Preparation method and application of high-expression p-AKT extracellular vesicle

Abstract

The invention relates to the technical field of biology, and particularly discloses a preparation method and application of high-expression p-AKT extracellular vesicles, the method comprises the following steps: culturing extracted human amniotic mesenchymal stem cells under low oxygen conditions of 37 DEG C, 5% CO2 and 1%-3% oxygen partial pressure to obtain Hypoxic-hAMSC, and subculturing the Hypoxic-hAMSC; when the convergence degree of the passage Hypoxic-hAMSC cells reaches 80%, discarding the culture medium, cleaning, adding the culture medium into a DMEM culture medium, culturing for 24-48 hours under a low-oxygen condition, collecting supernatant, and extracting the extracellular vesicles in the supernatant by using a high-speed centrifugation method to prepare the high-expression p-AKT extracellular vesicles. The high-expression p-AKT extracellular vesicles are prepared by culturing human amniotic mesenchymal stem cells under a low-oxygen condition (1%-3% oxygen partial pressure), and compared with extracellular vesicles obtained under a normal-oxygen condition (21% oxygen partial pressure), the high-expression p-AKT characteristic enables the extracellular vesicles to more effectively promote skin healing; the hAMSC-exo derivative has the characteristic of inhibiting inflammation of hAMSC-exo and the characteristic of promoting keratinocyte proliferation of p-AKT at the same time, and a new therapeutic drug is provided for better promoting skin wound healing.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method and application of extracellular vesicles that highly express p-AKT. Background Art

[0002] Skin wounds, particularly chronic wounds such as ulcers caused by venous hypertension, arterial ulcers, and diabetic foot ulcers, are slow to heal, prone to infection, and have a high recurrence rate. Furthermore, the pain associated with extensive wound ulceration severely impacts patients' physical and mental health and quality of life. Currently, treatments for skin wounds include traditional physical therapy, surgery, topical medications (ointments, gels), and biologics, but these have limited clinical efficacy, are costly, and carry the risk of drug resistance. Therefore, exploring methods and approaches to promote wound healing, particularly targeted therapies targeting its pathogenesis, has become a critical issue urgently needed in dermatology. Skin wound healing is a complex biological process involving the interplay of multiple cells, enzymes, factors, and signaling molecules, encompassing four phases: hemostasis, inflammation, proliferation, and remodeling. Clinically, the causes of chronic wounds vary, but they all share a common pathophysiological process: excessive inflammation. Therefore, developing agents that both inhibit inflammation and promote keratinocyte proliferation has significant clinical significance and application value.

[0003] Currently, stem cell therapy is a safe and effective treatment for skin wounds. Human amniotic mesenchymal stem cells (hAMSCs) offer advantages such as abundant sources, ease of acquisition, non-tumorigenicity, and no ethical barriers. Compared to hAMSCs, their extracellular vesicles (hAMSC-exo) contain multiple bioactive factors, are compact, stable, easily stored, easily permeate barriers, and suppress inflammation, making them more clinically valuable. Research has shown that p-AKT (phospho-protein kinase B) is involved in the growth of skin keratinocytes, promoting tissue repair and reconstruction. A search revealed Chinese patent application number 202510048606.0, which discloses the use of tobacco extracellular vesicles in Alzheimer's disease. This patent reveals that tobacco extracellular vesicles can significantly activate the PI3K-AKT signaling pathway in primary astrocytes, increasing the expression of p-PI3K and / or p-AKT proteins in astrocytes, promoting Aβ degradation, improving learning and memory, and alleviating cognitive impairment, thereby preventing or treating Alzheimer's disease. This existing patent describes tobacco extracellular vesicles as being useful in preventing or treating Alzheimer's disease. However, the patent only reveals that tobacco extracellular vesicles can activate the PI3K-AKT signaling pathway in astrocytes, not that the extracellular vesicles themselves overexpress p-AKT.

[0004] In summary, how to prepare hAMSC-exo with high expression of p-AKT and its application in wound healing have not been reported in existing literature.

[0005] Therefore, the present application provides a method for preparing extracellular vesicles with high p-AKT expression and its application. This hAMSC-exo with high p-AKT expression will have both the characteristics of hAMSC-exo in inhibiting inflammation and the characteristics of p-AKT in promoting keratinocyte proliferation, providing a new therapeutic drug for better promoting skin wound healing. Summary of the Invention

[0006] The purpose of the present invention is to solve the technical problems raised in the above background technology and provide a preparation method and application of extracellular vesicles with high expression of p-AKT.

[0007] The above-mentioned purpose of the present invention is achieved like this: The present invention provides a method for preparing extracellular vesicles that highly express p-AKT, comprising the following steps: S1. Extraction and hypoxic culture of primary hAMSCs: Under sterile conditions, discarded human amniotic membrane after childbirth was washed three times with D-Hank's solution. The membrane was then minced with ophthalmic scissors and digested with trypsin at 37°C for 90 minutes. The supernatant was discarded, and the remaining membrane was further digested with collagenase 4 until the tissue melted. The filtrate was filtered through a 200-mesh sieve, collected, and centrifuged. The precipitate was resuspended in hAMSC culture medium and cultured in a hypoxic incubator for 3-6 hours. The medium was then replaced and cultured again. Among them, hAMSCs obtained by culture under hypoxic conditions are Hypoxic-hAMSCs; S2. Passaging of Hypoxic-hAMSCs: When the confluence of Hypoxic-hAMSC cells reaches 80%-90%, they are passaged. The culture medium is discarded, the cells are washed three times with PBS, and 1 mL of 0.25% trypsin is added for digestion for 1-3 minutes. The cells are then digested with 2 mL of hAMSC culture medium, centrifuged, and the precipitate is collected and resuspended in hAMSC culture medium for culture. S3. Preparation of Hypoxic-hAMSC Extracellular Vesicles: When the confluence of the passaged Hypoxic-hAMSC cells in step S2 reaches 80%, the culture medium is discarded, the cells are washed three times with PBS, and DMEM medium is added. The cells are cultured under hypoxic conditions for 24-48 hours, and the supernatant is collected. The extracellular vesicles in the supernatant are extracted by high-speed centrifugation. After the supernatant is centrifuged and filtered multiple times, the resulting precipitate is the prepared Hypoxic-exo, which is the extracellular vesicles with high expression of p-AKT.

[0008] Furthermore, the hAMSC culture medium in step S1 consists of a-MEM liquid culture medium, 5%-10% fetal bovine serum, L-glutamine, sodium pyruvate, β-mercaptoethanol and epidermal growth factor.

[0009] Furthermore, the specific steps of extracting extracellular vesicles from the supernatant by high-speed centrifugation in step S3 include: 1) Centrifuge at least 50 mL of supernatant at 1000 g for 10 min at 4°C to obtain supernatant I. 2) Centrifuge supernatant I at 12,000 g for 20 min at 4°C to obtain supernatant II; 3) Centrifuge supernatant II at 12,000 g for 40 min at 4°C to obtain supernatant III; 4) Filter supernatant III through a 0.22 μm filter to obtain supernatant IV; 5) Centrifuge supernatant IV at 100,000 g for 1 h at 4°C to obtain the precipitate, which is Hypoxic-exo, i.e., extracellular vesicles that highly express p-AKT.

[0010] Furthermore, the hypoxic conditions in step S1 are: 37° C., 5% CO 2 , and 1%-3% low oxygen partial pressure.

[0011] Furthermore, the concentration of epidermal growth factor was 10 ng / mL.

[0012] Furthermore, in step S2, centrifugation is performed at 1000 rpm / min for 3 min.

[0013] Furthermore, in step S1, centrifugation is performed at 2500 rpm / min for 10 min.

[0014] Furthermore, the D-Hank's solution in step S1 contains 1% penicillin-streptomycin.

[0015] The solution of the present invention also provides an extracellular vesicle prepared by a method for preparing extracellular vesicles that highly express p-AKT.

[0016] The solution of the present invention also provides the use of extracellular vesicles in preparing a drug for promoting skin wound healing.

[0017] Furthermore, the drug is used to inhibit inflammatory response and promote keratinocyte proliferation and tissue repair.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. In the method of the present invention, extracellular vesicles (hypoxic-exo) that highly express p-AKT are prepared by culturing human amniotic mesenchymal stem cells (hAMSCs) under hypoxic conditions (1%-3% oxygen partial pressure) and extracting their extracellular vesicles. Compared with extracellular vesicles obtained under normoxic conditions (21% oxygen partial pressure) (normoxia-exo), the extracellular vesicles prepared by the method of the present invention highly express p-AKT, enabling them to effectively promote skin wound healing; 2. Hypoxic-exo prepared by the present invention has the characteristics of small extracellular vesicle size, good stability, convenient storage, easy barrier penetration, and inhibition of inflammatory response, and also has the characteristics of p-AKT promoting keratinocyte proliferation and tissue repair; 3. The preparation method of the present invention is used in the preparation of a drug for promoting skin wound healing, and the drug has an excellent therapeutic effect on chronic skin wounds caused by excessive inflammation; 4. The preparation method of Hypoxic-exo provided by the present invention is simple, the operation process is controllable, the stability is good, and it has important industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 3 is a morphological observation diagram of Normoxia-hAMSC and Hypoxic-hAMSC in an embodiment of the present invention; Figure 2 This is a graph showing the cell proliferation activity of Normoxia-hAMSC and Hypoxic-hAMSC detected by CCK8 in an embodiment of the present invention; Figure 3 Graph showing stemness gene expression levels of Normoxia-hAMSC and Hypoxic-hAMSC in an embodiment of the present invention; Figure 4 Graph showing the levels of extracellular vesicle marker proteins expressed by Normoxia-exo and Hypoxic-exo in the examples of the present invention; Figure 5 is a graph showing the expression levels of p-AKT in Normoxia-exo and Hypoxic-exo in the examples of the present invention; Figure 6 This is a graph showing the effects of Normoxia-exo and Hypoxic-exo on wound healing in mice according to an embodiment of the present invention; Figure 7 This is a graph showing the results of CCK8 detecting the effects of Normoxia-exo and Hypoxic-exo on Hacat cell proliferation in an embodiment of the present invention; Figure 81 is a graph showing the results of a scratch test to detect the effects of Normoxia-exo and Hypoxic-exo on Hacat cell migration ability in an embodiment of the present invention; Figure 9 This is a graph showing the results of a protein immunoblotting experiment in an embodiment of the present invention to detect the effects of Normoxia-exo and Hypoxic-exo on the secretion level of inflammatory factors. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0021] The implementation of the present invention is described in detail below with reference to specific embodiments.

[0022] Reference Figures 1-9 As shown, the following are preferred embodiments provided by the present invention.

[0023] Example 1: Example 1 of the present invention provides a method for preparing extracellular vesicles that highly express p-AKT and its use in the preparation of drugs that promote wound healing. The prepared hAMSC-exo that highly expresses p-AKT will have both the characteristics of hAMSC-exo in inhibiting inflammation and the characteristics of p-AKT in promoting keratinocyte proliferation, providing a new therapeutic drug for better promoting skin wound healing.

[0024] The method for preparing extracellular vesicles with high p-AKT expression comprises the following steps: 1) Isolation and Hypoxic Culture of Primary hAMSCs: Under sterile conditions, discarded human amniotic membrane after childbirth was washed three times with D-Hank's solution (containing 1% penicillin-streptomycin). The membrane was minced with ophthalmic scissors and digested with trypsin at 37°C for 90 min. The supernatant was discarded, and the remaining membrane was further digested with collagenase 4 until the tissue thawed. The filtrate was collected by filtration through a 200-mesh sieve and centrifuged at 2500 rpm / min for 10 min. The pellet was resuspended in hAMSC culture medium and cultured in a hypoxic incubator (37°C, 5% CO2, 1% hypoxic partial pressure) for 3-6 hours. After that, the medium was changed and culture continued. hAMSCs cultured under these hypoxic conditions are referred to as hypoxic-hAMSCs.

[0025] Among them, the components of hAMSC culture medium include: a-MEM liquid culture medium, 5%-10% fetal bovine serum, L-glutamine, sodium pyruvate, β-mercaptoethanol and epidermal growth factor (10ng / mL).

[0026] 2) Hypoxic-hAMSC Passaging: Passage the cells when they reach 80%-90% confluence. Discard the culture medium, wash three times with PBS, and digest with 1 mL of 0.25% trypsin (containing EDTA) for 1-3 minutes. Terminate the digestion with 2 mL of hAMSC culture medium. Centrifuge at 1000 rpm / min for 3 minutes, collect the pellet, resuspend in hAMSC culture medium, and culture.

[0027] 3) Obtaining Hypoxic-hAMSC Extracellular Vesicles (Hypoxic-exo): When the passaged Hypoxic-hAMSC cells reach 80% confluence, discard the culture medium, wash three times with PBS, add DMEM medium, and culture under hypoxic conditions for 24-48 hours. Collect the supernatant (at least 50 mL) and extract the extracellular vesicles by high-speed centrifugation. The specific steps are as follows: First, the collected supernatant (at least 50 mL) is centrifuged at 1000 g at 4°C for 10 minutes to obtain supernatant I. Then, supernatant I is centrifuged at 12,000 g at 4°C for 20 minutes to obtain supernatant II. Supernatant II is centrifuged at 12,000 g at 4°C for 40 minutes to obtain supernatant III. Supernatant III is filtered through a 0.22 μm filter to obtain supernatant IV. Finally, supernatant IV is centrifuged at 100,000 g at 4°C for 1 hour to obtain the precipitate of hypoxic-exosomes. These hypoxic-exosomes are extracellular vesicles that overexpress p-AKT.

[0028] Example 2: The difference between Example 2 and Example 1 is that in the preparation method of the extracellular vesicles with high p-AKT expression, the culture conditions in the incubator placed under hypoxic conditions are: 37°C, 5% CO2, and 2% low oxygen partial pressure.

[0029] Example 3: The difference between Example 3 and Example 1 is that in the preparation method of the extracellular vesicles with high p-AKT expression, the culture conditions in the incubator placed under hypoxic conditions are: 37°C, 5% CO2, and 3% low oxygen partial pressure.

[0030] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that, in the preparation method of the extracellular vesicles with high p-AKT expression, the culture conditions in the incubator placed under hypoxic conditions are: 37°C, 5% CO2, and 10% low oxygen partial pressure.

[0031] The specific experimental verification of the above embodiment of the present invention is as follows: 1. Characteristics of Hypoxic-hAMSCs Hypoxic-hAMSCs are hAMSCs obtained by culturing under hypoxic conditions (37°C, 5% CO2, 1% oxygen partial pressure); Normoxia-hAMSCs are hAMSCs obtained by culturing under normoxic conditions (37°C, 5% CO2, 21% oxygen partial pressure). Other experimental conditions are the same as those of Hypoxic-hAMSCs. Both Normoxia-hAMSCs and Hypoxic-hAMSCs are spindle-shaped and firmly attached to the wall (e.g. Figure 1 In this experiment, the cell proliferation ability under oxygen partial pressure of 1%, 2%, 3%, and 10% and normoxia conditions in the above-mentioned Examples 1 to 3 and Comparative Example 1 was tested. Among them, the hAMSC proliferation ability was stronger under oxygen partial pressure of 1%-3% (as shown). Figure 2 In addition, compared with Normoxia-hAMSCs, Hypoxic-hAMSCs showed higher expression of stemness marker genes SOX2 and Nanog (as shown in Figure 2). Figure 3 Maintaining good stem cell stemness is beneficial to its differentiation potential and survival ability.

[0032] 2. Characteristics of Hypoxic-exo Hypoxic-exo is the extracellular vesicle of Hypoxic-hAMSC; Normoxia-exo is the extracellular vesicle of Normoxia-hAMSC. Both Normoxia-exo and Hypoxic-exo express extracellular vesicle marker proteins, and the expression levels of CD9, CD63 and CD81 in Hypoxic-exo are higher than those in Normoxia-exo (e.g. Figure 4 At the same time, both can express p-AKT, but the expression level of p-AKT protein in Hypoxic-exo is higher than that in Normoxia-exo (as shown in Figure 5 ), which are extracellular vesicles with high expression of p-AKT, confirming that they are closely related to skin keratinocyte proliferation and tissue repair.

[0033] 3. Compared with Normoxia-exo, Hypoxic-exo is more conducive to promoting skin wound healing in mice Eight-week-old male C57BL / 6 mice were divided into four groups and anesthetized with 1% sodium pentobarbital (50 mg / kg). The central back was shaved and the skin was prepared. The skin was disinfected with 75% alcohol and a full-thickness skin excision was performed on the back using a sterile round biopsy punch to create a wound. Each group was then given the following treatments: ①Control group: local injection of 0.2 mL PBS; ②LPS group: subcutaneous injection of LPS (10 μg / mL) dissolved in 0.2 mL PBS; ③Normoxia-exo group: LPS (10 μg / mL) dissolved in 0.2 mL PBS was subcutaneously injected, followed by subcutaneous injection of Normoxia-exo (200 μg); ④Hypoxic-exo group: LPS (10 μg / mL) dissolved in 0.2 mL PBS was subcutaneously injected, followed by subcutaneous injection of Hypoxic-exo (200 μg).

[0034] The wound healing rate of mouse skin was evaluated on day 1, day 3, day 7, and day 15 (e.g. Figure 6 The results showed that compared with the control group, wound healing in the LPS group was significantly delayed, while both Normoxia-exo and Hypoxic-exo effectively accelerated wound healing, with the effect of Hypoxic-exo being more significant.

[0035] 4. Compared with Normoxia-exo, Hypoxic-exo more effectively promotes the proliferation of Hacat cells An inflammatory cell model was constructed by treating human immortalized keratinocytes (Hacat) with LPS. The experiment was divided into the following four groups: ①Control group: cells were treated with serum-free medium; ②LPS group: cells were treated with serum-free medium containing 1 μg / mL LPS; ③Normoxia-exo group: cells were treated with serum-free medium containing 1 μg / mL LPS, and then Normoxia-exo (30 μg / mL) was added; ④Hypoxic-exo group: cells were treated with serum-free medium containing 1 μg / mL LPS, and then Hypoxic-exo (30 μg / mL) was added.

[0036] CCK8 assay to detect Hacat cell proliferation (e.g. Figure 7 After treating cells according to the above groupings, 100 μL of the cell suspension was seeded into a 96-well plate and cultured in a pre-incubator. 10 μL of CCK8 reagent was added to each well and incubated at 37°C for 3 hours. The absorbance at a wavelength of 450 nm was measured using a microplate reader. The results showed that compared with the control group, the cell proliferation ability of the LPS group was significantly reduced. While both Normoxia-exo and Hypoxic-exo effectively promoted the proliferation of Hacat cells, the promoting effect of Hypoxic-exo was more pronounced.

[0037] 5. Compared with Normoxia-exo, Hypoxic-exo more effectively promotes Hacat cell migration Scratch assay to detect the migration ability of Hacat cells (such as Figure 8 After treating the cells according to the above groupings, a 200 μL pipette tip was used to vertically scratch the cells. The cells were then washed three times with PBS and cultured with culture medium. Images were taken at 0, 12, and 36 hours. The results showed that compared with the control group, the cell migration rate in the LPS group was slowed. However, both Normoxia-exo and Hypoxic-exo promoted Hacat cell migration, with the promoting effect of Hypoxic-exo being more pronounced.

[0038] 6. Compared with Normoxia-exo, Hypoxic-exo more effectively inhibits the high expression of inflammatory factors in Hacat cells induced by LPS Western blotting assay to detect inflammatory factors (such as Figure 9 After cells were treated as described above, proteins were extracted using RIPA lysis buffer containing phosphatase and protease inhibitors and heated in a metal bath at 95-100°C for 10 minutes. SDS-polyacrylamide gel electrophoresis was performed and the membranes were transferred to PVDF membranes. The membranes were blocked with 5% skim milk and incubated with primary antibodies (IL-1β, MMP3, NF-κB, TNFα, and β-actin) at 4°C overnight. The membranes were washed three times with TBST for 10 minutes each. Subsequently, HRP-linked secondary antibodies were incubated at room temperature for 1 hour. Protein bands were visualized using the ChemiDoc XRS+ system (Bio-Rad, USA). Results showed that compared with the control group, the protein expression levels of the inflammatory factors IL-1β, MMP3, NF-κB, and TNFα in the LPS group were significantly elevated. This phenomenon was reversed by the Normoxia-exo and Hypoxic-exo treatments, with Hypoxic-exo having a more pronounced inhibitory effect on the LPS-induced elevated expression of inflammatory factors.

[0039] In summary, through the above embodiments of the present invention, the present invention provides a method for preparing extracellular vesicles that highly express p-AKT and its use in the preparation of a drug for promoting skin wound healing, which overcomes the limitations of traditional treatment methods such as limited clinical efficacy, high cost, and the risk of drug resistance. The present application scheme provides an extracellular vesicle that is smaller in size, more stable, easier to store, easier to penetrate barriers, and inhibits inflammatory responses compared to the existing technology in this field; at the same time, it can better promote keratinocyte proliferation and tissue repair, and is more conducive to the healing of skin wounds. In addition, the high-expression p-AKT extracellular vesicles provided by the present invention refer to hAMSC extracellular vesicles prepared under hypoxic conditions provided by the present invention, with a higher expression of p-AKT protein than under normoxic conditions.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing extracellular vesicles that highly express p-AKT, characterized in that: The following steps are involved: S1. Extraction and hypoxic culture of primary hAMSCs: Under sterile conditions, discarded human amniotic membrane after childbirth was washed three times with D-Hank's solution. The membrane was then minced with ophthalmic scissors and digested with trypsin at 37°C for 90 minutes. The supernatant was discarded, and the remaining membrane was further digested with collagenase 4 until the tissue melted. The filtrate was filtered through a 200-mesh sieve, collected, and centrifuged. The precipitate was resuspended in hAMSC culture medium and cultured in a hypoxic incubator for 3-6 hours. The medium was then replaced and cultured again. Among them, hAMSCs obtained by culture under hypoxic conditions are Hypoxic-hAMSCs; S2. Passaging of Hypoxic-hAMSCs: When the confluence of Hypoxic-hAMSC cells reaches 80%-90%, they are passaged. The culture medium is discarded, and the cells are washed three times with PBS. 1 mL of 0.25% trypsin is added for digestion for 1-3 minutes. The cells are then digested with 2 mL of hAMSC culture medium. The cells are centrifuged, the precipitate is collected, and the cells are resuspended and cultured in hAMSC culture medium. S3. Preparation of Hypoxic-hAMSC Extracellular Vesicles: When the confluence of the passaged Hypoxic-hAMSC cells in step S2 reaches 80%, the culture medium is discarded, the cells are washed three times with PBS, and DMEM medium is added. The cells are cultured under hypoxic conditions for 24-48 hours, and the supernatant is collected. The extracellular vesicles in the supernatant are extracted by high-speed centrifugation. After the supernatant is centrifuged and filtered multiple times, the resulting precipitate is the prepared Hypoxic-exo, which is the extracellular vesicles with high expression of p-AKT. The hypoxic conditions in step S1 are: 37° C., 5% CO 2 , and a low oxygen partial pressure of 1%-3%.

2. The method for preparing extracellular vesicles with high p-AKT expression according to claim 1, characterized in that: The hAMSC culture medium in step S1 consists of a-MEM liquid culture medium, 5%-10% fetal bovine serum, L-glutamine, sodium pyruvate, β-mercaptoethanol and epidermal growth factor.

3. The method for preparing extracellular vesicles with high p-AKT expression according to claim 1, characterized in that: The specific steps of extracting extracellular vesicles from the supernatant by high-speed centrifugation in step S3 include: 1) Centrifuge at least 50 mL of supernatant at 1000 g for 10 min at 4°C to obtain supernatant I. 2) Centrifuge supernatant I at 12,000 g for 20 min at 4°C to obtain supernatant II; 3) Centrifuge supernatant II at 12,000 g for 40 min at 4°C to obtain supernatant III; 4) Filter supernatant III through a 0.22 μm filter to obtain supernatant IV; 5) Centrifuge supernatant IV at 100,000 g for 1 h at 4°C to obtain the precipitate, which is Hypoxic-exo, i.e., extracellular vesicles that highly express p-AKT.

4. The method for preparing extracellular vesicles with high p-AKT expression according to claim 2, characterized in that: The concentration of epidermal growth factor was 10 ng / mL.

5. The method for preparing extracellular vesicles with high p-AKT expression according to claim 1, characterized in that: In step S2, centrifuge at 1000 rpm / min for 3 min.

6. The method for preparing extracellular vesicles with high p-AKT expression according to claim 1, characterized in that: In step S1, centrifuge at 2500 rpm / min for 10 min.

7. The method for preparing extracellular vesicles with high p-AKT expression according to claim 1, characterized in that: The D-Hank's solution in step S1 contains 1% penicillin-streptomycin.

8. Extracellular vesicles prepared according to the method for preparing extracellular vesicles with high p-AKT expression according to any one of claims 1 to 7.

9. Use of the extracellular vesicles according to claim 8 in the preparation of a medicament for promoting skin wound healing.

10. The use according to claim 9, characterized in that The drug is used for inhibiting inflammatory response and promoting keratinocyte proliferation and tissue repair.

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