Thy1-coated mesenchymal stem cell 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 a Thy1-coated mesenchymal stem cell apoptosis vesicle as well as a preparation method and application thereof. According to the Thy1-coated mesenchymal stem cell apoptosis vesicle, the surface of a mesenchymal stem cell-derived apoptosis vesicle is coated with Thy1 membrane glycoprotein. According to the Thy1-coated mesenchymal stem cell apoptosis vesicle and the preparation method thereof disclosed by the invention, the prepared Thy1-coated mesenchymal stem cell apoptosis vesicle can better promote osteogenic differentiation of mesenchymal stem cells, so that bone loss can be safely and efficiently prevented, and the Thy1-coated mesenchymal stem cell apoptosis vesicle can be better used for treating osteoporosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biological tissue engineering, and relates to Thy1-coated mesenchymal stem cell apoptotic vesicles, and a preparation method and application thereof. Background Art

[0002] Osteoporosis is a common bone disease, which is divided into primary (unknown cause) and secondary (traceable cause) osteoporosis, which is characterized by quantitative and qualitative destruction of bone tissue, resulting in decreased bone density, thereby increasing the risk of fractures. Among women and men over 50 years old worldwide, 1 / 2 and 1 / 4, respectively, will suffer fractures due to osteoporosis in their remaining lives. The impaired osteogenic differentiation ability of mesenchymal stem cells (MSCs) will directly lead to weakened bone formation, which is an important cause of bone loss and an important pathogenesis of osteoporosis. Therefore, in patients with osteoporosis, how to activate the osteogenic signaling pathway of MSCs and efficiently induce their osteogenic differentiation is the key to bone tissue engineering technology, and it is also the key to preventing and treating bone loss-related diseases and maintaining bone tissue homeostasis.

[0003] Apoptotic vesicles (apoVs) are a heterogeneous subpopulation of extracellular vesicles (EVs) produced during apoptosis, with diameters ranging from 100nm to 1000nm, encapsulating a large amount of DNA, RNA, proteins, and even complete organelles. Previous studies have shown that most cells undergoing apoptosis are cleared by phagocytes in the form of apoVs; however, increasing evidence shows that apoVs-mediated transfer of biomolecules can occur between different types of cells, which is closely related to the maintenance of stem cell stemness and tissue regeneration. Currently, more and more studies are focusing on the application of apoVs in the diagnosis and treatment of diseases, including: diagnosis of gastrointestinal diseases, treatment of atherosclerosis, hemophilia, liver fibrosis and bacterial infections, bone, skin, hair and other tissue regeneration engineering, and immunotherapy. Currently, acellular bone tissue engineering technology with apoVs as the core is gaining attention. Studies have shown that apoVs derived from young MSCs can regulate the apoptosis balance and differentiation direction of host MSCs by transporting key signaling molecules, thereby significantly improving bone loss caused by ovarian castration, aging, trauma, etc. At the same time, studies have also shown that the use of apoVs derived from young donors can combat the aging of the body. Although studies have confirmed that apoVs are involved in regulating aging and bone metabolism, they still focus on the characteristics and therapeutic potential of apoVs derived from young cells. The changes in the characteristics of apoVs derived from MSCs during the natural aging of the body are still unclear, and the impact on the efficacy of bone diseases has not been evaluated.

[0004] It is known that the biological properties of EVs depend largely on the characteristics of donor cells, and the biological functions of EVs derived from senescent cells show similar trends of change as those of aging cells. A study showed that EVs from aged bone matrix are beneficial to MSCs adipogenesis and enhance vascular smooth muscle cell calcification. In vivo injection therapy can promote bone fat metabolism imbalance in mice and aggravate vascular calcification. As a subpopulation of EVs, the biological properties of apoVs may also be affected by donor cells. Therefore, indiscriminate use of apoVs in preclinical and / or clinical experiments may lead to unstable results and undermine the development of this emerging field. Exploring the biological properties and functions of apoVs derived from aged donors is conducive to improving the understanding of how apoVs participate in individual aging and bone metabolism, which is crucial for the engineering modification and clinical translation application of apoVs.

[0005] Thy1 is a 25-37 kDa glycosylphosphatidylinositol (GPI)-anchored cell surface protein. Thy1 plays an important role in cell migration, adhesion, differentiation, transdifferentiation, apoptosis, mechanical transduction and cell division, and its function has been widely studied in the fields of immunology, neurobiology, cancer, stem cell biology, tissue remodeling and aging. Summary of the invention

[0006] The primary purpose of the present invention is to provide a Thy1-coated mesenchymal stem cell apoptotic vesicle, which can better promote the osteogenic differentiation of mesenchymal stem cells, thereby safely and efficiently preventing bone loss, and thus can be better used for the treatment of osteoporosis.

[0007] To achieve this object, in a basic embodiment, the present invention provides a Thy1-coated mesenchymal stem cell apoptotic vesicle, wherein the Thy1-coated mesenchymal stem cell apoptotic vesicle is coated with Thy1 membrane glycoprotein on the surface of the apoptotic vesicle derived from mesenchymal stem cells.

[0008] In a preferred embodiment, the present invention provides a Thy1-coated mesenchymal stem cell apoptotic vesicle, wherein the mass ratio of the mesenchymal stem cell-derived apoptotic vesicle to the Thy1 membrane glycoprotein used to prepare the Thy1-coated mesenchymal stem cell apoptotic vesicle is 1:4-1:6.

[0009] In a preferred embodiment, the present invention provides a Thy1-coated mesenchymal stem cell apoptotic vesicle, wherein the Thy1-coated mesenchymal stem cell apoptotic vesicle is spherical, has a double-layer lipid membrane structure, and has a particle size of 100-300 nm (average particle size 120-180 nm).

[0010] The second object of the present invention is to provide a method for preparing the Thy1-coated mesenchymal stem cell apoptotic vesicles as described above, so as to better prepare the Thy1-coated mesenchymal stem cell apoptotic vesicles as described above. The prepared Thy1-coated mesenchymal stem cell apoptotic vesicles can better promote the osteogenic differentiation of mesenchymal stem cells, thereby safely and efficiently preventing bone loss, and thus can be better used for the treatment of osteoporosis.

[0011] To achieve this object, in a basic embodiment, the present invention provides a method for preparing the Thy1-coated mesenchymal stem cell apoptotic vesicles as described above, the preparation method comprising the following steps:

[0012] (1) preparing the apoptotic vesicles derived from mesenchymal stem cells;

[0013] (2) mixing the mesenchymal stem cell-derived apoptotic vesicles with the Thy1 membrane glycoprotein and incubating them for a certain period of time so that one or more layers of the Thy1 membrane glycoprotein are adsorbed on the surface of the mesenchymal stem cell-derived apoptotic vesicles, and isolating the Thy1-coated mesenchymal stem cell apoptotic vesicles.

[0014] In a preferred embodiment, the present invention provides a method for preparing the Thy1-coated mesenchymal stem cell apoptotic vesicles as described above, wherein the preparation process of step (1) comprises the following steps:

[0015] (11) Isolation of bone marrow mesenchymal stem cells from animal tissues;

[0016] (12) Induce apoptosis of bone marrow mesenchymal stem cells;

[0017] (13) Centrifugation and washing to obtain the apoptotic vesicles derived from mesenchymal stem cells.

[0018] In a preferred embodiment, the present invention provides a method for preparing Thy1-coated mesenchymal stem cell apoptotic vesicles as described above, wherein in step (12), bone marrow mesenchymal stem cells are resuspended in culture medium, and then staurosporine (STS, preferably at a concentration of 500 nM) is added to induce cell apoptosis.

[0019] In a preferred embodiment, the present invention provides a method for preparing Thy1-coated mesenchymal stem cell apoptotic vesicles 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.

[0020] In a preferred embodiment, the present invention provides a method for preparing Thy1-coated mesenchymal stem cell apoptotic vesicles as described above, wherein step (13) comprises the following steps:

[0021] (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;

[0022] (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 mesenchymal stem cell-derived apoptotic vesicles;

[0023] (133) The crude mesenchymal stem cell-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 mesenchymal stem cell-derived apoptotic vesicles.

[0024] In a preferred embodiment, the present invention provides a method for preparing Thy1-coated mesenchymal stem cell apoptotic vesicles as described above, wherein in step (2), the incubation temperature is 35-40° C. and the incubation time is 1-2 hours.

[0025] In a preferred embodiment, the present invention provides a method for preparing Thy1-coated mesenchymal stem cell apoptotic vesicles as described above, wherein in step (2), the Thy1-coated mesenchymal stem cell apoptotic vesicles are obtained by centrifugation, and the centrifugation conditions are: temperature 4°C, centrifugation at 15000-18000g for 25-35 minutes.

[0026] In a preferred embodiment, the present invention provides a method for preparing Thy1-coated mesenchymal stem cell apoptotic vesicles as described above, wherein step (2) comprises the following steps:

[0027] (21) resuspending the apoptotic vesicles derived from mesenchymal stem cells with sterile PBS (preferably making the final concentration of the apoptotic vesicles derived from mesenchymal stem cells to be 50-200 ng / ml, more preferably making the final concentration to be 100 ng / ml), and then adding a Thy1 membrane glycoprotein solution resuspended in 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;

[0028] (22) incubating the obtained mixture at 37° C. for 1-2 hours to form a protein corona on the surface of the mesenchymal stem cell-derived apoptotic vesicles;

[0029] (23) The Thy1-coated mesenchymal stem cell apoptotic vesicles are obtained by centrifugation.

[0030] The third object of the present invention is to provide the use of the Thy1-coated mesenchymal stem cell apoptotic vesicles as described above for preparing a medicament for promoting the osteogenic differentiation of mesenchymal stem cells, so as to better promote the osteogenic differentiation of mesenchymal stem cells, thereby safely and efficiently preventing bone loss, and thus being better used for the treatment of osteoporosis.

[0031] To achieve this object, in a basic embodiment, the present invention provides use of the Thy1-coated mesenchymal stem cell apoptotic vesicles as described above for preparing a medicament for promoting osteogenic differentiation of mesenchymal stem cells.

[0032] In a preferred embodiment, the present invention provides the use of the Thy1-coated mesenchymal stem cell apoptotic vesicles as described above for preparing a medicament for promoting osteogenic differentiation of mesenchymal stem cells, wherein the Thy1-coated mesenchymal stem cell apoptotic vesicles are further used to prepare a medicament for preventing, treating or alleviating osteoporosis.

[0033] The beneficial effect of the present invention is that by utilizing the Thy1-coated mesenchymal stem cell apoptotic vesicles and the preparation method thereof of the present invention, the prepared Thy1-coated mesenchymal stem cell apoptotic vesicles can better promote the osteogenic differentiation of mesenchymal stem cells, thereby safely and efficiently preventing bone loss, and thus can be better used for the treatment of osteoporosis.

[0034] The preparation method of the invention is simple in extracting apoptotic vesicles of mesenchymal stem cells, has low preparation cost, high yield and can be quickly detected.

[0035] The Thy1-coated mesenchymal stem cell apoptotic vesicles provided by the present invention have significantly improved abilities of promoting osteogenic differentiation of mesenchymal stem cells and inhibiting bone loss compared with single mesenchymal stem cell apoptotic vesicles, and have broad clinical application prospects in the treatment of senile osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 These are the detection results of the Thy1-coated apoptotic vesicles prepared in Example 1-2, wherein a is the detection result of cryo-transmission electron microscopy, b is the detection result of nanoparticle tracking analysis, c is the zeta potential detection result, and d is the flow cytometry detection result of Annexin V positivity.

[0037] Figure 2The results of the Thy1-coated apoptotic vesicles prepared in Example 1-2, the apoptotic vesicles prepared in Comparative Example 1-2, or the apoptotic vesicle mixture inhibiting the adipogenic differentiation of MSCs in vitro are shown in Figure 1, wherein a is the results of alizarin red (ARS) and alkaline phosphatase (ALP) staining, b is the results of PCR of osteogenic-related genes, c is the results of Oil Red O staining, and d is the results of PCR 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<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0038] Figure 3 The results of the Thy1-coated apoptotic vesicles prepared in Example 1-2, the apoptotic vesicles prepared in Comparative Example 1-2, or the apoptotic vesicle mixture inhibiting bone loss and fat accumulation in mice, i.e., treating aging-related bone-fat imbalance, are shown in Figure 1, wherein a is the micro-CT scan result of the femur, b is the change of bone density (BMD) and bone volume fraction (BV / TV), c and d are the H&E and Masson staining results of the femoral metaphysis tissue, e is the abdominal fat volume, and f is the H&E staining result of the liver tissue and the Oil Red O staining result. The data were compared among multiple groups by one-way ANOVA and Tukey's post hoc test, ns, no significant difference, ****p<0.0001. DETAILED DESCRIPTION

[0039] The specific embodiments of the present invention are further described below through examples and drawings. Comparative Example 1: Preparation of apoptotic vesicles of bone marrow mesenchymal stem cells from 1-week-old neonatal mice (1w apoVs)

[0040] Bone marrow mesenchymal stem cells were extracted from 1-week-old male C57B6J suckling mice and cultured in vitro (for specific methods, see the literature DOI:10.15252 / embr.202152576), and 500 nM STS was added to the bone marrow mesenchymal stem cells resuspended in proliferation medium (MEM-α medium containing 10% FBS and 1% penicillin-streptomycin double antibody) to induce apoptosis, and apoptotic vesicles were obtained by centrifugation. The specific method is as follows:

[0041] a) centrifuging the culture solution at 800 g for 10 minutes at 4°C to remove cell debris in the culture solution and obtain a centrifugal supernatant;

[0042] b) centrifuging the centrifuged supernatant at 17,000 g for 30 minutes at 4° C., taking the precipitate to obtain the crude apoptotic vesicles derived from mesenchymal stem cells;

[0043] c) The crude mesenchymal stem cell-derived apoptotic vesicles were washed with sterile PBS, and then centrifuged at 17,000 g for 30 minutes at 4° C. to obtain the pure mesenchymal stem cell-derived apoptotic vesicles.

[0044] Example 1: Preparation of Thy1-coated apoptotic vesicles (I)

[0045] 1) resuspending the apoptotic vesicles prepared in Comparative Example 1 with an appropriate amount of sterile PBS to make the final concentration of the apoptotic vesicles 100 ng / ml;

[0046] 2) Add Thy1 protein solution (concentration of 0.5 mg / ml) resuspended in PBS to the apoptotic vesicles, so that the mass ratio of apoptotic vesicles to Thy1 protein is 1:4;

[0047] 3) Incubate the resulting mixture at 37°C for 1-2 hours to form a protein corona on the surface of the apoptotic vesicles;

[0048] 4) Centrifugation (4°C, 17000 g for 30 minutes) to obtain apoptotic vesicles of mesenchymal stem cells coated with Thy1 membrane glycoprotein.

[0049] Example 2: Preparation of Thy1-coated apoptotic vesicles (II)

[0050] The mass ratio of apoptotic vesicles to Thy1 protein in step 2) was changed to 1:6, and the rest was the same as in Example 1. Comparative Example 2: Preparation of a mixture of Thy1 and apoptotic vesicles of bone marrow mesenchymal stem cells

[0051] Step 3) and step 4) in Example 1 were removed, that is, Thy1 and the apoptotic vesicles of bone marrow mesenchymal stem cells were simply mixed without incubation and subsequent centrifugation.

[0052] Example 3: Characterization of apoptotic vesicles The Thy1-coated apoptotic vesicles prepared in Example 1-2 were tested for their morphology, particle size, zeta potential, and Annexin V positivity by cryo-transmission electron microscopy, nanoparticle tracking analysis, potential detection, and flow cytometry. For some specific methods, see Example 2 of CN 114306384A.

[0053] like Figure 1 As shown in a, the extracellular vesicles prepared in Example 1 and Example 2 are spherical and have a lipid bilayer membrane structure.

[0054] like Figure 1 As shown in b, the particle size distribution of the apoptotic vesicles prepared in Example 1 and Example 2 is mainly concentrated in the range of 100-300 nm.

[0055] like Figure 1As shown in Figure c, the peak zeta potentials of the apoptotic vesicles prepared in Example 1 and Example 2 were both between -20 and -30 mV.

[0056] like Figure 1 As shown in Figure d, the positive rate of phosphatidylserine externalization of apoptotic vesicles prepared in Example 1 and Example 2 is as high as 70-80%, proving that they are derived from cell apoptosis.

[0057] Example 4: Effects of Thy1-coated apoptotic vesicles on osteogenic and adipogenic differentiation of MSCs in vitro

[0058] The effects of the Thy1-coated apoptotic vesicles prepared in Example 1-2, the apoptotic vesicles prepared in Comparative Example 1-2, or the apoptotic vesicle mixture on the osteogenic and adipogenic differentiation of MSCs in vitro were studied respectively. The specific methods are as follows:

[0059] Bone marrow mesenchymal stem cells were cultured under the following three culture conditions:

[0060] 1) Proliferation medium (PM): DMEM medium containing 10% FBS and 1% penicillin-streptomycin.

[0061] 2) Osteoinduction medium (OM): DMEM medium containing 10% FBS, 1% penicillin-streptomycin, 10 mM β-sodium glycerophosphate, 0.2 mM L-ascorbic acid and 100 nM dexamethasone.

[0062] 3) Extracellular vesicles (OM+apoVs: 200 ng / ml) were added to the osteogenic induction medium and cultured in an incubator (containing 95% air and 5% CO2 by volume, 100% humidity, and a constant temperature of 37°C).

[0063] After 10 days of osteogenic induction, the osteogenic differentiation of cells was examined by Alizarin red staining and alkaline phosphatase staining.

[0064] Alizarin red s (ARS) staining:

[0065] 1) Prepare the dye solution by weighing 1 g of powder (SIGMA, A5533-25G) and dissolving it in 100 ml of MilliQ water.

[0066] 2) Aspirate the culture medium, rinse three times with PBS, fix the cells with 95% ice-cold ethanol for 30 min, discard the ice-cold ethanol, rinse three times with MilliQ water, dry, add dye, and stain the mineralized nodules.

[0067] 3) After staining is completed, remove the dye solution, rinse with distilled water, and take photos under a microscope.

[0068] Alkaline phosphatase (ALP) staining:

[0069] 1) According to the instructions of the ALP staining kit, the culture medium was aspirated, and the cells were rinsed three times with PBS (be careful to prevent cell stripping), fixed with 95% ethanol aqueous solution at room temperature for 10 minutes, and rinsed three times with PBS.

[0070] 2) Mix BCIP, NBT and color developing solution in the kit at a volume ratio of 1:1:38, add to the fixed cells, place at room temperature away from light, and stain for 5-30 minutes until the color is obvious.

[0071] 3) Aspirate the color development solution and rinse with PBS three times to terminate the color development reaction. Scan or take photos under a microscope.

[0072] The effect of the apoptotic vesicles prepared in Example 1, Comparative Example 1 and Comparative Example 2 on the adipogenic differentiation of MSCs in vitro was studied, and the specific method was as follows:

[0073] MSCs were cultured under the following three culture conditions:

[0074] 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).

[0075] 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).

[0076] 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.

[0077] Oil Red O staining and quantification:

[0078] 1) Stain MSCs with Oil Red O working solution and observe the staining under a microscope;

[0079] 2) For quantitative analysis, the stained MSCs were immersed in 100% isopropanol for 10 minutes and the absorbance was measured at 500 nm.

[0080] like Figure 2 As shown, compared with osteogenic induction culture (OM) cells, the Thy1-coated apoptotic vesicles prepared in Example 1-2, the apoptotic vesicles prepared in Comparative Example 1-2, or the apoptotic vesicle mixture were added during OM culture, respectively. After 7-14 days of osteogenic induction, the alkaline phosphatase activity and the accumulation of mineralized nodules of MSCs were significantly increased; and after adding the Thy1-coated apoptotic vesicles prepared in Example 1-2, the in vitro osteogenic differentiation of MSCs was significantly higher than that of the apoptotic vesicles or the apoptotic vesicle mixture prepared in Comparative Example 1-2, indicating that the Thy1-coated apoptotic vesicles prepared in Example 1-2 have a significantly stronger effect on promoting osteogenesis than the apoptotic vesicles or the apoptotic vesicle mixture prepared in Comparative Example 1-2. Compared with adipogenic induced culture (AM) cells, the Thy1-coated apoptotic vesicles prepared in Example 1-2, the apoptotic vesicles prepared in Comparative Example 1-2, or a mixture of apoptotic vesicles were added to the AM culture respectively. After 14 days of adipogenic induction, the accumulation of MSCs lipid droplets was significantly reduced; and after adding the Thy1-coated apoptotic vesicles prepared in Example 1-2, the adipogenic differentiation of MSCs in vitro was significantly lower than that after adding the apoptotic vesicles prepared in Comparative Example 1-2 or a mixture of apoptotic vesicles, indicating that the adipogenic inhibitory effect of the Thy1-coated apoptotic vesicles prepared in Example 1-2 is significantly stronger than that of the apoptotic vesicles or a mixture of apoptotic vesicles prepared in Comparative Example 1-2.

[0081] Example 5: Therapeutic effect of Thy1-coated apoptotic vesicles on age-related bone-fat imbalance

[0082] The Thy1-coated 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 age-related bone fat imbalance in mice, and the specific methods are as follows:

[0083] (1) Tail vein injection

[0084] Male 18-month-old C57b6J mice (purchased from the Animal Department of Peking University Health Science Center) were injected with drugs via tail vein (PBS control, Thy1-coated apoptotic vesicles prepared in Example 1-2, apoptotic vesicles or apoptotic vesicle mixture prepared in Comparative Example 1-2, 5 groups in total, 3 mice in each group), and the weekly dosage for each mouse was 1 mg / kg. After 8 consecutive weeks of injection, the mice were killed by cervical dislocation and inhalation, and the main organs (heart, lung, spleen, liver, kidney) and femurs were taken and fixed in neutral formalin solution for 48 hours.

[0085] (2) Decalcification and sectioning

[0086] After micro-CT scanning, the femur tissue was placed in a 10% EDTA solution (pH = 7.4) and placed on a 37°C shaker. The solution was changed every other day. The decalcification lasted from one week to one month. When the needle could pierce the bone, the decalcification was completed. After decalcification, wax immersion, and paraffin embedding, a paraffin slicer was used to cut slices with a thickness of 5 μm. Afterwards, the slices were stained with hematoxylin-eosin (H&E) and Masson's trichrome.

[0087] (3) H&E staining

[0088] 1) Slices were washed with xylene solution twice, 5-10 minutes each time;

[0089] 2) Slices were washed with anhydrous ethanol twice, 3-5 minutes each time;

[0090] 3) Rinse the slices with 95% alcohol, twice, 5 minutes each time;

[0091] 4) The slices were washed with 90% alcohol, 80% alcohol, and 70% alcohol, once each for 2-3 minutes (hydration);

[0092] 5) Wash the slices with distilled water for 3 minutes;

[0093] 6) Stain the sections with hematoxylin for 10-15 minutes;

[0094] Color separation: insert the slice into 0.5-1% hydrochloric acid alcohol (70% alcohol) for a few seconds to more than ten seconds;

[0095] 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;

[0096] 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);

[0097] 8) Sections were washed with xylene twice, 3-5 minutes each time;

[0098] 9) The sections were sealed with neutral gum and images were collected and analyzed under a microscope.

[0099] (4) Masson trichrome staining

[0100] 1) Repeat the above H&E staining until the "blueing" step;

[0101] 2) Rinse the sections with distilled water;

[0102] 3) The sections were stained with Ponceau acid fuchsin solution for 5-10 minutes and quickly rinsed with distilled water;

[0103] 4) Treat the slices with phosphomolybdic acid aqueous solution for 3-5 minutes;

[0104] 5) The sections were stained with aniline blue solution for 5 minutes;

[0105] 6) Treat the sections with 1% glacial acetic acid for 1 minute (differentiation);

[0106] 7) Dehydration, sealing, and image acquisition and analysis under a microscope.

[0107] (5) Oil Red O staining

[0108] 1) Place the slices in Oil Red O working solution for staining, the staining time is usually 2-5 minutes;

[0109] 2) Differentiation and rinsing: After staining, use 60% isopropanol for differentiation to remove excess stain;

[0110] 3) After differentiation, rinse the sections thoroughly with distilled water to remove residual stain;

[0111] 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;

[0112] 5) After counterstaining, use hydrochloric acid alcohol for differentiation, usually for 1-5 seconds to remove excess hematoxylin;

[0113] 6) Rinse the sections with running water to turn the cell nuclei back to blue.

[0114] 7) Finally, use glycerol gelatin or other aqueous mounting media for sealing, and collect and analyze images under a microscope.

[0115] like Figure 3As shown, the aged mice had obvious bone loss. After the tail vein was injected with the Thy1-coated apoptotic vesicles prepared in Example 1-2, the apoptotic vesicles prepared in Comparative Example 1-2, or the apoptotic vesicle mixture, the bone density (BMD) was significantly increased, and the bone volume fraction (BV / TV) was significantly increased. Moreover, after the injection of the Thy1-coated apoptotic vesicles prepared in Example 1-2, the bone loss 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 Thy1-coated apoptotic vesicles prepared in Example 1-2 have better therapeutic efficacy on senile osteoporosis than the apoptotic vesicles or the apoptotic vesicle mixture prepared in Comparative Example 1-2. The accumulation of body fat and liver fat in aged mice increased significantly. After the tail vein was injected with the Thy1-coated apoptotic vesicles prepared in Example 1-2, the apoptotic vesicles prepared in Comparative Example 1-2, or the apoptotic vesicle mixture, the accumulation of abdominal fat was significantly reduced, and fatty liver was significantly improved. Moreover, after the injection of the Thy1-coated apoptotic vesicles prepared in Example 1-2, the reduction of body fat and liver fat was significantly better than that after the injection of the apoptotic vesicles prepared in Comparative Example 1-2 or the apoptotic vesicle mixture, indicating that the Thy1-coated apoptotic vesicles prepared in Example 1-2 have better therapeutic efficacy on senile obesity than the apoptotic vesicles prepared in Comparative Example 1-2 or the apoptotic vesicle mixture.

[0116] 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 Thy1-coated mesenchymal stem cell apoptotic vesicle, characterized in that: The Thy1-coated mesenchymal stem cell apoptotic vesicles are coated with Thy1 membrane glycoprotein on the surface of the apoptotic vesicles derived from mesenchymal stem cells.

2. The Thy1-coated mesenchymal stem cell apoptotic vesicle according to claim 1, characterized in that: The mass ratio of the apoptotic vesicle derived from the mesenchymal stem cells to the Thy1 membrane glycoprotein for preparing the Thy1-coated mesenchymal stem cell apoptotic vesicle is 1:4-1:

6.

3. The method for preparing Thy1-coated mesenchymal stem cell apoptotic vesicles according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: (1) preparing the apoptotic vesicles derived from mesenchymal stem cells; (2) mixing the mesenchymal stem cell-derived apoptotic vesicles with the Thy1 membrane glycoprotein and incubating them for a certain period of time so that one or more layers of the Thy1 membrane glycoprotein are adsorbed on the surface of the mesenchymal stem cell-derived apoptotic vesicles, and isolating the Thy1-coated mesenchymal stem cell apoptotic vesicles.

4. The preparation method according to claim 3, characterized in that: The preparation process of step (1) comprises the following steps: (11) Isolation of bone marrow mesenchymal stem cells from animal tissues; (12) Induce apoptosis of bone marrow mesenchymal stem cells; (13) Centrifugation and washing to obtain the apoptotic vesicles derived from mesenchymal stem cells.

5. The preparation method according to claim 4, characterized in that: In step (12), the bone marrow mesenchymal stem cells are resuspended in culture medium, and then staurosporine is added to induce cell 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 mesenchymal stem cell-derived apoptotic vesicles; (133) The crude mesenchymal stem cell-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 mesenchymal stem cell-derived apoptotic vesicles.

7. The preparation method according to claim 3, characterized in that: In step (2), the incubation temperature is 35-40° C. and the incubation time is 1-2 hours.

8. The preparation method according to claim 3, characterized in that: In step (2), the Thy1-coated mesenchymal stem cell apoptotic vesicles are obtained by centrifugation under the following conditions: temperature 4° C., centrifugation at 15000-18000 g for 25-35 minutes. 9 . Use of the Thy1-coated mesenchymal stem cell apoptotic vesicles according to claim 1 or 2 for preparing a medicament for promoting osteogenic differentiation of mesenchymal stem cells.

10. The use according to claim 9, characterized in that: The Thy1-coated mesenchymal stem cell apoptotic vesicles are further used to prepare a medicament for preventing, treating or alleviating osteoporosis.

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