CN114672456A

CN114672456A - Method for improving extracellular vesicle secretion efficiency of adipose-derived stem cells by utilizing ultrasonic stimulation and application

Info

Publication number: CN114672456A
Application number: CN202210195340.9A
Authority: CN
Inventors: 刘凯; 许�鹏; 郑毅
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2022-03-01
Filing date: 2022-03-01
Publication date: 2022-06-28

Abstract

The invention discloses a method for improving the extracellular vesicle secretion efficiency of adipose-derived stem cells by using ultrasonic stimulation and application thereof, wherein the method comprises the following steps of A, extracting the adipose-derived stem cells; B. ultrasonic stimulation of adipose-derived stem cells: after the third-generation adipose-derived stem cells are full of the cells, PBS is used for washing, serum-free low-sugar DMEM is replaced, a coupling agent with a certain thickness is uniformly smeared below a cell culture dish, then a transducer of ultrasonic generation equipment is tightly attached to the coupling agent to remove air, and the dosage of ultrasound is set to be 0.5-1.5W/cm²The exposure time is 30-120 s; C. extracting the fat stem cell extracellular vesicles. Proved by verification, after the adipose-derived stem cells are stimulated by the ultrasonic waves, the number of the secreted vesicles is larger than that of the vesicles which are not stimulated by the ultrasonic wavesThe fat stem cells are improved by more than 60 times; the protein content of the vesicle is increased by more than 3 times, and the vesicle specific marker is highly expressed, and the effect on promoting the healing of the skin wound surface is obvious.

Description

Method for improving extracellular vesicle secretion efficiency of adipose-derived stem cells by using ultrasonic stimulation and application

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a method for improving the extracellular vesicle secretion efficiency of adipose-derived stem cells by using ultrasonic stimulation and a specific application of the extracellular vesicle.

Background

The stem cells have the potential of multi-directional differentiation, and show great application prospects in regenerative medicine. Among them, the Adipose-derived-stem cells (ADSCs) have rich sources and are easy to obtain, and the liposuction process is minimally invasive and easy to be accepted by patients, so that the lipolysis-derived-stem cells are more suitable for conversion application in clinic.

Previous studies suggest that stem cells migrate to damaged sites in vivo and differentiate into damaged tissues and cells. However, numerous studies have followed stem cell findings in vivo: less stem cells survive and transform into damaged cells. Recent studies have shown that: the role of stem cells is primarily played by their secreted extracellular vesicles and growth factors, rather than being directly transformed into damaged tissue cells.

Extracellular Vesicles (EVs) are mixed particles secreted by cells, and comprise Microvesicles (MVs) and Exosomes (Exosomes), wherein the Microvesicles and Exosomes are respectively released to the outside of the cells after budding and forming of a multivesicular body, and the encapsulated contents of the Microvesicles act on receptor cells. Extracellular vesicles are signal transduction mediators secreted by cells, and are fundamentally different from cells. The use of outer vesicles avoids many of the safety issues involved in cell therapy: such as stem cell therapy, are subject to strong immunogenicity, risk of tumor formation, and may clog the tiny blood sinuses. And the immunogenicity of the extracellular vesicles is very low, so that allogeneic application can be carried out. Compared with the instability of the growth factor, the protein and nucleic acid in the extracellular vesicle are protected by the membrane structure and are not easy to degrade, and the extracellular vesicle can be used as a ready-made preparation and has a wide application transformation prospect.

Currently, for regenerative therapy purposes, the production of extracellular vesicles requires the culturing of large numbers of cells and the extraction and enrichment from the cell supernatant. However, the extracellular vesicles secreted from the cells are produced in low amounts, and it is difficult to meet the requirements of clinical treatment. Previous studies report that the secretion of extracellular vesicles by cells can be promoted to some extent by methods such as low-oxygen stimulation and the addition of chemical drugs, however, these methods still cannot meet the clinical transformation requirements. Also, exogenously added chemicals may have an effect on the composition and purity of the extracellular vesicles.

Disclosure of Invention

The invention is based on the research, and provides a method for improving the efficiency of secreting extracellular vesicles from adipose-derived stem cells by using ultrasonic stimulation and application of the extracellular vesicles in skin wound healing.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided a method for improving the efficiency of secreting extracellular vesicles from adipose stem cells by using ultrasonic stimulation, comprising the following steps:

A. adipose-derived stem cell extraction

Digesting the waste granular fat after liposuction by using an enzyme digestion method, extracting fat stem cells, and carrying out conventional passage to 3 rd generation after overgrowth;

B. Ultrasound stimulation of adipose-derived stem cells

After the third-generation adipose-derived stem cells are full, washing the cells twice by PBS (phosphate buffer solution), replacing serum-free low-sugar DMEM (DMEM), uniformly coating a coupling agent with a certain thickness below a cell culture dish, tightly attaching a transducer of an ultrasonic generation device and the coupling agent to remove air, causing the cells to obviously die due to excessive ultrasonic dose acting on the cells, and preferably setting the ultrasonic dose to be 0.5-1.5W/cm to reduce the cell apoptosis²The exposure time is 30 to 120s, and the most preferable is 1.5W/cm²、30S。

C. Extraction of adipose-derived stem cell extracellular vesicles

Stimulating the adipose-derived stem cells by using ultrasonic waves, continuously culturing for 48 hours, and collecting cell supernatants; extracellular vesicles in the supernatant were extracted by ultrafiltration and dissolved in PBS.

Preferably, in step a, the digestive enzyme used is collagenase type IV;

the digestion conditions were as follows: 0.2% (m/v) collagenase type IV was mixed with granular adipose tissue in a volume ratio of 1:1, sufficiently shaken on a shaker at 37 ℃ and digested for 2 hours.

Preferably, in step a, when the adipose-derived stem cells are routinely passaged to 3 rd generation, the adipose-derived stem cells of 3 rd generation are identified by flow cytometry and three-way induction of osteogenesis, adipogenesis and chondrogenesis.

Preferably, in step B, a pulse repetition frequency of 1MHz and 10Hz, a duty cycle of 60 percent and a duty cycle of 9mm are used²The ultrasonic generating device of the cross-sectional area performs ultrasonic stimulation on the cells.

When ultrasonic stimulation is carried out, 0.5-1cm of couplant is uniformly smeared below a 10cm cell culture dish, and then a transducer of ultrasonic generation equipment is tightly attached to the couplant to remove air.

Preferably, in step C, the supernatant is filtered by a 0.22 μm filter, added into an ultrafiltration centrifuge tube with a 100KD membrane, centrifuged and concentrated at 3000g and 4 ℃ until the volume of the inner tube is 500 μ L, transferred into a sterile freezing tube, and frozen at-80 ℃ in a refrigerator.

And (3) detecting the extracted outer vesicles: NTA detection shows that the number of the secreted vesicles is increased by more than 60 times by stimulating the adipose-derived stem cells under the ultrasonic stimulation condition; the protein quantitative result shows that the protein content of the vesicle secreted by the adipose-derived stem cells is improved by more than 3 times by the stimulation of the ultrasonic waves; the Western Blot results show that vesicles highly express vesicle-specific markers.

In a second aspect of the present invention, there is provided an adipose stem cell-derived outer vesicle extracted according to the above method, which exhibits an average diameter of 131nm and highly expresses CD63, CD81 and TSG101 without expressing GM 130.

In a third aspect of the invention, the application of the adipose-derived stem cell outer vesicle prepared by the method in preparing a medicine for promoting skin wound healing is provided.

Preferably, the bioactive preparation for promoting the skin wound healing is a bioactive preparation for promoting the vitality and proliferation of an epidermal cell line and a skin fibroblast, a bioactive preparation for improving the migration capability of the skin fibroblast, or a bioactive preparation for promoting the angioblast capability of a umbilical vein endothelial cell line.

In the fourth aspect of the invention, a bioactive preparation for promoting skin wound healing is provided, which contains the adipose-derived stem cell outer vesicle with the added mass of 200 μ g.

Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:

in the aspect of effect, after the adipose-derived stem cells are stimulated by ultrasound, the number of the secreted vesicles is increased by more than 60 times compared with that of the adipose-derived stem cells which are not stimulated by ultrasound; the protein content in the vesicle is increased by more than 3 times, and the vesicle specific marker is highly expressed. In vitro cell experiments show that the extracellular vesicles can effectively promote the activity and proliferation of epidermal cell lines and skin fibroblasts, improve the migration capability of the skin fibroblasts, or promote the hemangiogenesis capability of umbilical vein endothelial cell lines, and have remarkable effect in promoting the healing of skin wounds.

In the aspect of specific implementation, the ultrasonic stimulation is simple to operate and easy to popularize in a large range.

Drawings

Fig. 1 shows that the ultrasonic stimulation obviously promotes the fat stem cells to secrete extracellular vesicles: A. the appearance of the extracellular vesicles extracted after the adipose-derived stem cells are stimulated by ultrasonic waves under a transmission electron microscope; nta detection of extracellular vesicles showed an average diameter of 131 nm; western Blot results show: extracellular vesicles highly express CD63, CD81, and TSG101 and do not express GM 130; D. endocytosis experiments showed that: the vesicles can be taken up by skin fibroblasts; nta assay showed: at 1.5W/cm²30s, the number of secreted vesicles is increased by more than 60 times; F. the protein quantification results show that: 1.5W/cm²30s, the fat stem cells are stimulated, and the protein content of the secreted vesicles is increased by more than 3 times; western Blot results showed 1.5W/cm²And after 30s of conditions stimulate the adipose-derived stem cells, the secreted vesicles highly express the vesicle specific markers.

Fig. 2 shows that ultrasound-stimulated adipose stem cell-secreted vesicles (US-EVs) significantly promoted the viability and proliferation of epidermal cell lines (HACATs) and skin Fibroblasts (FBs): A. the influence of different concentrations of US-EVs and EVs on the cell viability of FBs and the effect comparison; B. the influence of different concentrations of US-EVs and EVs on the cell viability of HACATs and the effect comparison; C. d, comparing the cell proliferation capacity promoting effects of different concentrations of US-EVs and EVs on FBs; E. comparison of the cell proliferation potency-promoting effects of different concentrations of US-EVs and EVs on HACATs.

Fig. 3 shows that ultrasound-stimulated adipose stem cell-secreted vesicles (US-EVs) significantly promoted the migratory capacity of skin Fibroblasts (FBs): A. the migration promotion results of different concentrations of US-EVs and EVs on FBs; B. EVs and EVs with different concentrations have no obvious promotion effect on migration of HACATs; C. statistics of comparison of promotion effects of US-EVs and EVs on FBs migration; D. statistics of US-EVs and the effect of EVs on migratory capacity of HACATs.

Figure 4 shows that ultrasound-stimulated adipose stem cell-secreted vesicles (US-EVs) significantly promoted the angiogenic capacity of umbilical vein endothelial cell lines (HUVECs): A. influence and effect comparison of different concentrations of US-EVs and EVs on cell viability of HUVECs; B. promoting results of US-EVs and EVs with different concentrations on migration of HUVECs; C. microscopic images of the influence of different concentrations of US-EVs and EVs on HUVECs in vitro angiogenesis; D. counting the HUVECs cell migration rate by different concentrations of US-EVs and EVs; E. the influence of different concentrations of US-EVs and EVs on the number of blood vessels formed; F. different concentrations of US-EVs and EVs have an effect on the formation of vascular branch points.

Fig. 5 shows that vesicles secreted by adipose stem cells (US-EVs) stimulated by ultrasound significantly promoted healing of the skin wound in diabetic mice: a, pictures of wound surface size change of different experimental groups on days 0-14; b: and (4) comparing the statistical results of the wound surface areas of different experimental groups in different periods.

Detailed Description

In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

Example 1: method for improving extracellular vesicle secretion efficiency of adipose-derived stem cells by using ultrasonic stimulation

1. Cell extraction and identification

Extraction and identification of adipose-derived stem cells: digesting the waste granular fat after liposuction by using an enzyme digestion method, extracting fat stem cells, carrying out conventional passage after the lipolysis, and identifying the third-generation fat stem cells (P3) by respectively carrying out flow cytometry and three-way induction (osteogenesis, adipogenesis and chondrogenesis).

When the digestion treatment is carried out, the adopted digestive enzyme is collagenase IV; the digestion conditions were as follows: 0.2% (m/v) collagenase type IV was mixed with granular adipose tissue in a volume ratio of 1:1, sufficiently shaken on a shaker at 37 ℃ and digested for 2 hours.

2. Ultrasonic stimulation of adipose-derived stem cells

Using a pulse repetition frequency of 1MHz, 10Hz, a duty cycle of 60%, 9mm²The ultrasonic stimulation is carried out on the cells by the ultrasonic generating equipment with the cross section area, and the specific process is as follows: uniformly coating a couplant with the thickness of 0.5-1cm below a 10cm cell culture dish, and then closely attaching a transducer of an ultrasonic generating device to the couplant to remove air, wherein the adopted ultrasonic stimulation intensity is as follows: 0.5 to 1.5W/cm ²30 to 120s, preferably 1.5W/cm²、30s。

When the adipose-derived stem cells are subjected to ultrasonic stimulation, the stimulation intensity and the stimulation time are not too large or too strong so as to avoid inducing apoptosis or rupture, for example, when the ultrasonic stimulation intensity is 1.5W/cm²And over 120s, the cell undergoes obvious apoptosis.

3. Extraction of fat stem cell extracellular vesicle

After the third generation of adipose stem cells were confluent, the cells were washed twice with PBS and then replaced with serum free low sugar DMEM. (1) For cells without ultrasound stimulation: after incubation for 48 hours, cell supernatants were collected. (2) For ultrasonically stimulated cells: after sonication as in step 2 and continued culturing for 48 hours, cell supernatants were collected. Respectively extracting extracellular vesicles from the cell supernatants of the above different treatments by ultrafiltration, dissolving the extracted vesicles in PBS, and storing in a refrigerator at-80 deg.C.

Example 2: adipose-derived stem cell extracellular vesicle identification and analysis

1. Identification of adipose-derived stem cell extracellular vesicles

The morphology and the size of the vesicle are observed through a transmission electron microscope, the average diameter of the vesicle is calculated through Nanoparticle Tracking Analysis (NTA), the related specific markers of the vesicle are detected through Western Blot, and an endocytosis experiment verifies whether the extracellular vesicle can be taken up by receptor cells.

The endocytosis experiment is carried out by adopting skin fibroblasts, and the extraction method comprises the following steps: soaking and digesting foreskin with Dispase for 24 hours, removing epidermis, digesting residual dermis by collagenase digestion method, extracting skin fibroblast, carrying out passage after the cell overgrowth, and being used for in vitro research of influence of extracellular vesicles on functions of proliferation, migration and the like of the skin fibroblast.

2. Quantitative analysis of adipose-derived stem cell extracellular vesicles

Quantitative analysis of extracted vesicles by NTA and protein quantification methods respectively

(1) NTA quantification: a small amount of vesicle solution was taken, diluted to a volume of about 300. mu.L with PBS buffer, and the particle number and particle diameter distributions were analyzed, and the dilution factor on the machine was recorded for conversion with the protein quantification.

(2) Protein quantification (BCA method): the BCA protein quantitative kit of Biyuntian company is adopted for quantitative detection, protein standard substances with different concentrations are prepared, vesicles are diluted by a certain multiple, the protein standard substances and the vesicles with proper volumes are added into a 96-well plate, working liquid is added for reaction, and the protein concentration of the vesicles is calculated according to the light absorption value of each well.

The results are shown in fig. 1, and the transmission electron microscope shows that the extracellular vesicles extracted after the adipose-derived stem cells are stimulated by ultrasonic waves are irregular and round (fig. 1A); NTA detection of extracellular vesicles showed an average diameter of 131nm (fig. 1B); the Western Blot results show: extracellular vesicles highly expressed CD63, CD81 and TSG101 but not GM130 (FIG. 1C), 1.5W/cm ²30s, the secreted vesicle high-expresses the vesicle specific marker after stimulating the adipose-derived stem cells (FIG. 1G); endocytosis experiments showed that: vesicles can be taken up by skin fibroblasts (fig. 1D); NTA detection shows that: at 1.5W/cm²30s stimulation of adipose stem cells, the number of vesicles secreted was increased by more than 60-fold (FIG. 1E); the protein quantification results show that: 1.5W/cm²30s, the amount of protein contained in vesicles secreted by the cells was increased by 3-fold (FIG. 1F).

Example 3: effect verification

1. Cell viability assay

Respectively inoculating epidermal cell lines (HACATs) and skin Fibroblasts (FBs) into a 96-well plate, respectively adding untreated adipose stem cell Extracellular Vesicles (EVs) and ultrasonically stimulated adipose stem cell extracellular vesicles (US-EVs) with different concentrations (20, 50, 100, 150 and 200 mu g/mL), culturing for 72 hours, measuring a cell OD value at 450nm by using a CCK-8 kit according to the instruction steps, and calculating the cell viability by the following formula:

cell viability ═ 100% (treatment group cell OD value/control group OD value) ×

The dose of vesicles used in subsequent in vitro experiments was determined by the results of cell viability.

2. Cell proliferation assay

Epidermal cell lines (HACATs) and skin Fibroblasts (FBs) were seeded in 96-well plates, untreated adipose stem cell Extracellular Vesicles (EVs) and ultrasonically stimulated adipose stem cell extracellular vesicles (US-EVs) were added at different concentrations (determined according to the above cell viability assay), and after 48 hours of incubation, the cells were labeled and fixed with a fluorescent dye according to the instructions of the EdU assay kit, and the cell proliferation rate was observed and calculated by fluorescence microscopy. The cell proliferation rate was calculated according to the following formula:

Cell proliferation rate (EdU positive cells/total number of seeded cells). times.100%

The results of the above experiments are shown in fig. 2, and US-EVs significantly promoted the cell viability (fig. 2A) and cell proliferation capacity (fig. 2C, D) of FBs compared to EVs, which was dose-dependent; the US-EVs can promote the viability of HACATs cells, but has no obvious difference compared with EVs, and the different dosages of US-EVs have no obvious difference on the cell viability promoting effect, and the cell viability promoting effect of the US-EVs with high concentration (200 mu g/mL) is lower than that of the US-EVs with low concentration (figure 2B), but the cell proliferation capacity of the HACATs can be obviously promoted (figure 2E, F).

3. Cell migration assay

Cell migration is detected by a scratch experiment, epidermal cell lines (HACATs), skin Fibroblasts (FBs) and umbilical vein endothelial cell lines (HUVECs) are respectively inoculated into 6-pore plates, after the cells grow over the 6-pore plates, the cells are scratched, serum-free DMEM/1% serum-containing DMEM is added, the scratch area is measured to be 0h, EVs and US-EVs with different concentrations are added into an experimental group, PBS with the same volume is added into a control group, the scratch area is measured to be 24h after 24h, the scratch area is measured for 0h and 24h respectively, and the cell migration rate is calculated by the following formula:

cell mobility (0h scratch area-24 h scratch area)/0 h scratch area × 100%.

Results referring to fig. 3, US-EVs significantly promoted the migration ability of FBs compared to EVs, with higher concentrations of US-EVs promoting migration better than lower concentrations (fig. 3A, C); US-EVs did not significantly change the migratory capacity of HACATs compared to EVs (FIG. 3B, D).

4. Detection of umbilical vein endothelial cell tube forming ability

The Matrigel is paved at the bottom of a 96-well plate and placed in an incubator for 20min to be solidified, umbilical vein endothelial cell lines (HUVECs) are inoculated on the Matrigel, low-sugar DMEM is added, EVs and US-EVs with different concentrations are added in an experimental group, PBS with the same volume is added in a control group, tube forming conditions are observed under a rear mirror for 6h and pictures are taken, and the tube forming quantity is counted by software.

Results referring to fig. 4, US-EVs significantly promoted cell viability of HUVECs compared to EVs (fig. 4A); US-EVs significantly promoted the migratory capacity of HUVECs compared to EVs (fig. B, D) US-EVs significantly promoted angiogenesis of HUVECs compared to EVs (fig. 4C), with the number of vessels and branch points significantly superior to EVs (fig. 4E, F).

5. Construction and treatment steps of skin wound model of diabetic mouse

32 db knockout, female c57 mice (diabetic) were purchased at 7 weeks of age and routinely housed under SPF conditions for 1 week to acclimatize. Starting at week 8, mice were randomly divided into 4 groups:

(1) PBS group: making a wound on the back of the mouse, and treating the wound by using PBS;

(2) HA + PBS group: wound surfaces are made on the backs of the mice, and 100 mu L of hyaluronic acid gel (HA) +100 mu L of PBS are used for treatment;

(3) HA + EV group: making a wound surface on the back of a mouse, and treating by 100 mu L of HA +200 mu L of EVs (the volume of the solution is 100 mu L, and the final concentration of the EVs is 1 mu g/mu L);

(4) HA + US-EV group: making a wound surface on the back of a mouse, and treating 100 mu L of HA +200 mu g of US-EVs (the volume of the solution is 100 mu L, and the final concentration of the EVs is 1 mu g/mu L);

the skin wound model establishment process is as follows: anesthetizing a mouse with a small animal anesthetic, scribing a circular scratch on the back of the mouse by using a corneal trephine with the diameter of 6mm, shearing the skin along the scratch by using an ophthalmic scissors to reach the deep fascia layer, shearing off the whole skin, treating the skin wound according to the grouping, applying parts of different preparations on the surface of the wound, and injecting parts of the different preparations under the skin around the wound edge.

6. Evaluation of therapeutic effect on skin wounds:

the general appearance is as follows: the skin wound surfaces of 0 day, 3 day, 7 day, 10 day and 14 day are photographed respectively, the healing condition, the exudation condition and the infection condition of the skin wound surfaces are observed by naked eyes, the size of the wound surface area is calculated through Image Pro Plus 6.0, and the change condition of the wound surface size of each group is evaluated.

Results referring to fig. 5, the skin healing of the four experimental groups was similar at day 3, and the US-EVs group was significantly better than the other groups over time, with the skin of the group healing almost completely at day 14 and skin damage remaining in the other three groups (fig. 5A, B).

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A method for improving the extracellular vesicle secretion efficiency of adipose-derived stem cells by using ultrasonic stimulation is characterized by comprising the following steps:

A. adipose-derived stem cell extraction

B. Ultrasound stimulation of adipose-derived stem cells

After the third-generation adipose-derived stem cells are full of the cells, the cells are washed twice by PBS, then serum-free low-sugar DMEM is replaced, a coupling agent with a certain thickness is uniformly smeared below a cell culture dish, then a transducer of ultrasonic generation equipment is tightly attached to the coupling agent to remove air, and the dosage of ultrasonic is set to be 0.5-1.5W/cm²The exposure time is 30-120 s;

C. extraction of adipose-derived stem cell extracellular vesicles

Stimulating adipose-derived stem cells by using ultrasonic waves, continuously culturing for 48 hours, and collecting cell supernatants; extracellular vesicles in the supernatant were extracted by ultrafiltration and dissolved in PBS.

2. The method for improving the efficiency of secreting extracellular vesicles from adipose stem cells by ultrasonic stimulation according to claim 1, wherein the method comprises the following steps:

wherein, in the step A, the adopted digestive enzyme is collagenase IV;

the digestion conditions were as follows: collagenase type IV with a mass volume ratio of 0.2% is mixed with the granular adipose tissue according to a volume ratio of 1:1, and the mixture is fully shaken on a shaking table at 37 ℃ and digested for 2 hours.

3. The method for improving the efficiency of secreting extracellular vesicles from adipose stem cells by ultrasonic stimulation according to claim 1, wherein the method comprises the following steps:

In the step A, when the adipose-derived stem cells are routinely passaged to the 3 rd generation, identifying the adipose-derived stem cells of the 3 rd generation by adopting flow cytometry and three-way induction of osteogenesis, adipogenesis and chondrogenesis.

4. The method for improving the efficiency of secreting extracellular vesicles from adipose stem cells by using ultrasonic stimulation according to claim 1, wherein the method comprises the following steps:

wherein, in the step B, the pulse repetition frequency of 1MHz and 10Hz, the duty ratio of 60 percent and the pulse width of 9mm are used²The ultrasonic generating device of the cross-sectional area performs ultrasonic stimulation on the cells.

5. The method for improving the efficiency of secreting extracellular vesicles from adipose stem cells by using ultrasonic stimulation according to claim 4, wherein the method comprises the following steps:

wherein, when ultrasonic stimulation is carried out, 0.5-1cm thick couplant is uniformly smeared below a 10cm cell culture dish, then a transducer of an ultrasonic generating device is tightly attached to the couplant to remove air, and the ultrasonic dose is set to be 1.5W/cm²The exposure time was 30 s.

6. The method for improving the efficiency of secreting extracellular vesicles from adipose stem cells by ultrasonic stimulation according to claim 1, wherein the method comprises the following steps:

wherein, in the step C, the supernatant is filtered by a 0.22 μm filter and then added into an ultrafiltration centrifugal tube with a 100KD membrane, and the mixture is centrifugally concentrated to 500 μ L of the volume of an inner tube at 3000g and 4 ℃, transferred into a sterile freezing tube and frozen in a refrigerator at minus 80 ℃.

7. The adipose-derived stem cell outer vesicles extracted by the method according to any one of claims 1 to 6.

8. Use of the adipose stem cell outer vesicles of claim 7 for preparing a bioactive preparation for promoting skin wound healing.

9. Use according to claim 7, characterized in that:

the bioactive preparation for promoting the healing of the skin wound is a preparation for promoting the vitality and proliferation of an epidermal cell line and a skin fibroblast, a preparation for improving the migration capacity of the skin fibroblast or a preparation for promoting the vascularization capacity of an umbilical vein endothelial cell line.

10. A bioactive preparation for promoting healing of a skin wound, comprising the adipose stem cell-derived outer vesicle according to claim 7, wherein the added mass of the adipose stem cell-derived outer vesicle is 200 μ g.