Cell-microsphere co-assembly as well as preparation method and application thereof

Abstract

The invention provides a cell-microsphere co-assembly body. The cell-microsphere co-assembly body is formed by co-assembling hydrogel microspheres and cells, the ratio of the hydrogel microspheres to the cells is (0.0001-10) [mu] L: (101-7). The microsphere-cell co-assembly is prepared from the microspheres prepared from specific components and the cells in a co-assembly mode, an excellent microenvironment can be provided for the cells, the paracrine effect of the cells can be enhanced, and after the microsphere-cell co-assembly is injected to a damaged part in situ, the treatment efficiency of the cells can be improved, and damaged tissue repair can be promoted.

Description

Technical Field

[0001] The present invention specifically relates to a cell-microsphere co-assembly and a preparation method and application thereof. Background Art

[0002] Since most human tissues and organs do not have the ability to regenerate, it is crucial to promote the repair of damaged tissues and organs.

[0003] In recent years, stem cell therapy based on mesenchymal stem cells (MSCs) has shown ideal therapeutic effects in tissue and organ damage. MSCs have the advantages of low immunogenicity and wide sources, and can promote the repair of damaged tissues and organs through sustained paracrine effects. However, due to the harsh microenvironment of the damaged area, the local retention rate and survival rate of MSCs are low, which limits its therapeutic effect.

[0004] Hydrogel microspheres refer to spheres or spheroids with a particle size of micrometers, which can be used to deliver bioactive substances such as drugs, genes, proteins, and factors. After local injection, hydrogel microspheres will slowly degrade, and the loaded bioactive substances will diffuse locally and slowly release according to the degradation of the microspheres. They have the advantages of long-term, safety, and targeting, and have wide application potential in clinical treatment. In addition, compared with traditional hydrogels, the microsphere system has a larger specific surface area and can better interact with the surrounding microenvironment. Zhou Jian et al., Performance of methacryloyl gelatin / decellular meniscus extracellular matrix composite hydrogels with different cross-linking densities [J], Chinese Journal of Tissue Engineering Research, 2493-2499, disclosed a cell-loaded hydrogel microsphere, but it only carried cells through photocrosslinking, and the size of the obtained composite hydrogel was relatively fixed, and due to the limited cell viability, it could not interact well with the external environment, which affected its efficacy. It is necessary to develop a gel complex with controllable size, good interaction with the external environment, paracrine function, and continuous maintenance of the secretion of related repair factors. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a cell-microsphere co-assembly, which is co-assembled by hydrogel microspheres and cells; the ratio of the hydrogel microspheres to the cells is 0.0001-10 μL: 10 1～7 indivual;

[0006] The hydrogel microspheres are microspheres prepared by mixing any one or more of synthetic materials, natural materials or derivative materials with a photoinitiator;

[0007] The synthetic material includes polyethylene glycol, polyvinyl alcohol, povidone, polylactic acid, polycaprolactone, polyurethane or polyglycerol sebacate;

[0008] The natural material includes gelatin, sodium alginate, sodium hyaluronate, chitosan, fibrin, collagen or decellularized extracellular matrix;

[0009] The derivative material includes polyethylene glycol diacrylate, methacrylated polyvinyl alcohol, methacrylated polycaprolactone, methacrylated polylactic acid, polyurethane methacrylate, methacrylated gelatin, methacrylated sodium hyaluronate, methacrylated sodium alginate, methacrylated chitosan, methacrylated fibrin, methacrylated collagen or methacrylated decellularized extracellular matrix.

[0010] Furthermore, the hydrogel microspheres are microspheres prepared by mixing any one or more of synthetic materials, natural materials or derivative materials with a photoinitiator, and using microfluidics, solvent evaporation, phase separation, spray drying, electrostatic spraying, template method or suspension polymerization.

[0011] Furthermore, the hydrogel microspheres are microspheres prepared by mixing methacrylylated decellularized extracellular matrix with a photoinitiator through a microfluidic chip.

[0012] Furthermore, the methacrylated decellularized extracellular matrix is ​​prepared by reacting the decellularized extracellular matrix and methacrylic anhydride under alkaline conditions, dialyzing the reaction product, and freeze-drying it; and the photoinitiator includes phenyl-2.4.6-trimethylbenzoyl lithium phosphite.

[0013] Furthermore, the decellularized extracellular matrix is ​​a decellularized extracellular matrix derived from porcine myocardial tissue.

[0014] Furthermore, the ratio of the hydrogel microspheres to the cells is 10 μL:10 6 indivual.

[0015] Furthermore, the cells include any one or more of mesenchymal stem cells, pluripotent stem cells, cardiomyocytes, vascular endothelial cells, adipose stem cells, chondrocytes, and tendon cells, preferably mesenchymal stem cells.

[0016] Furthermore, the particle size of the cell-microsphere co-assembly is 1-2000 μm; and the particle size of the hydrogel microsphere is 1-1000 μm.

[0017] The present invention also provides a method for preparing the aforementioned cell-microsphere co-assembly, which comprises the following steps:

[0018] 1) taking any one or more of synthetic materials, natural materials or derivative materials, mixing them with a photoinitiator aqueous solution and passing them through a microfluidic chip to obtain hydrogel microspheres;

[0019] 2) The hydrogel microspheres obtained in step 1) are mixed with cells in the above ratio and co-assembled to obtain the product.

[0020] Further, in step 1), the decellularized extracellular matrix is ​​dissolved in water, methacrylic anhydride is added, and the reaction is carried out under alkaline conditions, the reaction product is dialyzed, and freeze-dried to obtain the methacrylated decellularized extracellular matrix, which is then mixed with a photoinitiator aqueous solution and passed through a microfluidic chip to obtain hydrogel microspheres;

[0021] Furthermore, in step 1), the mass volume ratio of the decellularized extracellular matrix, water, and methacrylic anhydride is 1 g: 100 mL: 1 mL; the mass volume ratio of the methacrylated decellularized extracellular matrix, photoinitiator, and water is 10 g: 100-600 mg: 100 mL; and the photoinitiator includes phenyl-2.4.6-trimethylbenzoyl lithium phosphite.

[0022] Furthermore, in step 1), the alkaline pH value is 8-12; the reaction temperature is 0°C, and the time is 1 to 48 hours; the dialysis is performed in 0 to 50°C water using a 3500 dialysis bag; the methacrylylated decellularized extracellular matrix is ​​mixed with a photoinitiator aqueous solution as a dispersed phase and a continuous phase through a microfluidic chip; the flow rate ratio of the dispersed phase to the continuous phase is 1:1-1:60, and the preferred flow rate ratio is 1:20; the continuous phase is a liquid paraffin solution containing 1 to 30% Span 80.

[0023] Furthermore, the co-assembly time of step 2) is 18 to 36 hours, the temperature is 35 to 40°C, the carbon dioxide concentration is 3 to 8%, and the culture medium is a DMEM low-glucose culture medium containing 10% FBS and 1% dual antibody. Furthermore, the dual antibody is 10,000 units / mL of penicillin and 10 mg / mL of streptomycin.

[0024] The present invention also provides a use of the aforementioned cell-microsphere co-assembly in preparing a medicine for tissue repair.

[0025] Furthermore, the tissue includes myocardium, skin, muscle, cartilage, blood vessels, tendons and / or nerves.

[0026] Furthermore, the drug is a drug for treating myocardial infarction.

[0027] Furthermore, the drug has the effect of improving cardiac function, reducing infarct size and / or reducing fibrosis in the infarct area.

[0028] Furthermore, the drug is a drug for local injection; the drug for local injection is locally injected into the damaged part, and utilizes the paracrine function of cells to promote angiogenesis, inhibit cell apoptosis, and promote tissue repair.

[0029] Furthermore, the local injection includes intramyocardial injection, pericardial cavity injection, transcatheter coronary artery injection, intradermal injection, subcutaneous injection, intramuscular injection, joint cavity injection, vascular cavity injection, tendon sheath injection, intrathecal injection and / or spinal nerve injection.

[0030] The microsphere-cell co-assembly of the present invention is prepared by co-assembling microspheres made of specific ingredients and mesenchymal stem cells, which can provide an excellent microenvironment for cells, enhance the paracrine effect of cells, and improve the therapeutic efficiency of stem cells after in situ injection into the damaged area.

[0031] Experiments have shown that the microsphere system of the present invention can promote cell proliferation and migration, promote angiogenesis and inhibit apoptosis of damaged cells in a paracrine manner, and provide a new option with better efficacy for promoting the repair of damaged tissues and the clinical treatment of tissue and organ damage.

[0032] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0033] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Microscope image of ECM microspheres (scale bar: 50 μm);

[0035] Figure 2 (A) Transmission electron microscopy (scale bar: 5 μm, 500 nm) and (B) microscopy (scale bar: 100 μm) images of ECM-MSCs;

[0036] Figure 3 Survival of MSC cells in ECM-MSCs;

[0037] Figure 4 The relationship between the amount of cells and microspheres and the size of cell-microsphere assemblies;

[0038] Figure 5 ECM-MSC angiogenesis assay (scale bar: 100 μm);

[0039] Figure 6 ECM-MSC cell migration assay (MSC: mesenchymal stem cells; EMB: control microspheres; EMA: ECM-MSC assembly);

[0040] Figure 7(A) Echocardiogram after ECM-MSC treatment, sham: control group, MI: myocardial infarction group, ECM-MSC: cell-microsphere assembly group, (B) ejection fraction and (C) fractional shortening;

[0041] Figure 8 TCC staining, sham: control group, MI: myocardial infarction group, ECM-MSC: cell-microsphere assembly group;

[0042] Fig. 9 (Top) H&E staining and (bottom) Masson staining images. DETAILED DESCRIPTION

[0043] Example 1 Preparation of the cell-microsphere co-assembly of the present invention

[0044] 1) 0.05 g of porcine myocardial tissue-derived decellularized extracellular matrix was dissolved in 5 mL of water, 0.05 mL of methacrylic anhydride was added under ice bath, and then the pH value was adjusted to alkaline (pH value 8) with 1 M sodium hydroxide, and reacted under ice bath for 8 h, dialyzed in water at room temperature for 5 days with a 3500 dialysis bag, and then freeze-dried, and then mixed with 5 mg of photoinitiator phenyl-2.4.6-trimethylbenzoyl lithium phosphite and 0.5 mL of water as the dispersed phase, and passed through a microfluidic chip with a continuous phase (liquid paraffin containing 10% Span 80), and the flow rate ratio of the dispersed phase to the continuous phase was controlled to be 1:20, and the hydrogel microspheres were collected, and then washed 3 times with a 10% Tween 80 aqueous solution and 3 times with PBS to obtain 50 μm hydrogel microspheres (ECM);

[0045] 2) Take 10 μL of ECM microspheres and 10 6 Bone marrow-derived mesenchymal stem cells (MSCs) were co-assembled and cultured in a cell well plate for 24 h (temperature: 37°C, carbon dioxide concentration: 5%, culture medium: DMEM low-glucose culture medium containing 10% FBS and 1% double antibody) to obtain cell-microsphere co-assemblies (ECM-MSCs).

[0046] Example 2 Preparation of the cell-microsphere co-assembly of the present invention

[0047] 1) 0.05 g of gelatin was dissolved in 5 mL of water, 0.05 mL of methacrylic anhydride was added under ice bath, and then the pH value was adjusted to alkaline (pH value 9) with 1 M sodium hydroxide, and the reaction was carried out under ice bath for 12 h, and the mixture was dialyzed in water at 30° C. for 5 days with a 3500 dialysis bag and freeze-dried, and then mixed with 3 mg of photoinitiator phenyl-2.4.6-trimethylbenzoyl lithium phosphite and 0.5 mL of water as a dispersed phase, and passed through a microfluidic chip with a continuous phase (liquid paraffin containing 10% Span 80), and the flow rate ratio of the dispersed phase to the continuous phase was controlled to be 1:15, and the hydrogel microspheres were collected, and then washed with a 10% Tween 80 aqueous solution for 3 times and PBS for 3 times to obtain hydrogel microspheres;

[0048] 2) Take 5 μL of microspheres and 10 7 Tendon cells were co-assembled and cultured in a cell well plate for 18 h (temperature: 37° C., carbon dioxide concentration: 5%, culture medium: tendon cell culture medium containing 10% FBS and 1% double antibody) to obtain a cell-microsphere co-assembly.

[0049] Example 3 Preparation of cell-microsphere co-assembly of the present invention

[0050] 1) 0.05 g of polyethylene glycol diacrylate was mixed with 5 mg of photoinitiator phenyl-2.4.6-trimethylbenzoyl lithium phosphite and 0.5 mL of water as a dispersed phase, and passed through a microfluidic chip with a continuous phase (liquid paraffin containing 10% Span 80), and the flow rate ratio of the dispersed phase to the continuous phase was controlled to be 1:25, and hydrogel microspheres were collected, and then washed with a 10% Tween 80 aqueous solution for 3 times and PBS for 3 times to obtain hydrogel microspheres;

[0051] 2) Take 1 μL of microspheres and 10 5 After the chondrocytes were co-assembled and cultured in a cell well plate for 24 h (temperature: 37° C., carbon dioxide concentration: 5%, culture medium: chondrocyte culture medium containing 10% FBS and 1% double antibody), a cell-microsphere co-assembly was obtained.

[0052] The beneficial effects of the present invention are further illustrated by test examples below.

[0053] Experimental Example 1 Study on the treatment of myocardial infarction with porcine myocardial tissue-derived acellular extracellular matrix-mesenchymal stem cell co-assembly.

[0054] 1. Test Materials

[0055] 1. Materials

[0056] Decellularized extracellular matrix (d-ECM) derived from porcine myocardial tissue, methacrylic anhydride (MMA), lithium phenyl-2.4.6-trimethylbenzoyl phosphite (LAP), PBS, Matrigel, etc.

[0057] The d-ECM is prepared by the following method: cut the pig myocardial tissue into small pieces with a thickness of 1 mm, stir in a PBS solution containing 1% SDS for 72 hours, change the solution every 24 hours, treat with 1% Triton X-100 solution for 30 minutes after the tissue turns white, wash with PBS solution for 3 days, and freeze-dry for use. Take 10 mg of the freeze-dried decellularized myocardial tissue and digest it in a hydrochloric acid solution (0.1M) containing 1 mg of pepsin for 48 hours, centrifuge to remove insoluble precipitates, adjust the pH value to 7.4 with 1M NaOH, and freeze-dry to obtain the pig myocardial tissue-derived decellularized extracellular matrix (d-ECM).

[0058] 2. Cells

[0059] Mesenchymal stem cells derived from rat bone marrow were cultured in DMEM low-glucose medium containing 10% fetal bovine serum and 1% double antibody.

[0060] 3. Animals

[0061] SPF male SD rats, 6-8 weeks old, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. All animal experiments were performed under the approval and supervision of the Animal Management Committee of West China Hospital, Sichuan University.

[0062] 2. Experimental Methods

[0063] 1. Preparation and characterization of microspheres and cell-microsphere co-assemblies

[0064] 1.1 Preparation of microspheres and cell-microsphere co-assemblies

[0065] Step 1: Preparation of microspheres. Using porcine myocardial tissue-derived decellularized extracellular matrix (d-ECM) as the material, 0.05 g of d-ECM was dissolved in 5 mL of water, 0.05 mL of MAA was added under ice bath, and then the pH value was adjusted to 8-9 with 1 M sodium hydroxide, and the reaction was allowed to proceed overnight under ice bath. The d-ECMMA was dialyzed in water at room temperature for 5 days using a 3500 dialysis bag and then freeze-dried to obtain d-ECMMA for use. d-ECMMA and LAP were prepared by microfluidic technology (i.e., d-ECMMA was mixed with LAP and water as the dispersed phase, and liquid paraffin containing 10% Span 80 was passed through a microfluidic chip as the continuous phase) to prepare hydrogel microspheres, and the flow rate ratio of the dispersed phase to the continuous phase was controlled to be 1:20. The hydrogel microspheres were collected, and then washed 3 times with an emulsifier aqueous solution of 10% Tween 80 aqueous solution and 3 times with PBS to obtain ECM hydrogel microspheres, and the prepared microspheres were characterized by microscopy.

[0066] Step 2: Preparation of cell-microsphere co-assembly. Using the prepared 50 μm microspheres (ECM) as the skeleton material, 10 μL of ECM microspheres and 10 6 Bone marrow-derived mesenchymal stem cells (MSCs) were co-assembled and cultured for 24 h (temperature: 37°C, carbon dioxide concentration: 5%, culture medium: DMEM low-glucose culture medium containing 10% FBS and 1% double antibody) to obtain cell-microsphere co-assemblies (ECM-MSCs).

[0067] 1.2 Characterization of microspheres and cell-microsphere co-assemblies

[0068] The prepared ECM-MSCs were characterized by transmission electron microscopy and microscopy, and the survival of MSCs in the co-assembly was investigated. In addition, the size of the cell-microsphere co-assembly was controlled by adjusting the amount of cells and microsphere system.

[0069] 2. In vitro and in vivo studies of cell-microsphere co-assemblies (ECM-MSC)

[0070] 2.1 Pro-angiogenic effect of ECM-MSCs

[0071] The angiogenesis-promoting effect of ECM-MSC was investigated by using vascular endothelial cells (HUVEC). The specific operation was as follows: Place the matrix gel in a 96-well plate and wait for it to solidify. Add 3*10 4 HUVEC cells were cultured and then the secretion of ECM-MSC was added, and the formation of tubules was observed at a set time.

[0072] 2.2 Migration effect of ECM-MSCs

[0073] MSC cells were loaded into the ECM hydrogel microspheres prepared in step 1 under “1.1 Preparation of microspheres and cell-microsphere co-assemblies” as a control, and the migration ability of the cells in the ECM hydrogel microspheres and the ECM-MSC prepared in step 2 under “1.1 Preparation of microspheres and cell-microsphere co-assemblies” was investigated using Transwell chambers.

[0074] The method for preparing the control microspheres is as follows: d-ECMMA, LAP and MSC cells are used to prepare MSC-loaded microspheres of the same size as ECM-MSC by microfluidics technology.

[0075] 2.3 In vivo animal experiments

[0076] SPF male SD rats were used as model animals to study the effect of ECM-MSCs on myocardial infarction. First, the rat myocardial infarction model was established by ligating the left anterior descending coronary artery. The treatment groups were as follows: (1) normal control group; (2) myocardial infarction group; (3) cell-microsphere assembly group. ECM-MSCs were injected into the infarcted myocardium at 3 points in the myocardium. The experimental endpoints were echocardiography, TCC staining, H&E and Masson staining to investigate the therapeutic effect of ECM-MSCs on myocardial infarction.

[0077] 3. Experimental results.

[0078] 1. Characterization of microspheres and cell-microsphere co-assemblies

[0079] 1.1 Characterization of microspheres.

[0080] Microspheres prepared by microfluidics technology Figure 1 As shown, the size is 50 μm and the particle size distribution is uniform.

[0081] 1.2 Characterization of cell-microsphere co-assemblies

[0082] like Figure 2As shown, the microspheres were co-assembled with MSCs to obtain cell-microsphere co-assemblies with a size of 500 μm, and the cells tightly wrapped the microspheres. The survival of MSC cells in the ECM-MSC assembly was tested by CCK-8 experiment. The experimental results are shown in Figure 3 As shown in Figure 2, MSC cells in ECM-MSC can proliferate as the culture time increases, indicating that the ECM in ECM-MSC can provide nutrients to promote the growth of MSC cells. Figure 4 As shown, microspheres of different sizes can be obtained by adjusting the amount of cells and microsphere system.

[0083] 2. In vitro and in vivo studies of cell-microsphere co-assemblies (ECM-MSC)

[0084] 2.1 Pro-angiogenic effect of ECM-MSCs

[0085] The angiogenesis-promoting effect of ECM-MSC was examined by tube formation experiment. The experimental results are as follows Figure 5 As shown, HUVEC cells stimulated by ECM-MSCs can form a vascular network structure, indicating that ECM-MSCs can release related factors that promote angiogenesis through paracrine effects and promote angiogenesis.

[0086] 2.2 Migration effect of ECM-MSCs

[0087] The experimental results are as follows Figure 6 As shown, compared with large microspheres in which cells are loaded into microspheres, the cell-microsphere assembly can better promote the migration of cells out of the microspheres and is more conducive to the growth and proliferation of cells.

[0088] 3. In vivo animal experiments

[0089] At the end of the experiment, the therapeutic effect of ECM-MSC on myocardial infarction in rats was investigated by small animal ultrasound. Figure 7 As shown in the figure, ECM-MSC treatment can effectively alleviate myocardial infarction in rats, and the ejection fraction and shortening fraction are significantly higher than those in the MI group. The TCC staining results show that ( Figure 8 ), the myocardium of the MI group had a large amount of infarct tissue, while the ECM-MSC treatment group had only a small amount of infarct tissue. H&E staining and Masson staining results ( Fig. 9 ) It can be further seen that ECM-MSC can effectively alleviate infarction and inhibit the occurrence of fibrosis. The experimental results further illustrate that the ECM-MSC system can improve cardiac function, reduce infarction area, and reduce fibrosis in the infarct area, providing new ideas and practical approaches for the clinical treatment of myocardial infarction to promote public health, and have positive social and economic benefits.

[0090] In summary, compared with the microsphere system in which cells are loaded into hydrogel microspheres by blending the materials of cells and hydrogel microspheres, the cell-microsphere co-assembly ECM-MSC of the present invention is more conducive to cell proliferation and migration, and exerts a sustained paracrine effect, and has a more significant effect in promoting myocardial tissue repair.

Claims

1. A cell-microsphere co-assembly, characterized in that: It is composed of hydrogel microspheres and cells co-assembled; the ratio of the hydrogel microspheres to the cells is 0.0001-10μL:10 1～7 indivual; The hydrogel microspheres are microspheres prepared by mixing any one or more of synthetic materials, natural materials or derivative materials with a photoinitiator; The synthetic material includes polyethylene glycol, polyvinyl alcohol, povidone, polylactic acid, polycaprolactone, polyurethane or polyglycerol sebacate; The natural material includes gelatin, sodium alginate, sodium hyaluronate, chitosan, fibrin, collagen or decellularized extracellular matrix; The derivative material includes polyethylene glycol diacrylate, methacrylated polyvinyl alcohol, methacrylated polycaprolactone, methacrylated polylactic acid, polyurethane methacrylate, methacrylated gelatin, methacrylated sodium hyaluronate, methacrylated sodium alginate, methacrylated chitosan, methacrylated fibrin, methacrylated collagen or methacrylated decellularized extracellular matrix.

2. The cell-microsphere co-assembly according to claim 1, characterized in that: The hydrogel microspheres are prepared by mixing any one or more of synthetic materials, natural materials or derivative materials with a photoinitiator, and using a microfluidics method, a solvent evaporation method, a phase separation method, a spray drying method, an electrostatic spray method, a template method or a suspension polymerization method; The photoinitiator includes phenyl-2.4.6-trimethylbenzoyl lithium phosphite.

3. The cell-microsphere co-assembly according to claim 1, characterized in that: The ratio of hydrogel microspheres to cells is 10 μL:10 6 indivual.

4. The cell-microsphere co-assembly of claim 1, characterized in that: The cells include any one or more of mesenchymal stem cells, pluripotent stem cells, cardiomyocytes, vascular endothelial cells, adipose stem cells, chondrocytes, and tendon cells, preferably mesenchymal stem cells.

5. The cell-microsphere co-assembly according to any one of claims 1 to 4, characterized in that: The particle size of the cell-microsphere co-assembly is 1-2000 μm; the particle size of the hydrogel microsphere is 1-1000 μm.

6. A method for preparing the cell-microsphere co-assembly according to any one of claims 1 to 5, characterized in that: It includes the following steps: 1) taking any one or more of synthetic materials, natural materials or derivative materials, mixing them with a photoinitiator aqueous solution and passing them through a microfluidic chip to obtain hydrogel microspheres; 2) The hydrogel microspheres obtained in step 1) are mixed with cells in the above ratio and co-assembled to obtain the product.

7. Use of the cell-microsphere co-assembly according to any one of claims 1 to 5 in the preparation of a medicament for tissue repair.

8. The use according to claim 7, characterized in that: The tissues include myocardium, skin, muscle, cartilage, blood vessels, tendons and / or nerves.

9. The use according to claim 7, characterized in that: The drug is a drug for local injection; the drug for local injection is locally injected into the damaged part, and utilizes the paracrine function of cells to promote angiogenesis, inhibit cell apoptosis, and promote tissue repair.

10. The use according to claim 9, characterized in that: The local injection includes intramyocardial injection, pericardial cavity injection, transcatheter coronary artery injection, intradermal injection, subcutaneous injection, intramuscular injection, joint cavity injection, vascular cavity injection, tendon sheath injection, intrathecal injection and / or spinal nerve injection.

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