APOA2-loaded platelet apoptosis vesicle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological tissue engineering, and relates to APOA2-loaded platelet apoptosis vesicles as well as a preparation method and application thereof. According to the platelet apoptosis vesicle loaded with APOA2, protein APOA2 is loaded into apoptosis vesicles derived from platelets. According to the APOA2-loaded platelet apoptosis vesicle and the preparation method thereof disclosed by the invention, the prepared APOA2-loaded platelet apoptosis vesicle can better inhibit adipogenic differentiation of mesenchymal stem cells, so that liver fat accumulation can be safely and efficiently prevented, and the APOA2-loaded platelet apoptosis vesicle can be better used for treating non-alcoholic fatty liver diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biological tissue engineering, and relates to platelet apoptotic vesicles loaded with APOA2, and a preparation method and application thereof. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is a liver disease syndrome without alcohol abuse. It can develop from simple fatty liver to hepatitis, then to liver fibrosis, and even to end-stage liver diseases such as cirrhosis, hepatocellular carcinoma, or liver failure. In adults, the prevalence of NAFLD is as high as 30%, which is associated with increased liver-specific and total mortality. The development of NAFLD is divided into two steps: first, the accumulation of triglycerides in hepatocytes develops into hepatic steatosis; second, oxidative stress and activation of proinflammatory cytokines, which eventually lead to fibrosis. Due to the complex pathophysiological mechanism of NAFLD, there is currently no FDA (food and drug administration) approved drug treatment. In recent years, mesenchymal stem cells (MSCs) have become a reliable therapeutic target for reducing liver tissue damage and reversing NAFLD because they have the potential to differentiate into hepatocytes, can regulate immune responses, and can participate in anti-fibrosis and lipid metabolism. Therefore, how to efficiently regulate MSCs lipid metabolism is the key to preventing and treating NAFLD.

[0003] Apoptotic vesicles (apoVs) are a heterogeneous subpopulation of extracellular vesicles (EVs) produced during apoptosis. They range in diameter from 100nm to 1000nm and contain a large amount of DNA, RNA, and protein. The transfer of biomolecules mediated by apoVs can occur between different types of cells. Currently, apoVs are used in the diagnosis and treatment of diseases, including: diagnosis of gastrointestinal diseases, treatment of atherosclerosis, hemophilia, bacterial infections, tissue regeneration engineering such as bone, skin, and hair, and immunotherapy. There has been no breakthrough in the treatment of inhibiting fat accumulation through apoVs.

[0004] Apolipoprotein A2 (APOA2) is mainly synthesized by the liver and small intestine. It includes an amino acid sequence encoded by four exons. Its C-terminus is composed of a variable number of lipid-binding amphipathic helices and can exist as a monomer or dimer. APOA2 is involved in a variety of biological processes, such as cytokine production, glucose metabolism, and neutrophil activation. Its normal function is important for preventing cardiovascular diseases such as atherosclerosis. However, it is still unknown how APOA2 affects MSC differentiation, and its role in NAFLD has rarely been studied. Summary of the invention

[0005] The primary purpose of the present invention is to provide a platelet apoptotic vesicle loaded with APOA2, so as to better inhibit the adipogenic differentiation of mesenchymal stem cells, thereby safely and efficiently preventing liver fat accumulation, so as to be better used in the treatment of non-alcoholic fatty liver disease.

[0006] To achieve this object, in a basic embodiment, the present invention provides a platelet apoptotic vesicle loaded with APOA2, wherein the platelet apoptotic vesicle loaded with APOA2 is loaded with protein APOA2 in a platelet-derived apoptotic vesicle.

[0007] In a preferred embodiment, the present invention provides a platelet apoptotic vesicle loaded with APOA2, wherein the mass ratio of the platelet-derived apoptotic vesicle to the protein APOA2 used to prepare the platelet apoptotic vesicle loaded with APOA2 is 1:5-1:10.

[0008] In a preferred embodiment, the present invention provides a platelet apoptotic vesicle loaded with APOA2, wherein the platelet apoptotic vesicle loaded with APOA2 is spherical, has a double-layer lipid membrane structure, and has a particle size of 100-300 nm (average particle size 120-180 nm).

[0009] The second object of the present invention is to provide a method for preparing the platelet apoptotic vesicles loaded with APOA2 as described above, so as to better prepare the platelet apoptotic vesicles loaded with APOA2 as described above. The prepared platelet apoptotic vesicles loaded with APOA2 can better inhibit the adipogenic differentiation of mesenchymal stem cells, thereby safely and efficiently preventing liver fat accumulation, and thus can be better used in the treatment of non-alcoholic fatty liver disease.

[0010] To achieve this object, in a basic embodiment, the present invention provides a method for preparing the platelet apoptotic vesicles loaded with APOA2 as described above, the preparation method comprising the following steps:

[0011] (1) preparing the platelet-derived apoptotic vesicles;

[0012] (2) The platelet-derived apoptotic vesicles are mixed with the protein APOA2 and then treated so that the protein APOA2 is loaded into the platelet-derived apoptotic vesicles, and the platelet apoptotic vesicles loaded with APOA2 are separated and obtained.

[0013] In a preferred embodiment, the present invention provides a method for preparing platelet apoptotic vesicles loaded with APOA2 as described above, wherein the preparation process of step (1) comprises the following steps:

[0014] (11) Separating and purifying platelets from blood samples;

[0015] (12) Induce platelet apoptosis;

[0016] (13) Centrifugation and washing to obtain the platelet-derived apoptotic vesicles.

[0017] In a preferred embodiment, the present invention provides a method for preparing platelet apoptotic vesicles loaded with APOA2 as described above, wherein in step (11), an anticoagulant reagent is added to the blood sample after it is collected to prevent coagulation.

[0018] In a preferred embodiment, the present invention provides a method for preparing platelet apoptotic vesicles loaded with APOA2 as described above, wherein the anticoagulant reagent is selected from one or more of acid citrate dextrose, potassium oxalate, sodium fluoride, heparin, and sodium citrate.

[0019] In a preferred embodiment, the present invention provides a method for preparing platelet apoptotic vesicles loaded with APOA2 as described above, wherein in step (12), the platelets are resuspended in culture medium, and then staurosporine (STS, preferably at a concentration of 5000 nM) is added to induce cell apoptosis.

[0020] In a preferred embodiment, the present invention provides a method for preparing platelet apoptotic vesicles loaded with APOA2 as described above, wherein the culture medium is selected from one or more of MEM culture medium, DMEM culture medium, and RPMI-1640 culture medium.

[0021] In a preferred embodiment, the present invention provides a method for preparing platelet apoptotic vesicles loaded with APOA2 as described above, wherein step (13) comprises the following steps:

[0022] (131) The culture medium was centrifuged at 700-900 g for 8-15 min at 4°C to remove cell debris in the culture medium and obtain a centrifugal supernatant;

[0023] (132) Centrifuging the centrifuged supernatant at 15,000-18,000 g for 25-35 minutes at 4° C., taking the precipitate to obtain the crude platelet-derived apoptotic vesicles;

[0024] (133) The crude platelet-derived apoptotic vesicles were washed with sterile PBS and then centrifuged at 15,000-18,000 g for 25-35 minutes at 4°C to obtain the pure platelet-derived apoptotic vesicles.

[0025] In a preferred embodiment, the present invention provides a method for preparing platelet apoptotic vesicles loaded with APOA2 as described above, wherein in step (2), the treatment is ultrasonic treatment.

[0026] In a preferred embodiment, the present invention provides a method for preparing platelet apoptotic vesicles loaded with APOA2 as described above, wherein in step (2), the platelet apoptotic vesicles loaded with APOA2 are obtained by centrifugation, and the centrifugation conditions are: temperature 4°C, centrifugation at 15000-18000g for 25-35 minutes.

[0027] In a preferred embodiment, the present invention provides a method for preparing platelet apoptotic vesicles loaded with APOA2 as described above, wherein step (2) comprises the following steps:

[0028] (21) resuspending the platelet-derived apoptotic vesicles with sterile PBS (preferably making the final concentration of the platelet-derived apoptotic vesicles 50-200 ng / ml, more preferably making the final concentration 100 ng / ml), and then adding the protein APOA2 solution resuspended with sterile PBS (preferably at a concentration of 0.3-1.0 mg / ml, more preferably at a concentration of 0.5 mg / ml), preferably the final concentration of the total protein in the obtained mixed solution is 0.05-0.2 mg / ml, more preferably the final concentration of the total protein in the obtained mixed solution is 0.1 mg / ml;

[0029] (22) The resulting mixture was sonicated using a Qsonica Sonicator Q700 (Fisher Scientific, Hampton, NH, USA) with the following parameters: 500 V, 2 kHz, 20% power, 6 cycles, 4 s pulse / 2 s pause; cooled on ice for 2 min, and then sonicated again with the same parameters;

[0030] (23) The platelet apoptotic vesicles loaded with APOA2 are obtained by centrifugation.

[0031] The third object of the present invention is to provide the use of the platelet apoptotic vesicles loaded with APOA2 as described above for the preparation of an agent for inhibiting the adipogenic differentiation of mesenchymal stem cells, so as to better inhibit the adipogenic differentiation of mesenchymal stem cells, thereby safely and efficiently preventing liver fat accumulation, and thus being better used for the treatment of non-alcoholic fatty liver disease.

[0032] To achieve this object, in a basic embodiment, the present invention provides use of the platelet apoptotic vesicles loaded with APOA2 as described above for preparing a medicament for inhibiting adipogenic differentiation of mesenchymal stem cells.

[0033] In a preferred embodiment, the present invention provides the use of the platelet apoptotic vesicles loaded with APOA2 as described above for preparing an agent for inhibiting the adipogenic differentiation of mesenchymal stem cells, wherein the platelet apoptotic vesicles loaded with APOA2 are further used to prepare an agent for preventing, treating or alleviating non-alcoholic fatty liver disease.

[0034] The beneficial effect of the present invention is that by utilizing the platelet apoptotic vesicles loaded with APOA2 and the preparation method thereof, the prepared platelet apoptotic vesicles loaded with APOA2 can better inhibit the adipogenic differentiation of mesenchymal stem cells, thereby safely and efficiently preventing liver fat accumulation, and thus can be better used for the treatment of non-alcoholic fatty liver disease.

[0035] The platelet apoptotic vesicles prepared by the method of the present invention can be extracted from the patient's own blood, thus avoiding ethical and immune issues, and the extraction method is simple, low in cost, high in yield, and can be rapidly detected.

[0036] The platelet apoptotic vesicles loaded with APOA2 provided by the present invention have significantly improved abilities of inhibiting the adipogenic differentiation of mesenchymal stem cells and inhibiting the accumulation of fat in the liver compared with single platelet apoptotic vesicles, and have broad clinical application prospects in the treatment of NAFLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 These are the test results of the APOA2-loaded apoptotic vesicles prepared in Example 1 and the apoptotic vesicles prepared in Comparative Example 1, wherein A is the cryo-transmission electron microscopy test result, B is the nanoparticle tracking analysis test result, C is the zeta potential test result, D is the flow cytometry test result of Annexin V, E is the protein immunoblot test result of biomarker content, and F is the flow cytometry test result of surface marker APOA2 protein.

[0038] Figure 2 The results of the detection of the inhibition of adipogenic differentiation of MSCs in vitro by the APOA2-loaded apoptotic vesicles prepared in Example 1-2, the apoptotic vesicles prepared in Comparative Example 1-2, or the apoptotic vesicle mixture, wherein A and B are Oil Red O staining and quantitative results, respectively, and C is the PCR result of adipogenic-related genes. The data were compared among multiple groups by one-way ANOVA and Tukey's post hoc test, ns no significant difference, **p<0.01, ***p<0.001, ****p<0.0001.

[0039] Figure 3The apoptotic vesicles loaded with APOA2 prepared in Example 1-2, the apoptotic vesicles prepared in Comparative Example 1-2, or the apoptotic vesicle mixture inhibited the accumulation of body fat and liver fat in mice, i.e., the test result of treating non-alcoholic fatty hepatitis, wherein A is the content of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the blood of mice, B and C are the results of H&E staining of liver tissue and Oil Red O staining, respectively. The data were compared among multiple groups by one-way ANOVA and Tukey's post hoc test, ns, no significant difference,

[0040] ****p<0.0001. DETAILED DESCRIPTION

[0041] The specific implementation modes of the present invention are further described below through examples and drawings.

[0042] Example 1: Preparation of platelet apoptotic vesicles loaded with APOA2 (I)

[0043] 1) isolating and purifying human platelets in vitro, adding STS to induce apoptosis, and obtaining platelet-derived apoptotic vesicles by gradient centrifugation. For specific methods, see Example 1 of CN 114306384A;

[0044] 2) resuspending the apoptotic vesicles prepared in step 1) with an appropriate amount of sterile PBS to make the final concentration of the apoptotic vesicles 100 ng / ml;

[0045] 3) Add the APOA2 protein solution (concentration of 0.5 mg / ml) resuspended in PBS to the apoptotic vesicles, so that the mass ratio of apoptotic vesicles to APOA2 protein is 1:5;

[0046] 4) The resulting mixture was sonicated using a Qsonica Sonicator Q700 (Fisher Scientific, Hampton, NH, USA) with the following parameters: 500 V, 2 kHz, 20% power, 6 cycles, 4 s pulse / 2 s pause; cooled on ice for 2 min, and then sonicated again with the same parameters;

[0047] 5) Centrifugation (4°C, 17000 g for 30 minutes) to obtain platelet apoptotic vesicles loaded with protein APOA2.

[0048] Example 2: Preparation of platelet apoptotic vesicles loaded with APOA2 (II)

[0049] The mass ratio of apoptotic vesicles to APOA2 protein in step 2) was changed to 1:10, and the rest was the same as in Example 1.

[0050] Comparative Example 1: Preparation of platelet apoptotic vesicles

[0051] Human platelets were separated and purified in vitro, STS was added to induce apoptosis, and platelet-derived apoptotic vesicles were obtained by gradient centrifugation. For the specific method, see Example 1 of CN 114306384A.

[0052] Comparative Example 2: Preparation of a mixture of APOA2 and platelet apoptotic vesicles

[0053] Step 4) and step 5) in Example 1 were removed, that is, APOA2 and platelet apoptotic vesicles were simply mixed without ultrasound and subsequent centrifugation.

[0054] Example 3: Characterization of apoptotic vesicles

[0055] The APOA2-loaded platelet apoptotic vesicles prepared in Example 1 and the platelet apoptotic vesicles prepared in Comparative Example 1 were tested for morphology, particle size, zeta potential, Annexin V positivity, biomarker content, and surface marker APOA2 protein positivity by cryo-transmission electron microscopy, nanoparticle tracking analysis, flow cytometry, and protein immunoblotting, respectively. For some specific methods, please refer to Example 2 of CN 114306384A.

[0056] like Figure 1 As shown in A, the extracellular vesicles prepared in Example 1 and Comparative Example 1 are spherical and have a lipid bilayer membrane structure.

[0057] like Figure 1 As shown in B, the particle size distribution of the apoptotic vesicles prepared in Example 1 and Comparative Example 1 is mainly concentrated in the range of 100-300 nm, with the medians being 141.4 nm and 141.5 nm, respectively.

[0058] like Figure 1 As shown in Figure C, the peak zeta potentials of the apoptotic vesicles prepared in Example 1 and Comparative Example 1 were -26.60 mV and -25.92 mV, respectively.

[0059] like Figure 1 As shown in D, the positive rates of phosphatidylserine externalization of apoptotic vesicles prepared in Example 1 and Comparative Example 1 were as high as 79.4% and 78.1%, respectively, proving that they originated from cell apoptosis.

[0060] like Figure 1 As shown in E, the apoptotic vesicles prepared in Example 1 and Comparative Example 1 both highly expressed apoptotic vesicle-specific markers (ITGA5, RhoA, RPS25, Lamin B1) and extracellular vesicle markers (TSG101, CD9).

[0061] like Figure 1 As shown in F, the APOA2 protein positive rate of the engineered vesicles prepared in Example 1 is significantly higher than that of the apoptotic vesicles prepared in Comparative Example 1.

[0062] Example 4: Effect of platelet apoptotic vesicles loaded with APOA2 on adipogenic differentiation of MSCs in vitro

[0063] The APOA2-loaded apoptotic vesicles prepared in Example 1-2, the apoptotic vesicles prepared in Comparative Example 1-2, or the apoptotic vesicle mixture were respectively studied for their effects on adipogenic differentiation of MSCs in vitro, and the specific methods are as follows:

[0064] MSCs were cultured under the following three culture conditions:

[0065] 1) Proliferation medium (PM): MEM medium containing 10% FBS and 1% penicillin-streptomycin, cultured in a carbon dioxide (CO2) incubator (containing 95% air and 5% CO2 by volume, 100% humidity, and a constant temperature of 37°C).

[0066] 2) Adipogenic induction medium (AM): MEM medium containing 50 nM insulin, 100 nM dexamethasone, 200 μM indolemycin, 500 μM isobutylmethylxanthine, 1% penicillin / streptomycin, and 10% fetal bovine serum, cultured in a carbon dioxide (CO2) incubator (containing 95% air and 5% CO2 by volume, 100% humidity, and a constant temperature of 37°C).

[0067] 3) Add apoptotic vesicles (AM+apoVs: 200 ng / ml) to the adipogenic induction medium and culture in an incubator (containing 95% air and 5% CO2 by volume, 100% humidity, and a constant temperature of 37°C). After 7-14 days of adipogenic induction, the effect of cell osteogenic differentiation is examined by Oil Red O staining.

[0068] Oil Red O staining and quantification:

[0069] 1) Stain MSCs with Oil Red O working solution and observe the staining under a microscope;

[0070] 2) For quantitative analysis, the stained MSCs were immersed in 100% isopropanol for 10 minutes and the absorbance was measured at 500 nm.

[0071] like Figure 2As shown, compared with adipogenic induced culture (AM) cells, when the apoptotic vesicles loaded with APOA2 prepared in Example 1-2, or the apoptotic vesicles or apoptotic vesicle mixture prepared in Comparative Example 1-2 were added during AM culture, the accumulation of MSCs lipid droplets was significantly reduced after 14 days of adipogenic induction; and after adding the apoptotic vesicles loaded with APOA2 prepared in Example 1-2, the adipogenic differentiation of MSCs in vitro was significantly lower than that after adding the apoptotic vesicles or apoptotic vesicle mixture prepared in Comparative Example 1-2, indicating that the apoptotic vesicles loaded with APOA2 prepared in Example 1-2 have a significantly stronger anti-adipogenic effect than the apoptotic vesicles or apoptotic vesicle mixture prepared in Comparative Example 1-2.

[0072] Example 5: Therapeutic effect of platelet apoptotic vesicles loaded with APOA2 on NAFLD

[0073] The APOA2-loaded apoptotic vesicles prepared in Example 1-2, the apoptotic vesicles prepared in Comparative Example 1-2, or the apoptotic vesicle mixture were respectively studied for their therapeutic effects on NAFLD in mice, and the specific methods are as follows:

[0074] (1) Tail vein injection

[0075] Male 6-8 week old C57b6J mice (purchased from the Animal Department of Peking University Health Science Center) were divided into 6 groups, 4 mice in each group, respectively: a group fed with a high-fat diet (HFD) and injected with the apoptotic vesicles loaded with APOA2 prepared in Example 1, a group fed with a high-fat diet and injected with the apoptotic vesicles loaded with APOA2 prepared in Example 2, a group fed with a high-fat diet and injected with the apoptotic vesicles prepared in Comparative Example 1, a group fed with a high-fat diet and injected with the apoptotic vesicle mixture prepared in Comparative Example 2, a control group fed with a high-fat diet and not administered, and a control group fed with a regular chowfeeding (RC) and not administered. Each group was fed for 16 consecutive weeks, and mice in the drug-administered group were injected once a week through the tail vein, and the dosage for each mouse was 1 mg / kg each time. After 16 consecutive weeks of injection, samples were collected, and mice were killed by cervical dislocation and inhalation, and abdominal fat and liver were taken and fixed in neutral formalin for 48 hours.

[0076] (2) Slicing

[0077] After the tissue samples were immersed in wax and embedded in paraffin, 5 μm thick sections were cut using a paraffin slicer, and then the sections were stained with hematoxylin-eosin (H&E).

[0078] The fixed tissue is frozen and sectioned, usually with a thickness of 10 μm, and the sections are stained with Oil Red O.

[0079] (3) H&E staining

[0080] 1) Slices were washed with xylene solution twice, 5-10 minutes each time;

[0081] 2) Slices were washed with anhydrous ethanol twice, 3-5 minutes each time;

[0082] 3) Rinse the slices with 95% alcohol, twice, 5 minutes each time;

[0083] 4) The slices were washed with 90% alcohol, 80% alcohol, and 70% alcohol, once each for 2-3 minutes (hydration);

[0084] 5) Wash the slices with distilled water for 3 minutes;

[0085] 6) Stain the sections with hematoxylin for 10-15 minutes;

[0086] Color separation: insert the slice into 0.5-1% hydrochloric acid alcohol (70% alcohol) for a few seconds to more than ten seconds;

[0087] Bluing: 0.5-1% ammonia water, 1-2 minutes, check under a microscope (judgment of adequacy based on the color of the cell nucleus), then rinse with running water for 3 minutes;

[0088] 7) The sections were stained in 1% eosin aqueous solution for 5-10 minutes, then quickly washed with distilled water, 70%, 80%, and 90% alcohol for a few seconds to tens of seconds each, rinsed with 95% alcohol for 1-2 minutes, examined under a microscope (red and blue color contrast), and rinsed with 100% alcohol twice, each for 2-3 minutes (dehydration);

[0089] 8) Sections were washed with xylene twice, 3-5 minutes each time;

[0090] 9) The sections were sealed with neutral gum and images were collected and analyzed under a microscope.

[0091] (4) Oil Red O staining

[0092] 1) Place the slices in Oil Red O working solution for staining, the staining time is usually 2-5 minutes;

[0093] 2) Differentiation and rinsing: After staining, use 60% isopropanol for differentiation to remove excess stain;

[0094] 3) After differentiation, rinse the sections thoroughly with distilled water to remove residual stain;

[0095] 4) Use hematoxylin to counterstain the sections to show the cell nuclei. The counterstaining time should be strictly controlled, generally within 1 minute, to avoid over-staining of the cell nuclei;

[0096] 5) After counterstaining, use hydrochloric acid alcohol for differentiation, usually for 1-5 seconds to remove excess hematoxylin;

[0097] 6) Rinse the sections with running water to turn the cell nuclei back to blue.

[0098] 7) Finally, use glycerol gelatin or other aqueous mounting media for sealing, and collect and analyze images under a microscope.

[0099] like Figure 3 As shown, the body fat and liver fat accumulation of mice in the HFD control group increased significantly, and after the mice were injected with the apoptotic vesicles loaded with APOA2 prepared in Example 1-2 and the apoptotic vesicles or the apoptotic vesicle mixture prepared in Comparative Example 1-2 through the tail vein, the abdominal fat accumulation was significantly reduced, and the fatty liver was significantly improved. Moreover, after the injection of the apoptotic vesicles loaded with APOA2 prepared in Example 1-2, the reduction in body fat and liver fat was significantly better than that after the injection of the apoptotic vesicles or the apoptotic vesicle mixture prepared in Comparative Example 1-2, indicating that the apoptotic vesicles loaded with APOA2 prepared in Example 1-2 have better therapeutic effects on NAFLD than the apoptotic vesicles or the apoptotic vesicle mixture prepared in Comparative Example 1-2.

[0100] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these changes and variations. The above embodiments or implementations are merely illustrative of the present invention, and the present invention may also be implemented in other specific ways or other specific forms without departing from the gist or essential features of the present invention. Therefore, the described implementations should be considered illustrative rather than restrictive in any respect. The scope of the present invention should be described by the appended claims, and any changes equivalent to the intent and scope of the claims should also be included within the scope of the present invention.

Claims

1. A platelet apoptotic vesicle loaded with APOA2, characterized in that: The platelet apoptotic vesicles loaded with APOA2 are loaded with protein APOA2 in the apoptotic vesicles derived from platelets.

2. The platelet apoptotic vesicle loaded with APOA2 according to claim 1, characterized in that: The mass ratio of the platelet-derived apoptotic vesicles to the protein APOA2 for preparing the platelet apoptotic vesicles loaded with APOA2 is 1:5-1:

10.

3. The method for preparing platelet apoptotic vesicles loaded with APOA2 according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: (1) preparing the platelet-derived apoptotic vesicles; (2) The platelet-derived apoptotic vesicles are mixed with the protein APOA2 and then treated so that the protein APOA2 is loaded into the platelet-derived apoptotic vesicles, and the platelet apoptotic vesicles loaded with APOA2 are separated and obtained.

4. The preparation method according to claim 3, characterized in that: The preparation process of step (1) comprises the following steps: (11) Separating and purifying platelets from blood samples; (12) Induce platelet apoptosis; (13) Centrifugation and washing to obtain the platelet-derived apoptotic vesicles.

5. The preparation method according to claim 4, characterized in that: In step (12), the platelets are resuspended in culture medium, and then staurosporine is added to induce platelet apoptosis.

6. The preparation method according to claim 5, characterized in that: Step (13) comprises the following steps: (131) The culture medium was centrifuged at 700-900 g for 8-15 min at 4°C to remove cell debris in the culture medium and obtain a centrifugal supernatant; (132) Centrifuging the centrifuged supernatant at 15,000-18,000 g for 25-35 minutes at 4° C., taking the precipitate to obtain the crude platelet-derived apoptotic vesicles; (133) The crude platelet-derived apoptotic vesicles were washed with sterile PBS and then centrifuged at 15,000-18,000 g for 25-35 minutes at 4°C to obtain the pure platelet-derived apoptotic vesicles.

7. The preparation method according to claim 3, characterized in that: In step (2), the treatment is ultrasonic treatment.

8. The preparation method according to claim 3, characterized in that: In step (2), the platelet apoptotic vesicles loaded with APOA2 are obtained by centrifugation under the following conditions: temperature 4° C., centrifugation at 15000-18000 g for 25-35 minutes.

9. Use of the platelet apoptotic vesicles loaded with APOA2 according to claim 1 or 2 for preparing a medicament for inhibiting adipogenic differentiation of mesenchymal stem cells.

10. The use according to claim 9, characterized in that: The platelet apoptotic vesicles loaded with APOA2 are further used to prepare a medicament for preventing, treating or alleviating non-alcoholic fatty liver disease.

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