, and protect β cells. Therefore, a sufficient dosage is a prerequisite for ensuring the intensity of the therapeutic signal, achieving effective regulation of blood glucose homeostasis, and substantially improving pancreatic islet function.

[0108] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A stem cell therapy method for treating type 2 diabetes, characterized in that, Includes the following steps: S1. Placental mesenchymal stem cells were extracted, and primary stem cell populations were obtained by protease digestion and gradient centrifugation. High-purity mesenchymal stem cells were then obtained by amplification culture in serum-free medium. S2. The mesenchymal stem cells obtained in S1 are subjected to multi-gene modification, and the GIP and FGF1 dual-factor expression sequences are introduced into them through a non-viral vector to obtain gene-modified mesenchymal stem cells. S3. The gene-modified mesenchymal stem cells obtained in step S2 were pretreated with a culture medium containing epigallocatechin gallate and sitagliptin. S4. Microvesicles were extracted from the mesenchymal stem cells pretreated in S3 to obtain active microvesicles; S5. The microvesicles extracted in S4 are mixed with baicalin, L-arginine and vitamin D in a buffer solution to form a microvesicle-drug complex formulation. S6. The compound preparation prepared in S5 is introduced into the body of a type 2 diabetic patient via an intraperitoneal intervention. S7. After infusion, synergistic treatment with oral SGLT2 inhibitors is administered, and pancreatic function indicators are monitored regularly.

2. The stem cell therapy method for treating type 2 diabetes according to claim 1, characterized in that, Before step S1, the tissue pretreatment is also included: the obtained placental tissue is rinsed with Hank's balanced salt solution until there is no residue, cut into tissue blocks with a particle size of 0.5-2 mm³, and then 0.15% proteinase K is added and the tissue is shaken and digested at 35°C for 25-40 min.

3. The stem cell therapy method for treating type 2 diabetes according to claim 1, characterized in that, In step S1, the amplification culture conditions are as follows: cultured in a 36.5℃, 4.5% CO2 incubator using DMEM high glucose medium containing 8% human serum substitute for 6 to 8 days, with the medium being changed every 36 hours until the cell confluence reaches 85% to 95%.

4. The stem cell therapy method for treating type 2 diabetes according to claim 1, characterized in that, The mesenchymal stem cells isolated in step S1 need to be identified by surface markers, requiring a positive rate of ≥95% for CD73, CD90, and CD105, and a positive rate of ≤2% for CD34 and CD45.

5. A stem cell therapy method for treating type 2 diabetes according to claim 1, characterized in that, In step S2, the non-viral vector is an electroporation transfection system with a transfection voltage of 120–150V and a transfection time of 15–25ms. The gene-modified cells need to have their GIP and FGF1 expression efficiency detected by ELISA, requiring a dual-factor secretion level of no less than 50 ng / 10⁻⁶. 6 cells / 24h.

6. The stem cell therapy method for treating type 2 diabetes according to claim 1, characterized in that, In step S3, the pretreatment medium is RPMI-1640 medium, wherein the concentration of epigallocatechin gallate is 5–10 μmol / L, the concentration of sitagliptin is 8–15 μmol / L, the pretreatment time is 18–36 h, and the cell density is 5 × 10⁶ cells / year. 4 ~5×10 5 cells / mL.

7. A stem cell therapy method for treating type 2 diabetes according to claim 1, characterized in that, In step S4, the microvesicle extraction is performed using size exclusion chromatography: first, cell debris is removed by centrifugation at 1500×g for 15 min, and then microvesicle components with a particle size of 50-200 nm are collected by column chromatography; the obtained microvesicles must meet the requirements of positive expression of marker proteins CD9 and CD81, and the concentration must not be less than 2×10⁻⁶. 10 particles / mL.

8. A stem cell therapy method for treating type 2 diabetes according to claim 1, characterized in that, In step S5, the amount of baicalein added is 1-3 g / L, L-arginine is 0.3-0.6 g / L, and vitamin D is 0.1-0.3 g / L; the order of addition is to first dissolve baicalein in the buffer solution, then add L-arginine and vitamin D, and finally mix with microvesicles.

9. A stem cell therapy method for treating type 2 diabetes according to claim 1, characterized in that, In step S6, the dosage of the intraperitoneal interventional microvesicles is 5 × 10⁻⁶. 10 ~8×10 10 The dosage is 0.5-1.5 mL / min, and the compound preparation should be brought to room temperature before infusion.

10. A stem cell therapy method for treating type 2 diabetes according to claim 1, characterized in that, In step S7, the oral dose of the SGLT2 inhibitor is 10-25 mg / day, and administration begins within 12 hours after infusion; efficacy monitoring is based on a reduction of glycated hemoglobin to below 6.0% and an increase of more than 2 times in fasting C-peptide levels.

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