Double-factor controlled release microsphere as well as preparation method and application thereof

Abstract

The invention provides a two-factor controlled release microsphere as well as a preparation method and application thereof, and belongs to the technical field of biomedicine. The double-factor controlled release microspheres provided by the invention are prepared from the following raw materials: PLGA (poly (lactic-co-glycolic acid)), quercetin, a dichloromethane-ethyl acetate solution, an insulin-like growth factor 1 (IGF-1), a heparin solution and a polyvinyl alcohol solution. According to the two-factor controlled release microspheres of IGF-1 synergistic quercetin and the MSCs aggregate mediated by the microspheres, adhesion of MSCs in the cell aggregate is promoted through three-dimensional culture, the anti-aging and anti-apoptosis performance of the MSCs in the cell aggregate is enhanced through quercetin released in the early degradation space of the material, and the anti-aging and anti-apoptosis performance of the MSCs in the cell aggregate is improved. The proliferation, migration and secretion functions of cells in the aggregate are further adjusted through IGF-1 space-time delivery, and the treatment effect is enhanced.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to a dual-factor controlled-release microsphere and a preparation method and application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of multipotent stem cells that are widely present in the microenvironment of tissue matrix and have the ability of self-renewal, multidirectional differentiation and immunoregulation. The study of MSCs not only helps to understand the human development process, but can also be used for disease models and drug screening. More importantly, it can also be used for disease treatment. It has become one of the frontiers and hot spots in life science research for nearly half a century. Due to the wide range of sources, low immunogenicity and non-tumorigenicity of MSCs, they have become "practical stem cells" in the field of stem cell therapy in recent years. Traditional in vitro culture systems are prone to problems such as low MSCs cell yield, insufficient proliferation ability and functional loss, resulting in poor therapeutic effects. How to increase cell yield, avoid cell aging and maintain cell function during in vitro culture has always been the focus and difficulty of research in the field of regenerative medicine and tissue engineering.

[0003] MSCs therapy is a cell therapy based on regulating inflammatory response and participating in tissue repair and regeneration. The therapeutic function of MSCs is closely related to the microenvironment in which it is located. At the site of tissue damage, MSCs will respond to inflammatory factors to produce a large number of immunomodulatory factors, cell chemokines and growth factors, and promote tissue repair by regulating the tissue immune microenvironment and in situ tissue stem cells. It is further pointed out that the immunomodulatory function of MSCs needs to be induced, that is, the plasticity of MSCs immunomodulatory function. Based on the concept of plasticity of MSCs immunomodulatory function, we can better understand the role of MSCs in different stages of the disease, and MSCs pretreatment strategy can simulate the pathological inflammatory microenvironment in the disease state, providing an important new method for MSCs-based cell therapy. Studies have shown that for specific inflammatory microenvironments, the characteristics of MSCs immunomodulatory function plasticity can be used to formulate personalized clinical treatment strategies, which will comprehensively optimize MSCs-related clinical methods and enable them to achieve optimal regulation of inflammatory responses at different stages of disease progression. How to prepare MSCs with high cell functions and characteristics in batches during in vitro culture has become a key technical issue in the current preparation of engineered stem cells. Traditional in vitro two-dimensional culture methods cannot simulate the in vivo microenvironment well. The differences in this microenvironment are sufficient to have a huge impact on cell morphology, stress state, receptor distribution, metabolism and gene expression, seriously affecting the therapeutic effect of MSCs. Summary of the invention

[0004] In view of this, one of the objectives of the present invention is to provide a dual-factor controlled release microsphere capable of improving the damage and repair ability of mesenchymal stem cells and a preparation method and application thereof.

[0005] The second object of the present invention is to provide a mesenchymal stem cell aggregate and its application in the preparation of a wound treatment product.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a dual-factor controllable release microsphere, the raw materials of which include: PLGA, quercetin, dichloromethane-ethyl acetate solution, insulin-like growth factor 1 (IGF-1), heparin solution and polyvinyl alcohol solution; the concentration of the polyvinyl alcohol solution is 2% to 4%.

[0008] Preferably, the mass volume ratio of the PLGA, quercetin and dichloromethane-ethyl acetate solution is 120 mg:1.5 mg:3 mL; the mass ratio of quercetin and IGF-1 is 1.5 mg:20 μg; and the volume ratio of dichloromethane and ethyl acetate in the dichloromethane-ethyl acetate solution is 1:2.

[0009] Preferably, the concentration of the heparin solution is 5 mg / mL, and the mass volume ratio of the IGF-1 to the heparin solution is 20 μg:300 μL.

[0010] The present invention also provides a method for preparing the above-mentioned microspheres, comprising the following steps: mixing PLGA, quercetin and dichloromethane-ethyl acetate solution to obtain a quercetin solution; mixing IGF-1 with a heparin solution to obtain an IGF-1 solution; mixing the IGF-1 solution with the quercetin solution to obtain an oil phase; mixing the oil phase with a polyvinyl alcohol solution to generate droplets in a fluid focusing chip, collecting the droplets with a polyvinyl alcohol solution, removing the dichloromethane, and freeze-drying to obtain the microspheres.

[0011] Preferably, when the IGF-1 solution and the quercetin solution are mixed, the mixing method is ice bath homogenization; and the method of removing the dichloromethane is stirring and volatilization.

[0012] The present invention also provides the use of the microspheres or the preparation method in preparing products for inhibiting aging and apoptosis of mesenchymal stem cells or in three-dimensional culture of mesenchymal stem cells.

[0013] The present invention also provides the use of the above microspheres or the above preparation method in preparing a product for promoting differentiation of mesenchymal stem cells, wherein the differentiation includes one or more of osteogenic differentiation, chondrogenic differentiation and adipogenic differentiation.

[0014] The present invention also provides the use of the above microspheres or the above preparation method in preparing products that promote the proliferation, secretion and / or migration of mesenchymal stem cells.

[0015] The present invention also provides a mesenchymal stem cell aggregate obtained by mixed culturing of the mesenchymal stem cells and the above-mentioned microspheres.

[0016] The present invention also provides the use of the above-mentioned mesenchymal stem cell aggregates in preparing a product for treating wounds, wherein the wounds include diabetic wounds.

[0017] Beneficial effects of the present invention:

[0018] The present invention constructs dual-factor controlled release microspheres of IGF-1 and quercetin, and adopts MSCs aggregates mediated by the microspheres, promotes the adhesion of MSCs inside the cell aggregates through three-dimensional culture, enhances the anti-aging and anti-apoptosis properties of MSCs inside the cell aggregates through the spatial release of quercetin by early degradation of the material, and further regulates the proliferation, migration and secretion functions of cells inside the aggregates through the spatiotemporal delivery of IGF-1 to enhance the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The morphological characterization and release performance results of the dual-factor controlled release microspheres prepared by microfluidics, wherein A is the light microscope image of the droplets of the dual-factor controlled release microspheres; B is the SEM image of the dual-factor controlled release microspheres; C is the confocal microscope image of the dual-factor controlled release microspheres; D is the particle size image of the dual-factor controlled release microspheres prepared by microfluidics; E is the quercetin (Qur) and IGF-1 release image of the dual-factor controlled release microspheres;

[0020] Figure 2 The effects of dual-factor controlled release microspheres on stem cell function, where A is a morphology diagram of dual-factor controlled release microspheres regulating mesenchymal stem cells; B is a β-galactosidase staining diagram of dual-factor controlled release microspheres on the anti-aging performance of mesenchymal stem cells; C is the WB detection result of dual-factor controlled release microspheres on the anti-aging proteins of mesenchymal stem cells;

[0021] Figure 3 The results of preparing cell aggregates with dual-factor controlled-release microspheres and mesenchymal stem cells, from left to right are HE staining of aggregates, pure cell aggregates (control group) and osteogenic, chondrogenic and adipogenic differentiation of cell aggregates containing dual-factor controlled-release microspheres (IGF-1 / Qur-Mps);

[0022] Figure 4 The results of migration ability test of pure cell aggregates (control group) and cell aggregates containing dual-factor controlled release microspheres (IGF-1 / Qur-Mps);

[0023] Figure 5These are the test results of the healing ability of pure cell aggregates (control group) and cell aggregates containing dual-factor controllable release microspheres (IGF-1 / Qur-Mps) on the wounds of diabetic mice. From left to right are the wound pictures at 0, 3, 7, and 14 days after wound formation and the wound area tracking analysis pictures corresponding to the pictures. DETAILED DESCRIPTION

[0024] The invention provides a dual-factor controllable release microsphere, the raw materials of which include: PLGA, quercetin, dichloromethane-ethyl acetate solution, IGF-1, heparin solution and polyvinyl alcohol solution; the concentration of the polyvinyl alcohol solution is 2%-4%.

[0025] The present invention does not specifically limit the specific sources of the above-mentioned various raw materials, and conventional commercial products in this field can be used. In the present invention, the quercetin is preferably quercetin powder. In the present invention, the mass volume ratio of the PLGA, quercetin and dichloromethane-ethyl acetate solution is preferably 120mg:1.5mg:3mL; the mass ratio of the quercetin and IGF-1 is preferably 1.5mg:20μg; the volume ratio of dichloromethane and ethyl acetate in the dichloromethane-ethyl acetate solution is preferably 1:2. In the present invention, the concentration of the heparin solution is preferably 5mg / mL, and the mass volume ratio of the IGF-1 to the heparin solution is preferably 20μg:300μL.

[0026] The present invention also provides a method for preparing the above-mentioned microspheres, comprising the following steps: mixing PLGA, quercetin and dichloromethane-ethyl acetate solution to obtain a quercetin solution; mixing IGF-1 with a heparin solution to obtain an IGF-1 solution; mixing the IGF-1 solution with the quercetin solution to obtain an oil phase; mixing the oil phase with a polyvinyl alcohol solution to generate droplets in a fluid focusing chip, collecting the droplets with a polyvinyl alcohol solution, removing the dichloromethane, and freeze-drying to obtain the microspheres.

[0027] In the present invention, when the IGF-1 solution and the quercetin solution are mixed, the mixing method is preferably ice bath homogenization, and more preferably homogenization is performed using an ultrasonic cell crusher. In the present invention, a polyvinyl alcohol solution is used as the aqueous phase, and the oil phase and the aqueous phase are added to a syringe to generate droplets in a fluid focusing chip. The present invention does not specifically limit the specific source of the fluid focusing chip, and conventional commercial products in the field can be used. The present invention does not specifically limit the specific operation of generating droplets in the fluid focusing chip, and conventional operations in the field can be used. In the present invention, the method for removing dichloromethane is preferably stirring and volatilizing, and the stirring is preferably stirred using a magnetic stirrer, and the stirring time is preferably 4 hours. The present invention does not specifically limit the specific method of freeze-drying.

[0028] The present invention also provides the use of the above-mentioned microspheres or the above-mentioned preparation method in the following aspects: (1) preparing products for inhibiting senescence and / or apoptosis of mesenchymal stem cells; (2) three-dimensional culture of mesenchymal stem cells; (3) preparing products for promoting differentiation of mesenchymal stem cells, wherein the differentiation preferably includes one or more of osteogenic differentiation, chondrogenic differentiation and adipogenic differentiation; (4) preparing products for promoting proliferation, secretion and / or migration of mesenchymal stem cells. In the present invention, the types of the products preferably include reagents, kits or drugs.

[0029] The present invention also provides a mesenchymal stem cell aggregate obtained by mixed culturing of the mesenchymal stem cells and the above-mentioned microspheres.

[0030] In the present invention, it is preferred that 6×10 5 The mesenchymal stem cells are fully mixed with the microspheres at a ratio of 3:1 between the number of cells and the number of microspheres. In the present invention, the culture temperature is preferably 37° C., and the culture time is preferably 12 hours.

[0031] The present invention also provides the use of the above-mentioned mesenchymal stem cell aggregates in the preparation of a product for treating wounds, wherein the wounds include diabetic wounds. In the present invention, the types of the product preferably include reagents, kits or drugs.

[0032] The MSCs of the present invention can simulate the in vivo microenvironment to maintain the cell phenotype and biological function by self-assembly into cell aggregates to achieve three-dimensional culture of cells. Compared with monolayer culture, the cell behavior of MSCs cultured under three-dimensional conditions is often completely different. In order to solve the randomness of cell structure and function inside cell aggregates in three-dimensional culture and improve the cell therapy effect, the present invention constructs MSCs aggregates mediated by a double-factor controlled release functional microsphere system of cell growth factor coordination quercetin based on the basic principle of functional biomaterial biomimetic construction of extracellular microenvironment and regulation of stem cell fate, and constructs a high-quality three-dimensional aggregate cell batch culture technology mediated by functional microspheres, promotes the adhesion of MSCs inside cell aggregates through three-dimensional culture, releases quercetin through the space of early material degradation, and enhances the anti-aging and anti-apoptosis properties of MSCs inside cell aggregates, and further regulates the proliferation, migration, and secretion functions of cells inside aggregates through IGF-1 spatiotemporal delivery to enhance the therapeutic effect. The present invention also studies the preparation of a functionalized microsphere system for the spatiotemporal ordered controllable release of cell growth factors and quercetin, and the coordinated effects of its physicochemical properties on the cellular bioinformation molecules in MSCs aggregates, explores the bionic construction of the internal cellular microenvironment of three-dimensional MSCs aggregates mediated by functionalized biomaterials, reveals the mechanism and law of three-dimensional culture and IGF-1 and quercetin regulating the immune regulation function of MSCs, develops batch preparation technology of engineered stem cells mediated by functionalized biomaterials, and explores its mechanism and application in the field of chronic inflammatory diseases.

[0033] 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.

[0034] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] Example 1

[0037] Preparation of dual-factor controlled release microspheres using microfluidic technology

[0038] A fluid focusing chip was designed, and 120 mg of PLGA (purchased from Jinan Daigang Bioengineering Co., Ltd.) and 1.5 mg of quercetin powder (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were dissolved in 3 mL of dichloromethane-ethyl acetate solution (the dichloromethane-ethyl acetate solution was obtained by mixing 1 mL of dichloromethane (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and 2 mL of ethyl acetate (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.)), and the quercetin solution was obtained by fully stirring and dissolving;

[0039] 20 μg of IGF-1 was added to 300 μL of heparin solution (the concentration of the heparin solution was 5 mg / mL) and fully dissolved to obtain an IGF-1 solution;

[0040] The IGF-1 solution was added to the quercetin solution, stirred thoroughly, and homogenized using an ultrasonic cell disruptor (20 KHz) in an ice bath to serve as the oil phase;

[0041] 2 g of polyvinyl alcohol was dissolved in 100 mL of distilled water to prepare a 2% polyvinyl alcohol solution as the aqueous phase;

[0042] The oil phase and the water phase were added to the syringe respectively, and droplets were generated in a fluid focusing chip (purchased from Dalian Tuowei Chip Technology Co., Ltd.) (the oil phase flow rate was 0.4 mL / h, and the water phase flow rate was 1.2 mL / h). The droplets were collected with a 2% polyvinyl alcohol solution, stirred with a magnetic stirrer for 4 hours to evaporate the dichloromethane, and then freeze-dried with a freeze dryer after solidification to obtain dual-factor controlled release microspheres.

[0043] Example 2

[0044] Morphology detection of dual-factor controlled release microspheres obtained in Example 1

[0045] The droplets collected in Example 1 were placed under a microscope for observation to obtain the morphology of the microdroplets, as shown in FIG. Figure 1 As shown in A in the figure, the microdroplets have uniform particle size and are monodispersed.

[0046] The freeze-dried microspheres of Example 1 were resuspended in distilled water, blown evenly with a pipette, and then dropped onto a clean silicon wafer. After natural drying, they were fixed to the stage with conductive glue, sprayed with gold for 30 seconds, and observed on a SEM. Figure 1 As shown in B, the prepared microspheres have smooth surfaces and uniform particle sizes. The particle size results of the microspheres measured by the SEM image using nanomeasure software and then plotted using origin are shown in Figure 1 As shown in D, the particle size of the microspheres prepared by the present invention is 24.25±1.05 μm.

[0047] The internal structure of the microspheres was observed using a laser confocal microscope. IGF-1 was labeled with FITC. The specific method was as follows: 40 μg IGF-1 was fully dissolved in 500 μL PBS, and 10 μL 1 mg / mL FITC solution was added while stirring. The reaction was protected from light for 24 hours. After the reaction, the solution was transferred to a dialysis bag, and the water was changed every 3 days. The dialysis was fully performed for 2 weeks. After the dialysis, FITC-labeled IGF-1 was obtained for the preparation of dual-factor controlled release microspheres (the preparation method of microspheres is the same as in Example 1). Figure 1 As shown in C, IGF-1 is located inside the microspheres, and quercetin (Qur) is located on the outer shell of the microspheres, indicating that the microspheres prepared by the present invention contain two factors, IGF-1 and Qur.

[0048] Example 3

[0049] Example 1 Release performance test of the dual-factor controlled release microspheres obtained

[0050] 2 mg of the dual-factor controlled release microspheres obtained in Example 1 were suspended in 0.5 mL PBS (0.02% Tween 20 and 10 mg / mL BSA), placed in a 37°C constant temperature incubator for incubation, and the growth factor release was continuously detected. 10 μL of supernatant was collected by centrifugation every 3 days, and 10 μL of PBS (0.02% Tween 20 and 10 mg / mL BSA) was added for continued incubation. The supernatant was stored at -20°C. After collection, the content of the factor was measured using an enzyme-linked immunosorbent assay kit (ELISA). The concentration of the growth factor was obtained by comparison with the standard curve. All experiments were repeated three times. The results are shown in Table 1. Figure 1 As shown in Figure E, both factors were slowly released, with the release of quercetin reaching 84.67±2.13% and IGF-1 reaching 87.66±1.65% on the 18th day.

[0051] Example 4

[0052] Effect of the dual-factor controlled release microspheres obtained in Example 1 on cell morphology

[0053] Mesenchymal stem cells were cultured at a rate of 1×105 The cells were inoculated onto a 6-well plate and cultured for 24 hours. 2 μg of the dual-factor controlled release microspheres obtained in Example 1 (denoted as IGF-1 / Qur-Mps) were added. After 48 hours of co-culture, the cells were washed three times with PBS and fixed with 4% paraformaldehyde. The cell nuclei and cytoskeleton were stained with DAPI and FITC, respectively, to observe the cell morphology. Figure 2 As shown in A, it was found that after co-culture with microspheres, the cells still maintained a typical spindle-shaped structure, indicating that the microspheres have good biological activity.

[0054] Example 5

[0055] Effect of the dual-factor controlled release microspheres obtained in Example 1 on the anti-aging function of cells

[0056] The microspheres obtained in Example 1 (denoted as IGF-1 / Qur-Mps group) were co-cultured with mesenchymal stem cells for 7 days. After induction with 2% hydrogen peroxide, the cells were stained with β-galactosidase and the anti-aging effect was observed under an optical microscope. The mesenchymal stem cells without microspheres were used as the control group. The results are as follows: Figure 2 As shown in B, the addition of dual-factor controlled release microspheres can inhibit cell senescence. After 7 days of co-culture, the cells were washed three times with PBS, 100 μL RIPA cell lysis buffer was added, and the cells were repeatedly blown on ice until they were completely lysed. The cells were centrifuged at 12000×g for 15 minutes. The upper liquid was the extracted protein, 5×SDS-PAGE loading buffer was added, and the cells were boiled for 10 minutes for Western blotting to detect the expression of aging-related proteins. β-actin was used as an internal reference. Figure 2 As shown in C, the dual-factor controlled-release microsphere group expressed low P16 (a pro-aging marker), indicating that the dual-factor controlled-release microsphere can inhibit the aging of mesenchymal stem cells by releasing dual factors.

[0057] Example 6

[0058] Preparation of dual-factor controlled release microspheres and cell aggregates

[0059] 6×10 5 The mesenchymal stem cells and the dual-factor controlled release microspheres obtained in Example 1 were fully mixed at a ratio of 3:1 between the number of mesenchymal stem cells and the number of microspheres. The mixed cell and microsphere suspension (500 cells / microwell) was added to the treated Aggrewell TMThe plate was centrifuged at 1000g for 5 minutes at room temperature, and then placed in a 37°C incubator for 12 hours. The formation of cell aggregates was observed under a microscope. MSC aggregates with dual-factor controlled release microspheres (IGF-1 / Qur-Mps) and MSC aggregates without microspheres (expressed as MSC) were used as control groups. The aggregates were fixed with 4% paraformaldehyde, dehydrated with gradient alcohol, and sectioned with HE staining to observe the distribution of microspheres in the aggregates. Figure 3 As shown in the leftmost column, IGF-1 / Qur-Mps is evenly distributed inside the cells.

[0060] Example 7

[0061] Dual-factor controlled release microspheres to regulate the multidirectional differentiation potential of cell aggregates

[0062] Aggregates were prepared according to the method of Example 6. After 7 days of culture, the culture medium was replaced with an induction medium for differentiation into osteogenic, chondrogenic and adipogenic directions. After 14 days of induction culture, the aggregates were taken out, rinsed three times with PBS, fixed with 4% paraformaldehyde, dehydrated with gradient alcohol, and sliced. Alizarin red, Alcian blue and Oil red O staining were used for observation. Figure 3 As shown, the aggregates in the IGF-1 / Qur-Mps group were stained darker than those in the pure cell aggregates group, indicating that they had stronger differentiation potential toward osteogenic, chondrogenic, and adipogenic directions.

[0063] Example 8

[0064] Dual-factor controlled release microspheres to regulate cell aggregate migration performance detection

[0065] Aggregates were prepared according to the method of Example 6. After 7 days of culture, the aggregates were added to the matrix gel. After 48 hours, the migration of the aggregates was observed. Figure 4 As shown, the number of cells migrating out of the aggregates in the IGF-1 / Qur-Mps group was greater, and their morphology was more stretched, showing a typical spindle-shaped structure of mesenchymal cells.

[0066] Example 9

[0067] Example 6 Effect of the dual-factor controlled release microsphere-mediated aggregates on treating wounds in diabetic mice

[0068] 20 male C57BL / 6 mice (6-8 weeks, 20-25g) were purchased from Spef (Beijing) Biotechnology Co., Ltd. for the experiment. A high-sugar mouse model was constructed by intraperitoneal injection of streptozotocin (100mg / kg). The blood glucose level of the mice was verified after 1 week. When the random blood glucose of the mice increased to 11mmol / L, it indicated that the modeling was successful. After the mice were anesthetized, a puncher with a diameter of 8mm was used to punch holes in the symmetrical parts of the back with the spine as the axis. Ophthalmic scissors were used to assist in cutting off the discarded skin to form two full-thickness excision skin wounds with a diameter of 8mm. The mice were randomly divided into a control group and an IGF-1 / Qur-Mps group. The microsphere cell aggregates without microspheres and the microsphere cell aggregates prepared in Example 6 were transplanted into the bodies of the wound model animals. The microsphere cell aggregates without microspheres were used as the control. The wounds were photographed and recorded on days 0, 3, 7, and 14 to observe the healing of the wounds. The results are as follows Figure 5 As shown, it is shown that the microsphere-containing cell aggregates of the present invention have a better effect of promoting wound healing.

[0069] 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 dual-factor controlled release microsphere, characterized in that: The raw materials of the microspheres include: PLGA, quercetin, dichloromethane-ethyl acetate solution, IGF-1, heparin solution and polyvinyl alcohol solution; the concentration of the polyvinyl alcohol solution is 2% to 4%.

2. The microsphere according to claim 1, characterized in that The mass volume ratio of the PLGA, quercetin and dichloromethane-ethyl acetate solution is 120 mg:1.5 mg:3 mL; the mass ratio of quercetin and IGF-1 is 1.5 mg:20 μg; the volume ratio of dichloromethane and ethyl acetate in the dichloromethane-ethyl acetate solution is 1:

2.

3. The microsphere according to claim 1, characterized in that The concentration of the heparin solution is 5 mg / mL, and the mass volume ratio of the IGF-1 to the heparin solution is 20 μg:300 μL.

4. The method for preparing microspheres according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: mixing PLGA, quercetin and dichloromethane-ethyl acetate solution to obtain quercetin solution; mixing IGF-1 and heparin solution to obtain IGF-1 solution; mixing the IGF-1 solution and quercetin solution to obtain an oil phase; mixing the oil phase and polyvinyl alcohol solution to generate droplets in a fluid focusing chip, collecting the droplets with the polyvinyl alcohol solution, removing the dichloromethane, and freeze-drying to obtain the microspheres.

5. The preparation method according to claim 4, characterized in that: When the IGF-1 solution and the quercetin solution are mixed, the mixing method is ice bath homogenization; and the method of removing the dichloromethane is stirring and volatilization.

6. Use of the microspheres according to any one of claims 1 to 3 or the preparation method according to any one of claims 4 to 5 in preparing products for inhibiting aging and apoptosis of mesenchymal stem cells or in three-dimensional culture of mesenchymal stem cells.

7. Use of the microspheres according to any one of claims 1 to 3 or the preparation method according to any one of claims 4 to 5 in preparing a product for promoting differentiation of mesenchymal stem cells, characterized in that: The differentiation includes one or more of osteogenic differentiation, chondrogenic differentiation and adipogenic differentiation.

8. Use of the microspheres according to any one of claims 1 to 3 or the preparation method according to any one of claims 4 to 5 in the preparation of products that promote the proliferation, secretion and / or migration of mesenchymal stem cells.

9. A mesenchymal stem cell aggregate, characterized in that: The product is obtained by mixing and culturing mesenchymal stem cells with the microspheres according to any one of claims 1 to 3.

10. Use of the mesenchymal stem cell aggregates according to claim 9 in preparing a product for treating wounds, characterized in that: The wound surface includes a diabetic wound surface.

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