CN114224917A

CN114224917A - Application of mesenchymal stem cell apoptosis microvesicle combined hydrogel in preparation of mammal skin repair product

Info

Publication number: CN114224917A
Application number: CN202111588344.5A
Authority: CN
Inventors: 邱新毓; 轩昆; 郭静; 韩冰; 关玲霞; 班晶浩; 王毅萱
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2022-03-25

Abstract

The invention provides application of mesenchymal stem cell apoptosis microvesicle combined hydrogel in preparation of a mammalian skin repair product, belonging to the technical field of regenerative medicine. In the invention, the mesenchymal stem cell apoptosis microvesicle can promote vascularization of a new tissue and regulate local immune microenvironment of a receptor tissue so as to accelerate epithelialization of a skin wound to promote healing. The hydrogel with good biocompatibility can effectively protect the loss of the mesenchymal stem cell apoptosis microvesicle and can ensure the biological activity of the mesenchymal stem cell apoptosis microvesicle. The application of the mesenchymal stem cell apoptosis microvesicle and the hydrogel in a combined manner can improve the utilization efficiency and the skin repair effect of the mesenchymal stem cell apoptosis microvesicle and better play the biological function of the mesenchymal stem cell apoptosis microvesicle, thereby accelerating the healing of wounds and promoting the repair and regeneration of damaged skin.

Description

Application of mesenchymal stem cell apoptosis microvesicle combined hydrogel in preparation of mammal skin repair product

Technical Field

The invention belongs to the technical field of regenerative medicine, and particularly relates to an application of mesenchymal stem cell apoptosis microvesicle combined hydrogel in preparation of a mammalian skin repair product.

Background

The skin is located on the surface of the body and is in direct contact with the external environment, has the functions of protecting, excreting, regulating body temperature, sensing external stimulation and the like, and is the largest organ in the human body. Skin protects various tissues and organs in the body from external stimuli and is a very important natural barrier for the human body. The skin is divided into epidermis and dermis. Under normal conditions, the epidermal stratum corneum cells are constantly shed, being supplemented by basal cell proliferation, which is physiological regeneration. If the skin is injured and healed, it is called reparative regeneration, and the regeneration process and repair time vary according to the area and depth of injury. Small-area injuries can heal for several days without scars, and large-area injuries can cause scars, cause functional limitation and the like.

The process of skin regeneration is: the wound surface is coagulated, fibroblasts in the lower connective tissue proliferate, capillaries are increased to form granulation tissue, then collagen fibers and stroma are generated to form new connective tissue to repair the wound, meanwhile, basal cells of the epidermis around the wound and residual hair follicle and sweat gland epithelial cells are continuously divided to cover the wound surface, and finally, a multiple layer of epithelium is formed, and the wound is completely healed. If the skin injury area is large and deep, epidermis repair is difficult, a skin grafting method is needed to help wound repair, and scars are often left after wound repair. At present, the exploration of means capable of promoting the regeneration of damaged skin tissues has important scientific and medical significance.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of a mesenchymal stem cell apoptosis microvesicle combined hydrogel in preparation of a mammalian skin repair product, wherein the mesenchymal stem cell apoptosis microvesicle combined hydrogel can effectively promote regeneration of damaged skin tissues.

The invention provides application of mesenchymal stem cell apoptosis microvesicles in preparing a mammalian skin repair product in combination with hydrogel.

Preferably, the hydrogel comprises a temperature-sensitive hydrogel.

Preferably, the mesenchymal stem cells comprise bone marrow mesenchymal stem cells.

Preferably, the preparation method of the apoptotic microvesicle comprises the following steps:

(1) after subculturing the mesenchymal stem cells, adding a staurosporine solution for apoptosis induction culture, and collecting supernatant containing apoptotic cells;

(2) performing differential centrifugation on the supernatant obtained in the step (1), and collecting precipitates obtained after the differential centrifugation; the differential centrifugation comprises: carrying out first centrifugation on the supernatant under the condition of 800-1000 g, and collecting the first centrifuged supernatant; performing second centrifugation on the first centrifugation supernatant under the condition of 15000-16000 g;

(3) and (3) resuspending the precipitate obtained by the differential centrifugation in the step (2), performing third centrifugation, and collecting the precipitate of the third centrifugation to obtain the apoptotic microvesicle.

Preferably, the mesenchymal stem cells are 2-4 generation mesenchymal stem cells.

Preferably, the centrifugal force of the third centrifugation in the step (3) is 15000-16000 g; and the time of the third centrifugation is 25-30 min.

Preferably, the mass ratio of the mesenchymal stem cell apoptosis microvesicle to the hydrogel is 1: (1-3).

Preferably, the product comprises a pharmaceutical product; the dosage form of the medicine comprises injection.

Preferably, the mammal includes a mouse, rat, rabbit, dog, sheep, cow, pig or human.

The invention provides a product for promoting the repair of skin injury of mammals, which comprises mesenchymal stem cell apoptosis micro vesicles and hydrogel; the mass ratio of the mesenchymal stem cell apoptosis microvesicle to the hydrogel is 1: (1-3).

The invention provides application of mesenchymal stem cell apoptosis microvesicles in preparing a mammalian skin repair product in combination with hydrogel. In the invention, the mesenchymal stem cell apoptosis microvesicle can promote vascularization of a new tissue and regulate local immune microenvironment of a receptor tissue so as to accelerate epithelialization of a skin wound to promote healing. The hydrogel with good biocompatibility can effectively protect the loss of the mesenchymal stem cell apoptosis microvesicle and can ensure the biological activity of the mesenchymal stem cell apoptosis microvesicle. The application of the mesenchymal stem cell apoptosis microvesicle and the hydrogel in a combined manner can improve the utilization efficiency and the skin repair effect of the mesenchymal stem cell apoptosis microvesicle and better play the biological function of the mesenchymal stem cell apoptosis microvesicle, thereby accelerating the healing of wounds and promoting the repair and regeneration of skin injuries.

Drawings

FIG. 1 is a scanning electron microscope morphology of apoptotic microvesicles of mesenchymal stem cells of mice according to an embodiment of the present invention;

FIG. 2 is a graph of apoptosis-related specific proteins contained within apoptotic microvesicles in accordance with embodiments of the present invention;

FIG. 3 is a graph showing H & E staining results of skin tissues of a control group (PBS buffer) according to an embodiment of the present invention;

FIG. 4 is a graph showing H & E staining results of skin tissues of experimental groups (combination preparations) according to an example of the present invention;

FIG. 5 is a graph showing the results of Masson's staining of skin tissue in a control group (PBS buffer) according to an embodiment of the present invention;

FIG. 6 is a graph showing Masson's staining results of skin tissues of experimental groups (combination preparations) according to an example of the present invention;

FIG. 7 is a photograph taken by confocal laser microscopy of immunofluorescent staining of skin tissue Cytokeratin-14 in a control group (PBS buffer) according to an embodiment of the present invention;

FIG. 8 is a confocal laser microscopy photograph of immunofluorescent staining of skin tissue Cytokeratin-14 from the experimental group (combination preparation) according to an embodiment of the present invention;

FIG. 9 is a histogram of Cytokeratin-14 staining versus fluorescence intensity for control and experimental groups in accordance with the present invention;

FIG. 10 is a photograph taken by confocal laser microscopy of a CD206 immunofluorescent stain of skin tissue from a control group (PBS buffer) according to an embodiment of the present invention;

FIG. 11 is a photograph taken by a confocal laser microscope of the CD206 immunofluorescent staining of skin tissue of an experimental group (combined preparation) according to an embodiment of the present invention;

FIG. 12 is a statistical graph of CD206 staining versus fluorescence intensity for control and experimental groups in accordance with the present invention.

Detailed Description

In the present invention, the mesenchymal stem cell preferably comprises a bone marrow mesenchymal stem cell. In the present invention, the method for preparing apoptotic microvesicles preferably comprises the steps of:

The method comprises the steps of firstly carrying out subculture on the mesenchymal stem cells, then adding a staurosporine solution for apoptosis induction culture, and collecting supernatant. In the invention, the mesenchymal stem cells are preferably the 2 nd-4 th generation mesenchymal stem cells, and are more preferably the 2 nd generation mesenchymal stem cellsAnd 3 generation of bone marrow mesenchymal stem cells. When the cell generation is too small, other miscellaneous cells are easily mixed, the accuracy of the experiment is influenced, and when the cell generation is too large, the biological performance of the cell is reduced, and the effect of the experiment is influenced. In the invention, the cell liquid for subculturing the bone marrow mesenchymal stem cells is preferably added with the following components in concentration on the basis of a DMEM basic culture medium: 2mM/L glutamine, 100U/mL penicillin, 100g/mL streptomycin and 20% volume concentration fetal bovine serum. In the present invention, the temperature of the subculture is 37 ℃; said subcultured CO₂The concentration is preferably 5%; the subculture was performed on a standard six-well plate; the seeding density of the mesenchymal stem cells of the cells in the standard six-well plate is preferably 1.5 multiplied by 10⁶Each of the holes; the subculture time is based on that the fusion density of the cultured bone marrow mesenchymal stem cells reaches 80-90%. In the present invention, the solvent of the staurosporine solution is dimethyl sulfoxide, the concentration of the staurosporine solution is preferably 0.5 μ M, and the staurosporine solution is used for inducing apoptosis; the time for induction culture is preferably 9-16 h, and more preferably 12 h.

After collecting the supernatant containing the apoptotic cells, the invention carries out differential centrifugation on the supernatant, and collects the sediment after the differential centrifugation; the differential centrifugation comprises: carrying out first centrifugation on the supernatant under the condition of 800-1000 g, and collecting the first centrifuged supernatant; performing second centrifugation on the first centrifugation supernatant under the condition of 15000-16000 g; the temperature of the differential centrifugation is preferably 1-4 ℃. In the present invention, the first centrifugation is used to remove dead cells and residual cell debris; the second centrifugation serves to collect apoptotic microvesicles.

After differential centrifugation sediment is obtained, the sediment obtained by the differential centrifugation is resuspended, then third centrifugation is carried out, and the sediment of the third centrifugation is collected to obtain the apoptosis micro vesicle. In the invention, the centrifugal force of the third centrifugation is preferably 15000-16000 g; the time of the third centrifugation is preferably 25-30 min.

In the invention, the diameter of the apoptosis micro vesicle is 0.5-1 μm.

In the present invention, the reagent used for resuspension is preferably PBS buffer; the resuspension is used for cleaning precipitates obtained by differential centrifugation to remove impurities and drug residues.

In the present invention, the hydrogel preferably includes a temperature-sensitive type hydrogel. In the invention, the mass concentration of the temperature-sensitive hydrogel is preferably 30%; the temperature-sensitive hydrogel is preferably prepared by the following method: mixing the temperature-sensitive gel powder with a solvent to obtain temperature-sensitive hydrogel; the solvent is preferably PBS buffer solution; the temperature of the mixing is preferably 4 ℃. The temperature-sensitive hydrogel adopted by the invention has the following advantages: the operation is simple, and the solid state is quickly changed after the preparation is placed at the temperature of 37 ℃ close to the body temperature, so that the sustained-release effect is favorably exerted locally; in addition, the stem cell apoptosis micro vesicle is dissolved in liquid, the hydrogel is in a liquid state at a relatively low temperature, and then the vesicle is mixed with the hydrogel and placed on local tissues of injured skin, so that the vesicle can be changed into a solid hydrogel under the action of the temperature of living skin, and the functions of fixing and releasing the vesicle are realized.

The invention also provides a product for promoting the repair of the skin injury of the mammal, which comprises the mesenchymal stem cell apoptosis micro vesicle and the hydrogel; the mass ratio of the mesenchymal stem cell apoptosis microvesicle to the hydrogel is 1: (1-3). In the present invention, the hydrogel serves as a carrier; the mesenchymal stem cell apoptotic microvesicles function as an active ingredient.

In the invention, the hydrogel serving as a carrier can prevent a great amount of apoptotic microvesicles from losing, ensure the biological activity of the apoptotic microvesicles and realize the effective utilization of the apoptotic microvesicles.

In the present invention, the mass ratio of the mesenchymal stem cell apoptotic microvesicle to the hydrogel is preferably 1: 2.

In the present invention, the product preferably comprises a pharmaceutical product; the dosage form of the drug preferably includes an injection. In the present invention, the pharmaceutical product is preferably in the form of an injection; the method of use of the medicament preferably comprises: the medicine is injected to the affected part of the defect or the injured skin, and because of the temperature-sensitive hydrogel, the medicine can be changed into a solid state from a liquid state after encountering skin tissues and then acts on the tissues; the dosage of the medicine is preferably 0.5 mL. The shape of the defective skin was circular and the diameter was 1 cm.

In the present invention, the mammal preferably includes a mouse, a rat, a rabbit, a dog, a sheep, a cow, a pig or a human.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Example 1: separation and purification culture of mouse bone marrow mesenchymal stem cells

The 8-week-old mice are killed by dislocation of the neck, and are firstly soaked in 75% absolute ethyl alcohol solution for 5 minutes, and the shin bones and the thighbones at the two sides are separated by using sterile ophthalmic scissors and curved forceps and are soaked in PBS buffer solution at the temperature of 4 ℃. The muscle and tendon tissue attached to the tibia and femur was then carefully removed and transferred to sterile pre-cooled PBS buffer. The two ends of the long bone are cut off by an ophthalmologic scissors to expose the marrow cavity, and then the long bone is placed into the mesenchymal stem cell culture solution. The needle is inserted into the marrow cavity by using a disposable syringe with the specification of 1ml, and the red marrow in the marrow cavity is repeatedly washed until the marrow cavity is white. After the bone marrow is completely flushed, the culture solution containing the mesenchymal stem cells is collected into a sterile glass centrifugal tube, and the liquid is repeatedly blown by using a glass dropper until the red bone marrow is completely blown away. Inoculating the above culture solution into sterile plastic culture dish, supplementing mesenchymal stem cell culture solution, and placing at 37 deg.C and 5% CO₂In a cell culture incubator. And after 72h, completely removing the culture solution, gently washing the culture solution for 2 times by using sterile PBS buffer solution, and adding a proper amount of complete culture solution of the mesenchymal stem cells to continue culturing. The culture medium was then changed every 3 days until the cell density reached 90% for cell passaging. Taking the mesenchymal stem cells of the 3 rd generation for subsequent experiments.

Example 2: extraction of mouse bone marrow mesenchymal stem cell apoptosis micro vesicle and preparation of combined preparation

Removing extracellular vesicles from fetal calf serum by ultracentrifugation (100000g, 12h)Then adding the mixture into a complete culture solution of the bone marrow mesenchymal stem cells for culture. Passing the mesenchymal stem cells to the 3 rd generation according to the 2 x 10⁶The density of each well was plated in six-well cell culture plates, which were placed at 37 ℃ and contained 5% CO₂Culturing in a cell culture incubator. When the cell fusion rate is 90%, adding 0.5 mu M staurosporine for overnight induction for 12h, then collecting the supernatant of apoptotic cells and carrying out differential centrifugation, and the specific steps are as follows: centrifuging at 1000g for 10min to remove cell debris and impurities in the collected liquid, centrifuging at 16000g for 30min to collect the apoptotic microvesicles, washing with PBS buffer solution for 2 times, centrifuging in the same method to separate the apoptotic microvesicles, finally suspending in 200 μ l PBS buffer solution, quantifying by BCA protein, packaging in sterile EP tube, and freezing and storing in a refrigerator at-80 deg.C for later use.

50 mu g of apoptosis micro-vesicles are added into 100 mu g of temperature-sensitive hydrogel to prepare a combined preparation.

Example 3: identification of apoptotic microvesicles

And (3) resuspending the collected apoptotic microvesicles by using a PBS buffer solution, drying at a critical point, spraying gold, drying, and putting the prepared sample into a scanning electron microscope for observation and photographing for recording (figure 1).

After the apoptotic microvesicles were washed 2 times with PBS, protein lysate + PMSF (99:1) was added and collected by cell scraping, and subjected to 8% SDS-PAGE electrophoresis, followed by transferring to a PVDF membrane. The membrane was blocked with 3% skim milk for 2h, rabbit anti-Caspase-3 and cleared Caspase-3 primary antibodies, rabbit HRP secondary antibodies were added, ECL chemiluminescence detected and recorded by photography (FIG. 2).

Example 4: establishment and treatment of mouse skin defect model

Mice of 8 weeks old were selected, anesthetized by intraperitoneal injection with 1% sodium pentobarbital, and fixed on a mouse plate to ensure their backs to face upward stably. Removing 1/2 mouse hair from its back with a hand-held electric hair pusher and exposing the skin, applying appropriate amount of depilatory cream on its surface, standing for 1min, and lightly wiping with 0.9% sodium chloride solution in wet gauze. After the skin preparation is finished, a circle with the diameter of 1cm is drawn at the center of the back skin surface, after the disinfection of the iodophor and the alcohol cotton ball is finished, the whole layer of skin is slightly cut along the drawn track by using an ophthalmic scissors and a curved forceps, and meanwhile, the integrity of the wound edge is ensured and the notch is avoided. Subsequently, 100. mu.g of the combined preparation was placed, and the control group was injected with an equal volume of PBS buffer. The wound surface is lightly dressed by adopting 3M skin dressing, the dressing is placed into an animal feeding room in groups, padding is replaced every other day, and whether the back of the rat is infected or not or other health problems are observed. On day 14, materials were drawn from wound healing and normal tissues after anesthesia with 1% sodium pentobarbital. All the tissues obtained were fixed in 4% paraformaldehyde solution and treated separately according to the purpose of the subsequent experiment.

Example 5: h & E and Masson staining of skin tissue

Taking the skin tissue of the healing part on the 14 th day, and putting the skin tissue into a dehydrator for gradient dehydration after fixation is completed. Paraffin embedding and sectioning were then performed, each paraffin section having a thickness of about 4 μm.

In H & E staining, paraffin sections are sequentially subjected to gradient dewaxing by 100%, 95%, 90%, 80% and 75% ethanol, hematoxylin staining is washed after 5min, hydrochloric acid alcohol differentiation is carried out for 30s, eosin staining is carried out for 2min, and gradient dehydration is carried out by 75%, 95% and 100% ethanol. And finally, sealing the neutral gum after the xylene is transparent, observing by using a body type microscope, and photographing and recording.

In Masson staining, paraffin sections were deparaffinized sequentially by a 100%, 95%, 90%, 80%, 75% ethanol gradient. The staining was carried out for 5min with a Weigart iron hematoxylin mixture (Weigart iron hematoxylin A, B mixture at a ratio of 1: 1), and the cells were washed slightly with running water. Alcohol differentiation with 1% hydrochloric acid was performed for 3s, followed by rinsing with running water. The ponceau acid fuchsin dyeing solution is dyed for 10min, and is slightly washed by running water. And (3) treating the phosphomolybdic acid solution for about 5min, and directly re-dyeing the phosphomolybdic acid solution for 5min by using aniline blue dye solution without washing. And finally, dehydrating according to the sequence of 75%, 80%, 95% and absolute alcohol, carrying out xylene transparent treatment, then sealing neutral gum, observing by using a body type microscope, and taking a picture and recording.

The H & E staining results are shown in fig. 3 and fig. 4, wherein fig. 3 is the staining result of the control group; FIG. 4 shows the staining results of the combination preparation group. The results of Masson's trichrome staining are shown in FIGS. 5 and 6, where FIG. 5 is the result of staining of the control group; FIG. 6 shows the staining results of the combination preparation group. From the results, compared with a PBS buffer solution control group, the combined preparation group can obviously reduce the area of scar tissues and improve the healing effect after treating the skin-defect mouse, so that the combined preparation of the mesenchymal stem cell apoptosis microvesicle and the temperature-sensitive hydrogel prepared by the invention can be effectively applied to the field of skin tissue injury repair.

Example 6: staining of the epithelialization associated protein Cytokeratin-14 in skin tissue

After the skin is completely sampled, fixing the skin with 4% paraformaldehyde solution for 24h, removing the fixing solution, and washing with PBS buffer solution for 2 times, each time for 10 min. Then dehydration treatment is carried out for 12h by using 30% sucrose solution, and after dehydration is finished, OCT embedding agent is used for embedding. After the sample is completely frozen at-25 ℃ in the embedding machine, the sample is fixed on a base at the slicing position to start slicing. The thickness of the initial rough cut was about 50 μm, and after the sample was exposed to about half of the thickness, the sample was finely cut to a thickness of 25 μm, and after the sample was completely exposed, the thickness was adjusted to 10 μm to prepare a sheet. The final section was 8 μm thick and placed under a glass plate, followed by adhesion using a cation-releasing sheet, after left to air at room temperature for about 2 hours, the residual OCT-embedding agent was gently washed off with PBS buffer, and finally the residual liquid around the sample was removed by blotting with absorbent paper or a cotton swab, and the section was frozen for use. At the beginning of staining, the cut cryosections were allowed to air at room temperature for 30min, then washed gently 1 time with PBS buffer, and after wiping off excess fluid around the sample, immunohistochemical strokes were used to make a circle of appropriate size. Then 0.05% TritonX-100 was added and permeabilized at room temperature for 10min, followed by 3 washes with PBS buffer, 5min each. Further blocking with 12% goat serum at room temperature for 1 h. After discarding the blocking solution, the diluted Cytokeratin-14 primary antibody solution (diluted with PBS buffer at a ratio of 1: 200) was added directly dropwise, and the wet box was placed in a refrigerator at 4 ℃ and incubated overnight. After the incubation was completed, the primary antibody solution was discarded, washed with PBS buffer for 3 times, each time for 5min, and goat anti-rabbit red fluorescent secondary antibody (diluted with PBS buffer at a ratio of 1: 200) was added for incubation at room temperature for 1 h. After incubation was complete, the cells were washed 3 times with PBS buffer for 5min each. And finally adding Hoechst dye solution to perform cell nucleus lining dyeing, washing for 5min at room temperature for 3 times by using PBS buffer solution, sealing the cell nucleus by using 80% glycerite after washing, and observing and photographing under a laser confocal microscope for recording.

The detection results are shown in figures 7-9, figure 7 shows the condition that the control group expresses the epithelialization related protein Cytokeratin-14 after the defect skin is healed, and figure 8 shows the result that the experimental group expresses the epithelialization related protein Cytokeratin-14 after the defect skin is treated. FIG. 9 is the relative fluorescence intensity values of the expression levels of the two Cytokeratin-14 proteins. From the above results, it can be seen that the expression level of the epithelialization protein Cytokeratin-14 in the experimental group is significantly increased, the epithelialization effect of the experimental group is better, and the healing of the defective skin tissue is facilitated, compared with the control group.

Example 7: skin tissue macrophage polarization-associated protein CD206 staining

After preparing the cryosection by mounting the above method, it was left to air at room temperature for 30min, then washed gently 1 time with PBS buffer, wiped to dry excess fluid around the sample, and immunoblotted to make a circle of appropriate size. Then 0.05% TritonX-100 was added and permeabilized at room temperature for 10min, followed by 3 washes with PBS buffer, 5min each. Further blocking with 12% goat serum at room temperature for 1 h. After discarding the blocking solution, the diluted CD206 primary antibody solution (diluted with PBS buffer at a ratio of 1: 200) was added directly dropwise, and the wet box was placed in a refrigerator at 4 ℃ and incubated overnight. After the incubation was completed, the primary antibody solution was discarded, washed with PBS buffer for 3 times, each time for 5min, and goat anti-rabbit green fluorescent secondary antibody (diluted with PBS buffer at a ratio of 1: 200) was added for incubation at room temperature for 1 h. After incubation was complete, the cells were washed 3 times with PBS buffer for 5min each. And finally adding Hoechst dye solution to perform cell nucleus lining dyeing, washing for 3 times by PBS buffer solution at room temperature for 5min each time, sealing the cell nucleus by using 80% glycerite after washing, and observing and photographing under a laser confocal microscope for recording.

The detection results are shown in fig. 10-12, fig. 10 is the expression condition of macrophage-related protein CD206 after healing of the defective skin of the control group, and fig. 11 is the expression result of macrophage polarization-related protein CD206 after treating the defective skin of the experimental group. FIG. 12 is the relative fluorescence intensity values of the expression levels of both CD206 proteins. The results show that compared with a control group, the combined preparation in the experimental group can effectively promote the change of immune microenvironment and obviously increase the expression level of macrophage polarization related protein CD206, thereby improving the conversion of anti-inflammatory phenotype and being beneficial to the healing of defective skin tissues.

In conclusion, the mesenchymal stem cell apoptosis microvesicle and temperature-sensitive hydrogel combined preparation provided by the invention can effectively promote the healing of defective skin.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims

1. The application of the mesenchymal stem cell apoptosis microvesicle combined with hydrogel in preparing a mammalian skin repair product.

2. The use of claim 1, wherein the hydrogel comprises a temperature-sensitive hydrogel.

3. The use of claim 1, wherein the mesenchymal stem cells comprise bone marrow mesenchymal stem cells.

4. The use according to claim 1, wherein the process for the preparation of apoptotic microvesicles comprises the steps of:

5. The use of claim 3 or 4, wherein the mesenchymal stem cells are passage 2-4 mesenchymal stem cells.

6. The use according to claim 4, wherein the centrifugal force of the third centrifugation in step (3) is 15000-16000 g; and the time of the third centrifugation is 25-30 min.

7. The use of claim 1, wherein the mesenchymal stem cell apoptotic microvesicle and hydrogel are present in a mass ratio of 1: (1-3).

8. The use of claim 1, wherein the product comprises a pharmaceutical product; the dosage form of the medicine comprises injection.

9. The use of claim 1, wherein the mammal comprises a mouse, rat, rabbit, dog, sheep, cow, pig, or human.

10. A product for promoting repair of skin injury in mammals comprises mesenchymal stem cell apoptosis microvesicles and hydrogel; the mass ratio of the mesenchymal stem cell apoptosis microvesicle to the hydrogel is 1: (1-3).