Exosome-like nanovesicles of Acanthopanax cortex osteogenic induction medium, its preparation method, osteogenic induction and differentiation method, and uses

Abstract

An osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex, a preparation method of the osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex, an osteogenic induction and differentiation method, the use of exosome-like nanovesicles of Acanthopanax cortex in the preparation of an osteogenic induction medium, and the use of the osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex in the preparation of an osteogenic induction drug. In the present invention, an exosome-like nanovesicle solution of Acanthopanax cortex is used as an additive component of the osteogenic induction medium, which can greatly improve the osteogenic differentiation efficiency of mesenchymal stem cells, has a significant osteogenic differentiation effect, and the exosome-like nanovesicles of Acanthopanax cortex have good cell compatibility. The exosome-like nanovesicles of Acanthopanax cortex can also be effectively taken up by human bone marrow mesenchymal stem cells, thereby enhancing their osteogenic induction effect. The osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex can be prepared into an osteogenic induction drug for preventing and treating osteoporosis, bone defect, nonunion or delayed bone healing.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells in biomedical technology, and particularly relates to a cortex acanthopanacis exosome-like nanovesicle osteogenic induction medium, a preparation method of the cortex acanthopanacis exosome-like nanovesicle osteogenic induction medium, an osteogenic induction and differentiation method, the use of cortex acanthopanacis exosome-like nanovesicles in the preparation of an osteogenic induction medium, and the use of the cortex acanthopanacis exosome-like nanovesicle osteogenic induction medium in the preparation of an osteogenic induction drug. Background Art

[0002] Osteoporosis is a major health problem faced by the middle-aged and elderly population. Epidemiological data shows that the prevalence of osteoporosis in people over 40 years old in China is as high as 25.6%, and it climbs to 32.0% in people over 65 years old. Osteoporosis is a systemic skeletal disease caused by an imbalance between osteoclasts and osteoblasts, and its main characteristics are bone mass reduction and decreased mineral density. This pathological state leads to systemic bone loss and a significant increase in bone fragility. The decrease in bone density not only exacerbates the deterioration of the bone microstructure but also promotes the degradation of matrix proteins, thus significantly increasing the risk of fractures. Currently, the treatment options for osteoporosis include bisphosphonates, hormone replacement therapy (HRT), selective estrogen receptor modulators (SERM), and denosumab, but the efficacy of these methods is limited, and long-term use can cause serious side effects.

[0003] Human bone marrow mesenchymal stem cells (hBM-MSCs) have attracted much attention due to their multi-directional differentiation potential and can differentiate into various tissue cells such as bone, cartilage, muscle, tendon, and ligament. Currently, studies at home and abroad have widely confirmed the osteogenic differentiation ability of hBM-MSCs, making them ideal cells in the bone tissue regeneration and repair project. This discovery provides new strategies and ideas for the treatment of refractory bone diseases such as bone defects, non-unions, and delayed bone healing.

[0004] Currently, the formulations of osteogenic induction media are diverse, and common components include basal media (such as α-MEM, low-glucose DMEM, etc.), fetal bovine serum, antibiotics (penicillin and streptomycin), dexamethasone, ascorbic acid, and sodium β-glycerophosphate, etc., but the specific component contents vary. However, there are still many problems in the osteogenic induction process, such as low induction efficiency, unstable effects, and long induction time, etc. These problems limit the application of hBM-MSCs in the clinical prevention and treatment of osteoporosis. There is an urgent need to study and develop a safe and efficient osteogenic differentiation method to promote the research on the osteogenic differentiation mechanism of hBM-MSCs and to promote its clinical application in the prevention and treatment of diseases such as osteoporosis.

[0005] Therefore, in view of the deficiencies of the prior art, it is highly necessary to provide an osteogenic induction medium of exosome-like nanovesicles of Acanthopanax gracilistylus W. W. Smith, a preparation method of the osteogenic induction medium of exosome-like nanovesicles of Acanthopanax gracilistylus W. W. Smith, an osteogenic induction and differentiation method, the use of exosome-like nanovesicles of Acanthopanax gracilistylus W. W. Smith in the preparation of an osteogenic induction medium, and the use of the osteogenic induction medium of exosome-like nanovesicles of Acanthopanax gracilistylus W. W. Smith in the preparation of an osteogenic induction drug to solve the deficiencies of the prior art. Summary of the Invention

[0006] One of the objectives of the present invention is to provide an osteogenic induction medium of exosome-like nanovesicles of Acanthopanax gracilistylus W. W. Smith, which can avoid the deficiencies of the prior art and improve the osteogenic differentiation effect.

[0007] The above objective of the present invention is achieved by the following technical measures:

[0008] Provide an osteogenic induction medium of exosome-like nanovesicles of Acanthopanax gracilistylus W. W. Smith, containing an exosome-like nanovesicle solution of Acanthopanax gracilistylus W. W. Smith, dexamethasone, L-ascorbic acid, β-glycerophosphate sodium, and a complete medium.

[0009] Preferably, the preparation method of the above exosome-like nanovesicle solution of Acanthopanax gracilistylus W. W. Smith is as follows: Soak Acanthopanax gracilistylus W. W. Smith in PBS solution to obtain a soaking solution; filter the soaking solution and perform multiple centrifugations to obtain a supernatant; perform a first resuspension and precipitation operation on the supernatant to obtain a resuspended solution; purify the resuspended solution by sucrose gradient density centrifugation to obtain a purified solution; then perform a dilution and precipitation operation on the purified solution with PBS solution to obtain a precipitate; perform a second resuspension and precipitation operation on the precipitate to obtain the exosome-like nanovesicle solution of Acanthopanax gracilistylus W. W. Smith.

[0010] The content of the osteogenic induction medium of exosome-like nanovesicles of Acanthopanax gracilistylus W. W. Smith of the present invention is as follows:

[0011] Exosome-like nanovesicle solution of Acanthopanax gracilistylus W. W. Smith: 1 μg / mL - 40 μg / mL;

[0012] Dexamethasone: 80 nM - 120 nM;

[0013] L-ascorbic acid: 40 μM - 60 μM;

[0014] β-glycerophosphate sodium: 8 mM - 12 mM;

[0015] The complete medium is a low-glucose DMEM medium containing 5%Vol. - 15%Vol. fetal bovine serum and 0.5%Vol. - 2%Vol. penicillin-streptomycin.

[0016] Furthermore, the content of the osteogenic induction medium of exosome-like nanovesicles of Acanthopanax gracilistylus W. W. Smith of the present invention is as follows:

[0017] Acanthopanax Cortex exosome-like nanovesicle solution: 2.5 μg / mL to 5.0 μg / mL;

[0018] Dexamethasone: 100 nM;

[0019] L-Ascorbic acid: 50 μM;

[0020] β-Glycerophosphate disodium: 10 mM;

[0021] The complete medium is a low-glucose DMEM medium containing 10% Vol. fetal bovine serum and 1% Vol. penicillin-streptomycin.

[0022] Preferably, the above Acanthopanax Cortex exosome-like nanovesicle solution is prepared by the following steps:

[0023] S1. Cut the Acanthopanax Cortex into pieces and proceed to S2;

[0024] S2. Immerse the cut Acanthopanax Cortex in PBS solution for 8 h to 12 h to obtain an immersion solution;

[0025] S3. Filter the immersion solution obtained in S2 with medical gauze to obtain a crude filtrate;

[0026] S4. Centrifuge the crude filtrate obtained in S3 at a centrifugal force of 1000 g to 2000 g for 15 min to 30 min to obtain a first supernatant; then filter the first supernatant with a filter mesh with a pore size of 70 μm to 40 μm to obtain a first filtrate;

[0027] S5. Centrifuge the first filtrate obtained in S4 at a centrifugal force of 3000 g to 5000 g for 30 min to 60 min to obtain a second supernatant; then filter the second supernatant with a filter mesh with a pore size of 0.7 μm to 0.4 μm to obtain a second filtrate;

[0028] S6. Centrifuge the second filtrate obtained in S5 at a centrifugal force of 8000 g to 12000 g for 30 min to 60 min to obtain a third supernatant; then filter the third supernatant with a filter mesh with a pore size of 0.7 μm to 0.4 μm to obtain a third filtrate;

[0029] S7. Centrifuge the third filtrate obtained in S6 at a centrifugal force of 80000 g to 120000 g for 50 min to 100 min to obtain a first precipitate, and then resuspend the first precipitate with sterile PBS solution to obtain a resuspended solution;

[0030] S8. Sequentially add sucrose solutions with different mass concentrations from low to high in a centrifuge tube, and then add the resuspended solution obtained in S7. Centrifuge at a centrifugal force of 100,000 g to 150,000 g for 100 min to 150 min, and collect the separation layer between the middle sucrose solution layers to obtain a purified solution;

[0031] S9. Dilute the purified solution obtained in S8 with a sterile PBS solution, centrifuge at a centrifugal force of 100,000 g to 150,000 g for 100 min to 150 min, and collect the precipitate to obtain a second precipitate;

[0032] S10. Resuspend the second precipitate obtained in S9 with a sterile PBS solution, and then filter the resuspended solution through a 0.22 μm filter. The obtained solution is the Acanthopanax bark exosome-like nanovesicle solution.

[0033] Further preferably, the above Acanthopanax bark exosome-like nanovesicle solution is prepared by the following steps:

[0034] S1. Cut the root bark of Acanthopanax bark into small pieces of Acanthopanax bark with a cross-sectional area of 0.2 cm 2 to 1.0 cm 2 , and proceed to S2;

[0035] S2. Immerse the small pieces of Acanthopanax bark obtained in S1 in a PBS solution at a weight-to-volume ratio (g / mL) of 1:10 to 20 for 10 h to obtain an immersion solution;

[0036] S3. Filter the immersion solution obtained in S2 with medical gauze to obtain a crude filtrate;

[0037] S4. Centrifuge the crude filtrate obtained in S3 at a centrifugal force of 1500 g for 30 min to obtain a first supernatant; filter the first supernatant through a filter mesh with a pore size of 70 μm, and then filter the filtered filtrate through a filter mesh with a pore size of 40 μm to finally obtain the first filtrate;

[0038] S5. Centrifuge the first filtrate obtained in S4 at a centrifugal force of 4000 g for 30 min to 30 min to obtain a second supernatant; filter the second supernatant through a filter mesh with a pore size of 0.7 μm to obtain the second filtrate;

[0039] S6. Centrifuge the second filtrate obtained in S5 at a centrifugal force of 10,000 g for 60 min to obtain a third supernatant, and filter the third supernatant through a filter mesh with a pore size of 0.45 μm to obtain a third filtrate;

[0040] S7. Centrifuge the third filtrate obtained in S6 at 4°C under vacuum conditions at a centrifugal force of 100,000 g for 70 min to obtain a first precipitate, and then resuspend the first precipitate with a sterile PBS solution to obtain a resuspended solution.

[0041] S8. Sequentially add 2 mL of sucrose solutions with mass concentrations of 15%, 30%, 45%, and 60% to a centrifuge tube, then add the resuspended solution obtained in S7, and centrifuge at 4°C under vacuum conditions at a centrifugal force of 120,000 g for 120 min. Collect the separation layer between the 30% to 45% sucrose solution layers to obtain a purified solution.

[0042] S9. Dilute the purified solution obtained in S8 with a sterile PBS solution, centrifuge at 4°C under vacuum conditions at a centrifugal force of 120,000 g for 150 min, and collect the precipitate to obtain a second precipitate.

[0043] S10. Resuspend the second precipitate obtained in S9 with a sterile PBS solution, and then filter the resuspended solution using a 0.22 μm filter. The obtained solution is the acanthopanax cortex exosome-like nanovesicle solution.

[0044] The second object of the present invention is to provide a preparation method of an osteogenic induction medium to avoid the deficiencies of the prior art. The preparation method can prepare the osteogenic induction medium through a simple method, and the prepared osteogenic induction medium can improve the osteogenic differentiation effect.

[0045] The above object of the present invention is achieved by the following technical measures:

[0046] Provide a preparation method of the above osteogenic induction medium, which is prepared by the following steps:

[0047] Step (1). Dissolve dexamethasone in absolute ethanol to obtain a dexamethasone solution.

[0048] Dissolve L-ascorbic acid in a PBS buffer solution to obtain an L-ascorbic acid solution.

[0049] Dissolve β-glycerophosphate in a PBS buffer solution to obtain a β-glycerophosphate solution.

[0050] Soak acanthopanax cortex in a PBS solution to obtain a soaking solution; after filtering and centrifuging the soaking solution multiple times, obtain a supernatant; perform a first resuspension and precipitation operation on the supernatant to obtain a resuspended solution; purify the resuspended solution by sucrose density gradient centrifugation to obtain a purified solution; then perform a dilution and precipitation operation on the purified solution with a PBS solution to obtain a precipitate; the precipitate is subjected to a second resuspension and precipitation operation to obtain the acanthopanax cortex exosome-like nanovesicle solution.

[0051] Step (2): Add fetal bovine serum, penicillin, and streptomycin to low-sugar DMEM to obtain a complete medium. Then, add the dexamethasone solution, the L-ascorbic acid solution, the β-glycerophosphate solution, and the exosome-like nanovesicles solution of Acanthopanax cortex to the complete medium and mix evenly to obtain the osteogenic induction medium.

[0052] The third object of the present invention is to avoid the deficiencies of the prior art and provide an osteogenic induction and differentiation method. The osteogenic induction and differentiation method is based on an osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex, and the osteogenic differentiation effect of mesenchymal stem cells (hBM-MSCs) can be improved through this osteogenic induction medium.

[0053] The above object of the present invention is achieved by the following technical measures:

[0054] Provide an osteogenic induction and differentiation method, and culture mesenchymal stem cells with the above-mentioned osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex, and the mesenchymal stem cells are human bone marrow mesenchymal stem cells (hBM-MSCs).

[0055] The fourth object of the present invention is to avoid the deficiencies of the prior art and provide the use of exosome-like nanovesicles of Acanthopanax cortex in the preparation of an osteogenic induction medium. The exosome-like nanovesicles of Acanthopanax cortex promote the osteogenic induction and differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs), and adding exosome-like nanovesicles of Acanthopanax cortex to the osteogenic induction medium has a better osteogenic differentiation effect.

[0056] The above object of the present invention is achieved by the following technical measures:

[0057] Provide the use of exosome-like nanovesicles of Acanthopanax cortex in the preparation of an osteogenic induction medium. The exosome-like nanovesicles of Acanthopanax cortex promote the osteogenic induction and differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs).

[0058] The fifth object of the present invention is to avoid the deficiencies of the prior art and provide the use of an osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex in the preparation of an osteogenic induction drug. The osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex can improve the osteogenic differentiation effect, so it can be used in osteogenic induction drugs.

[0059] The above object of the present invention is achieved by the following technical measures:

[0060] Provide the use of exosome-like nanovesicles of Acanthopanax cortex in the preparation of an osteogenic induction medium. The exosome-like nanovesicles of Acanthopanax cortex promote the osteogenic induction and differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs).

[0061] The fifth object of the present invention is to avoid the deficiencies of the prior art and provide a use of acanthopanax exosome-like nanovesicle osteogenic induction medium in the preparation of osteogenic induction drugs. Since the acanthopanax exosome-like nanovesicle osteogenic induction medium can improve the osteogenic differentiation effect, the osteogenic induction drug prepared from the acanthopanax exosome-like nanovesicle osteogenic induction medium has an osteogenic differentiation effect.

[0062] The above object of the present invention is achieved by the following technical measures:

[0063] Provide a use of acanthopanax exosome-like nanovesicle osteogenic induction medium in the preparation of osteogenic induction drugs.

[0064] The acanthopanax exosome-like nanovesicle osteogenic induction medium promotes the osteogenic induction differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs).

[0065] The use of the acanthopanax exosome-like nanovesicle osteogenic induction medium in the preparation of at least one bone disease such as osteoporosis, bone defect, nonunion or delayed union.

[0066] An acanthopanax exosome-like nanovesicle osteogenic induction medium of the present invention, a preparation method of the acanthopanax exosome-like nanovesicle osteogenic induction medium, an osteogenic induction differentiation method, a use of acanthopanax exosome-like nanovesicles in the preparation of an osteogenic induction medium, and a use of the acanthopanax exosome-like nanovesicle osteogenic induction medium in the preparation of an osteogenic induction drug, wherein the acanthopanax exosome-like nanovesicle osteogenic induction medium contains an acanthopanax exosome-like nanovesicle solution, dexamethasone, L-ascorbic acid, β-glycerophosphate sodium and a complete medium. The preparation method of the acanthopanax exosome-like nanovesicle solution is to soak acanthopanax in a PBS solution to obtain a soaking solution, and the supernatant is obtained after the soaking solution is filtered and centrifuged multiple times; the first resuspension precipitation operation is performed on the supernatant to obtain a resuspended solution; the resuspended solution is purified by sucrose gradient density centrifugation to obtain a purified solution; then the purified solution is diluted and precipitated with a PBS solution to obtain a precipitate; the precipitate is finally obtained by the second resuspension precipitation operation to obtain the acanthopanax exosome-like nanovesicle solution. The present invention uses the acanthopanax exosome-like nanovesicle solution as an additive component of the osteogenic induction medium, which can greatly improve the osteogenic differentiation efficiency of mesenchymal stem cells, has a significant osteogenic differentiation effect, and the acanthopanax exosome-like nanovesicles have no obvious inhibitory effect on the proliferation of human bone marrow mesenchymal stem cells (hBM-MSCs), and have good cell compatibility. The acanthopanax exosome-like nanovesicles can also be effectively taken up by human bone marrow mesenchymal stem cells (hBM-MSCs), thereby enhancing their osteogenic induction effect. The acanthopanax exosome-like nanovesicle osteogenic induction medium can be prepared into an osteogenic induction drug for preventing and treating osteoporosis, bone defect, nonunion or delayed union. Description of the Drawings

[0067] The present invention will be further described with reference to the accompanying drawings, but the content in the drawings does not constitute any limitation to the present invention.

[0068] Figure 1 It is a transmission electron microscope photograph of the exosome-like nanovesicle solution of Acanthopanax cortex.

[0069] Figure 2 It is a particle size range and particle concentration distribution diagram of the exosome-like nanovesicle solution of Acanthopanax cortex.

[0070] Figure 3 It is a chart showing the effect of exosome-like nanovesicle solutions of different concentrations of Acanthopanax cortex on the proliferation activity of human bone marrow mesenchymal stem cells (hBM-MSCs).

[0071] Figure 4 It is a microscope photograph of the endocytosis of the exosome-like nanovesicle solution of Acanthopanax cortex by human bone marrow mesenchymal stem cells (hBM-MSCs).

[0072] Figure 5 It is an alizarin red staining picture of osteogenic induction differentiation of exosome-like nanovesicle solutions of different concentrations of Acanthopanax cortex.

[0073] Figure 6 It is a chart showing the expression of the related gene ALP in the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) detected by real-time fluorescence quantitative PCR method.

[0074] Figure 7 It is a chart showing the expression of the related gene RUNX2 in the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) detected by real-time fluorescence quantitative PCR method.

[0075] Figure 8 It is a chart showing the protein expression of osteogenic differentiation markers of human bone marrow mesenchymal stem cells (hBM-MSCs). Detailed Description of the Invention

[0076] The technical solution of the present invention will be further described in conjunction with the following embodiments. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following embodiments can be obtained from conventional biochemical reagent stores or pharmaceutical enterprises unless otherwise specified. Among them, dexamethasone was purchased from Sigma, USA; L-ascorbic acid was purchased from Sigma, USA. β-glycerophosphate was purchased from Sigma, USA. Ficoll-Histopaque®-1077 was purchased from Sigma, USA. Sterile PBS buffer was purchased from Geno, China. Fetal bovine serum (FBS) was purchased from Gibco, USA. 0.25%-EDTA trypsin was purchased from Gibco, USA. Low-glucose DMEM medium was purchased from Gibco, USA. α-MEM medium was purchased from Gibco, USA. Penicillin / Streptomycin (P / S) was purchased from Gibco, USA. BCA protein concentration assay kit (GENSTAR) was purchased from Kangrun, Beijing, China. Cell Counting Kit-8 (CCK8) kit was purchased from Zeta Life, USA. PKH26 fluorescent labeling dye was purchased from Sigma, USA. Anti-Runx2 antibody (AF5186) was purchased from Qinke Biotech, China. Anti-ALP antibody (R23427) was purchased from Zhengneng, China. Anti-Osteopontin antibody (sc-21742) was purchased from Santa Cruz, USA. Anti-β-actin antibody (Cat No. 66009-1-Ig) was purchased from Sanying, Wuhan, China. NucleoZol RNA extraction reagent was purchased from MACHEREY-NAGEL, Germany. Reverse transcription kit was purchased from Novizan, China. SYBR fluorescence quantitative reagent was purchased from Novizan, China. Human bone marrow mesenchymal stem cells were approved by the Ethics Committee of the Affiliated Hospital of Guangdong Medical University. Under the informed consent of the patients, 10 mL of bone marrow was aseptically collected from the proximal femur of patients undergoing hip replacement surgery. Mononuclear cells were isolated using Ficoll-Histopaque (d = 1.077 g / mL), and the cells were resuspended in α-MEM complete medium (containing 10% Vol. fetal bovine serum and 1% P / S) and passaged in a 37°C, 5% CO2 constant temperature incubator. Acanthopanax cortex is from Heilongjiang.

[0077] Example 1

[0078] An osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex contains exosome-like nanovesicle solution of Acanthopanax cortex, dexamethasone, L-ascorbic acid, β-glycerophosphate and complete medium.

[0079] The contents are as follows:

[0080] Exosome-like nanovesicle solution of Acanthopanax cortex (also abbreviated as AgELNs): 1 μg / mL to 40 μg / mL;

[0081] Dexamethasone: 80 nM to 120 nM;

[0082] L-ascorbic acid: 40 μM to 60 μM;

[0083] β-glycerophosphate: 8 mM to 12 mM.

[0084] The complete medium is a low-glucose DMEM medium containing 5% - 15% Vol. fetal bovine serum and 0.5% - 2% Vol. penicillin-streptomycin (also known as penicillin and streptomycin).

[0085] The preparation method of the exosome-like nanovesicle solution of Acanthopanax cortex in the present invention is as follows: Soak Acanthopanax cortex in PBS solution to obtain a soaking solution; After filtration and multiple centrifugations of the soaking solution, obtain a supernatant; Perform the first resuspension and precipitation operation on the supernatant to obtain a resuspended solution; Purify the resuspended solution by sucrose density gradient centrifugation to obtain a purified solution; Then perform a dilution and precipitation operation on the purified solution with PBS solution to obtain a precipitate; The precipitate is subjected to the second resuspension and precipitation operation to obtain the exosome-like nanovesicle solution of Acanthopanax cortex. The specific preparation is as follows:

[0086] S1. Wash the soil on the surface of fresh traditional Chinese medicine Acanthopanax cortex with double-distilled water, and then cut the root bark part of Acanthopanax cortex into small pieces of Acanthopanax cortex with a cross-sectional area of 0.2 cm 2 ~1.0 cm 2 and enter S2;

[0087] S2. Soak the small pieces of Acanthopanax cortex obtained in S1 in PBS solution at a weight-to-volume ratio (g / mL) of 1:10 - 20 for 8 h - 12 h to obtain a soaking solution;

[0088] S3. Filter the soaking solution obtained in S2 with medical gauze to obtain a crude filtrate;

[0089] S4. Centrifuge the crude filtrate obtained in S3 at a centrifugal force of 1000 g - 2000 g for 15 min - 30 min to obtain a first supernatant; Then filter the first supernatant with a filter mesh with a pore size of 70 μm - 40 μm to obtain a first filtrate;

[0090] S5. Centrifuge the first filtrate obtained in S4 at a centrifugal force of 3000 g - 5000 g for 30 min - 60 min to obtain a second supernatant; Then filter the second supernatant with a filter mesh with a pore size of 0.7 μm - 0.4 μm to obtain a second filtrate;

[0091] S6. Centrifuge the second filtrate obtained in S5 at a centrifugal force of 8000g - 12000g for 30 min - 60 min to obtain a third supernatant; then filter the third supernatant through a filter mesh with a pore size of 0.7μm - 0.4μm to obtain a third filtrate;

[0092] S7. Centrifuge the third filtrate obtained in S6 under vacuum conditions at 4°C and at a centrifugal force of 80000g - 120000g for 50 min - 100 min to obtain a first precipitate, and then resuspend the first precipitate with a sterile PBS solution to obtain a resuspended solution;

[0093] S8. Sequentially add sucrose solutions with different mass concentrations from low to high in a centrifuge tube, then add the resuspended solution obtained in S7, centrifuge under vacuum conditions at 4°C and at a centrifugal force of 100000g - 150000g for 100 min - 150 min, and collect the separation layer between the middle sucrose solution layers to obtain a purified solution;

[0094] S9. Dilute the purified solution obtained in S8 with a sterile PBS solution, centrifuge under vacuum conditions at 4°C and at a centrifugal force of 100000g - 150000g for 100 min - 150 min, and collect the precipitate to obtain a second precipitate;

[0095] S10. Resuspend the second precipitate obtained in S9 with a sterile PBS solution, and then filter the resuspended solution through a 0.22μm filter. The obtained solution is the acanthopanax exosome - like nanovesicle solution, where the concentration of the acanthopanax exosome - like nanovesicle solution is 4.31×10¹¹ particles / mL and the average particle size range is 89nm.

[0096] Among them, the preparation method of the acanthopanax exosome - like nanovesicle osteogenic induction medium in this example is as follows:

[0097] Step (1). Preparation of dexamethasone: Take dexamethasone, dissolve it with absolute ethanol to prepare a 1mM dexamethasone solution, filter and sterilize it through a 0.22μm filter membrane, dispense it into EP tubes, and store it at -20°C;

[0098] Preparation of L - ascorbic acid: Take L - ascorbic acid, dissolve it with PBS buffer to prepare a 50mM L - ascorbic acid solution, filter and sterilize it through a 0.22μm filter membrane, dispense it into EP tubes, and store it at -20°C;

[0099] Preparation of β - glycerophosphate: Take β - glycerophosphate, dissolve it with PBS buffer to prepare a 1M β - glycerophosphate solution, filter and sterilize it through a 0.22μm filter membrane, dispense it into EP tubes, and store it at -20°C.

[0100] Step (2), Preparation of osteogenic induction medium: In low-glucose DMEM, according to the addition amounts of fetal bovine serum, penicillin, and streptomycin in the complete medium of the present invention, the complete medium is obtained; then, according to the corresponding concentration amounts of dexamethasone, L-ascorbic acid, β-glycerophosphate sodium, and exosome-like nanovesicles solution of Acanthopanax cortex in the osteogenic induction medium of the present invention, the amounts are measured and then mixed evenly to obtain the required osteogenic induction medium.

[0101] This osteogenic induction medium with exosome-like nanovesicles of Acanthopanax cortex uses the exosome-like nanovesicles solution of Acanthopanax cortex as an additive component of the osteogenic induction medium, which can greatly improve the osteogenic differentiation efficiency of mesenchymal stem cells, has a significant osteogenic differentiation effect, and the exosome-like nanovesicles of Acanthopanax cortex have no obvious inhibitory effect on the proliferation of human bone marrow mesenchymal stem cells and have good cell compatibility. The exosome-like nanovesicles of Acanthopanax cortex can also be effectively taken up by human bone marrow mesenchymal stem cells, thereby enhancing its osteogenic induction effect. This osteogenic induction medium with exosome-like nanovesicles of Acanthopanax cortex can be prepared into an osteogenic induction drug for preventing and treating osteoporosis, bone defect, nonunion, or delayed bone healing.

[0102] Example 2

[0103] An osteogenic induction medium with exosome-like nanovesicles of Acanthopanax cortex, having the same other features as in Example 1, except that the contents are as follows:

[0104] Exosome-like nanovesicles solution of Acanthopanax cortex: 1.0 μg / mL;

[0105] Dexamethasone: 80 nM;

[0106] L-ascorbic acid: 40 μM;

[0107] β-glycerophosphate sodium: 8 mM.

[0108] The complete medium is a low-glucose DMEM medium containing 5%Vol. fetal bovine serum and 0.5%Vol. penicillin-streptomycin.

[0109] The exosome-like nanovesicles solution of Acanthopanax cortex in this example is prepared through the following steps:

[0110] S1. Use double-distilled water to wash the soil on the surface of fresh traditional Chinese medicine Acanthopanax cortex, and then cut the root bark part of Acanthopanax cortex into small pieces of Acanthopanax cortex with a cross-sectional area of 0.2 cm 2 and enter S2;

[0111] S2. Soak the small pieces of Acanthopanax cortex obtained in S1 in PBS solution at a weight-volume ratio (g / mL) of 1:10 for 12 h to obtain a soaking solution;

[0112] S3. Use medical gauze to filter the soaking solution obtained in S2 to obtain a crude filtrate;

[0113] S4. Centrifuge the crude filtrate obtained in S3 at a centrifugal force of 1000 g for 30 min to obtain a first supernatant; filter the first supernatant using a filter mesh with a pore size of 60 μm, and then filter the filtered filtrate using a filter mesh with a pore size of 40 μm to finally obtain a first filtrate;

[0114] S5. Centrifuge the first filtrate obtained in S4 at a centrifugal force of 3000 g for 60 min to obtain a second supernatant; then filter the second supernatant successively using a filter mesh with a pore size of 0.7 μm to obtain a second filtrate;

[0115] S6. Centrifuge the second filtrate obtained in S5 at a centrifugal force of 8000 g for 60 min to obtain a third supernatant; then filter the third supernatant using a filter mesh with a pore size of 0.6 μm to obtain a third filtrate;

[0116] S7. Centrifuge the third filtrate obtained in S6 at 4°C under vacuum conditions at a centrifugal force of 80000 g for 100 min to obtain a first precipitate, and then resuspend the first precipitate with a sterile PBS solution to obtain a resuspended solution;

[0117] S8. Sequentially add 2 mL of sucrose solutions with mass concentrations of 20%, 30%, 45%, and 60% from low to high in a centrifuge tube, then add the resuspended solution obtained in S7, centrifuge at 4°C under vacuum conditions at a centrifugal force of 100000 g for 150 min, and collect the separation layer between the 30% to 45% sucrose solution layers to obtain a purified solution;

[0118] S9. Dilute the purified solution obtained in S8 with a sterile PBS solution, centrifuge at 4°C under vacuum conditions at a centrifugal force of 100000 g for 150 min, and collect the precipitate to obtain a second precipitate;

[0119] S10. Resuspend the second precipitate obtained in S9 with a sterile PBS solution, and then filter the resuspended solution using a 0.22 μm filter. The obtained solution is the exosome-like nanovesicle solution of Acanthopanax cortex.

[0120] The preparation method of the exosome-like nanovesicle osteogenic induction medium of Acanthopanax cortex in this example is the same as that in Example 1.

[0121] Example 3

[0122] An exosome-like nanovesicle osteogenic induction medium of Acanthopanax cortex, with other characteristics being the same as those in Example 1, except that the contents are as follows:

[0123] Exosome-like nanovesicle solution of Acanthopanax cortex: 2.5 μg / mL;

[0124] Dexamethasone: 100 nM;

[0125] L - Ascorbic acid: 50 μM;

[0126] β - Glycerophosphate disodium: 10 mM.

[0127] Among them, the complete medium is low - sugar DMEM medium containing 10% Vol. fetal bovine serum and 1% Vol. penicillin - streptomycin.

[0128] The exosome - like nanovesicle solution of Acanthopanax cortex in this example is prepared through the following steps:

[0129] S1. Use double - distilled water to wash the soil on the surface of fresh traditional Chinese medicine Acanthopanax cortex, and then cut the root bark of Acanthopanax cortex into small pieces of Acanthopanax cortex with a cross - sectional area of 0.5 cm 2 and enter S2;

[0130] S2. Soak the small pieces of Acanthopanax cortex obtained in S1 in PBS solution at a weight - to - volume ratio (g / mL) of 1:15 for 8 h to obtain an immersion solution;

[0131] S3. Filter the immersion solution obtained in S2 with medical gauze to obtain a crude filtrate;

[0132] S4. Centrifuge the crude filtrate obtained in S3 at a centrifugal force of 1500 g for 30 min to obtain a first supernatant; filter the first supernatant with a filter net with a pore size of 70 μm, and then filter the filtered filtrate with a filter net with a pore size of 40 μm to finally obtain a first filtrate;

[0133] S5. Centrifuge the first filtrate obtained in S4 at a centrifugal force of 4000 g for 30 min to obtain a second supernatant; filter the second supernatant with a filter net with a pore size of 0.7 μm to obtain a second filtrate;

[0134] S6. Centrifuge the second filtrate obtained in S5 at a centrifugal force of 10000 g for 60 min to obtain a third filtrate, and filter the third supernatant with a filter net with a pore size of 0.45 μm to obtain a third filtrate;

[0135] S7. Centrifuge the third filtrate obtained in S6 at 4°C under vacuum conditions at a centrifugal force of 100000 g for 70 min to obtain a first precipitate, and then resuspend the first precipitate with sterile PBS solution to obtain a resuspended solution;

[0136] S8. Sequentially add 2 mL of sucrose solutions with mass concentrations of 15%, 30%, 45% and 60% from low to high in a centrifuge tube, then add the resuspended solution obtained in S7, and centrifuge at 4°C under vacuum conditions at a centrifugal force of 120000 g for 120 min, and collect the separation layer between the 30% to 45% sucrose solution layers to obtain a purified solution;

[0137] S9. Dilute the purified solution obtained in S8 with sterile PBS solution, centrifuge it at 120,000 g under vacuum conditions at 4°C for 150 min, collect the precipitate to obtain the second precipitate;

[0138] S10. Resuspend the second precipitate obtained in S9 with sterile PBS solution, and then filter the resuspended solution through a 0.22-μm filter. The resulting solution is the acanthopanax cortex exosome-like nanovesicle solution.

[0139] The preparation method of the acanthopanax cortex exosome-like nanovesicle osteogenic induction medium in this example is the same as that in Example 1.

[0140] Example 4

[0141] An acanthopanax cortex exosome-like nanovesicle osteogenic induction medium, with other features being the same as those in Example 1, except that the contents are as follows:

[0142] Acanthopanax cortex exosome-like nanovesicle solution: 5.0 μg / mL;

[0143] Dexamethasone: 100 nM;

[0144] L-Ascorbic acid: 50 μM;

[0145] β-Glycerophosphate disodium: 10 mM.

[0146] Among them, the complete medium is a low-glucose DMEM medium containing 10% Vol. fetal bovine serum and 1% Vol. penicillin-streptomycin.

[0147] The acanthopanax cortex exosome-like nanovesicle solution in this example is prepared through the following steps:

[0148] S1. Wash the soil on the surface of fresh traditional Chinese medicine acanthopanax cortex with double-distilled water, and then cut the root bark of acanthopanax cortex into small pieces of acanthopanax cortex with a cross-sectional area of 0.5 cm 2 and proceed to S2;

[0149] S2. Immerse the small pieces of acanthopanax cortex obtained in S1 in PBS solution at a weight-to-volume ratio (g / mL) of 1:15 for 10 h to obtain an immersion solution;

[0150] S3. Filter the immersion solution obtained in S2 with medical gauze to obtain a crude filtrate;

[0151] S4. Centrifuge the crude filtrate obtained in S3 at 1500 g for 30 min to obtain the first supernatant; filter the first supernatant through a filter mesh with a pore size of 70 μm, and then filter the filtered filtrate through a filter mesh with a pore size of 40 μm to finally obtain the first filtrate;

[0152] S5. Centrifuge the first filtrate obtained in S4 at a centrifugal force of 4000 g for 30 min to obtain a second supernatant; filter the second supernatant using a filter with a pore size of 0.7 μm to obtain a second filtrate.

[0153] S6. Centrifuge the second filtrate obtained in S5 at a centrifugal force of 10000 g for 60 min to obtain a third filtrate, and filter the third supernatant using a filter with a pore size of 0.45 μm to obtain a third filtrate.

[0154] S7. Centrifuge the third filtrate obtained in S6 at 4°C under vacuum conditions at a centrifugal force of 100000 g for 70 min to obtain a first precipitate, and then resuspend the first precipitate with a sterile PBS solution to obtain a resuspended solution.

[0155] S8. Sequentially add 2 mL of sucrose solutions with mass concentrations of 15%, 30%, 45%, and 60% from low to high in a centrifuge tube, then add the resuspended solution obtained in S7, and centrifuge at 4°C under vacuum conditions at a centrifugal force of 120000 g for 120 min. Collect the separation layer between the 30% and 45% sucrose solution layers to obtain a purified solution.

[0156] S9. Dilute the purified solution obtained in S8 with a sterile PBS solution, and centrifuge at 4°C under vacuum conditions at a centrifugal force of 120000 g for 150 min. Collect the precipitate to obtain a second precipitate.

[0157] S10. Resuspend the second precipitate obtained in S9 with a sterile PBS solution, and then filter the resuspended solution using a 0.22-μm filter. The obtained solution is the Acanthopanax cortex exosome-like nanovesicle solution.

[0158] The preparation method of the Acanthopanax cortex exosome-like nanovesicle osteogenic induction medium in this example is the same as that in Example 1.

[0159] Example 5

[0160] An Acanthopanax cortex exosome-like nanovesicle osteogenic induction medium, with other characteristics being the same as those in Example 1, except that the contents are as follows:

[0161] Acanthopanax cortex exosome-like nanovesicle solution: 10.0 μg / mL;

[0162] Dexamethasone: 120 nM;

[0163] L-Ascorbic acid: 60 μM;

[0164] β-Glycerophosphate disodium: 12 mM.

[0165] Among them, the complete medium is a low-glucose DMEM medium containing 15% Vol. fetal bovine serum and 2% Vol. penicillin-streptomycin.

[0166] The exosome-like nanovesicle solution of Acanthopanax cortex in the examples is prepared by the following steps:

[0167] S1. Wash the soil on the surface of fresh traditional Chinese medicine Acanthopanax cortex with double-distilled water, and then cut the root bark of Acanthopanax cortex into small pieces of Acanthopanax cortex with a cross-sectional area of 0.2 cm 2 and enter S2;

[0168] S2. Immerse the small pieces of Acanthopanax cortex obtained in S1 in a PBS solution at a weight-to-volume ratio (g / mL) of 1:20 for 9 h to obtain an immersion solution;

[0169] S3. Filter the immersion solution obtained in S2 with medical gauze to obtain a crude filtrate;

[0170] S4. Centrifuge the crude filtrate obtained in S3 at a centrifugal force of 2000 g for 15 min to obtain a first supernatant; filter the first supernatant with a filter mesh with a pore size of 60 μm, and then filter the filtered filtrate with a filter mesh with a pore size of 40 μm to finally obtain a first filtrate;

[0171] S5. Centrifuge the first filtrate obtained in S4 at a centrifugal force of 5000 g for 30 min to obtain a second supernatant; filter the second supernatant with a filter mesh with a pore size of 0.6 μm to obtain a second filtrate;

[0172] S6. Centrifuge the second filtrate obtained in S5 at a centrifugal force of 12000 g for 30 min to obtain a third filtrate, and filter the third supernatant with a filter mesh with a pore size of 0.5 μm to obtain a third filtrate;

[0173] S7. Centrifuge the third filtrate obtained in S6 at 4°C under vacuum conditions at a centrifugal force of 120000 g for 50 min to obtain a first precipitate, and then resuspend the first precipitate with a sterile PBS solution to obtain a resuspended solution;

[0174] S8. Add 2 mL of sucrose solutions with mass concentrations of 10%, 30%, 45%, and 70% to the centrifuge tube in sequence from low to high, then add the resuspended solution obtained in S7, and centrifuge at 4°C under vacuum conditions at a centrifugal force of 150000 g for 100 min, and collect the separation layer between the 30% to 45% sucrose solution layers to obtain a purified solution;

[0175] S9. Dilute the purified solution obtained in S8 with a sterile PBS solution, centrifuge at 4°C under vacuum conditions at a centrifugal force of 150000 g for 100 min, and collect the precipitate to obtain a second precipitate;

[0176] S10. Resuspend the second precipitate obtained in S9 with sterile PBS solution, and then filter the resuspended solution through a 0.22 μm filter. The resulting solution is the exosome-like nanovesicle solution of Acanthopanax cortex.

[0177] The preparation method of the osteogenic induction medium of the exosome-like nanovesicles of Acanthopanax cortex in this example is the same as that in Example 1.

[0178] Example 6

[0179] An osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex, with other characteristics the same as those in Example 3, except that the exosome-like nanovesicle solution of Acanthopanax cortex is 20.0 μg / mL, and other components are the same as those in Example 3.

[0180] The exosome-like nanovesicle solution of Acanthopanax cortex in this example is prepared by the following steps:

[0181] S1. Wash the soil on the surface of fresh traditional Chinese medicine Acanthopanax cortex with double-distilled water, and then cut the root bark part of Acanthopanax cortex into small pieces of Acanthopanax cortex with a cross-sectional area of 1.0 cm 2 and enter S2;

[0182] S2. Soak the small pieces of Acanthopanax cortex obtained in S1 in PBS solution at a weight-to-volume ratio (g / mL) of 1:18 for 10 h to obtain a soaking solution;

[0183] S3. Filter the soaking solution obtained in S2 with medical gauze to obtain a crude filtrate;

[0184] S4. Centrifuge the crude filtrate obtained in S3 at a centrifugal force of 1500 g for 20 min to obtain a first supernatant; filter the first supernatant with a filter mesh with a pore size of 60 μm, and then filter the filtered filtrate with a filter mesh with a pore size of 50 μm to finally obtain a first filtrate;

[0185] S5. Centrifuge the first filtrate obtained in S4 at a centrifugal force of 4000 g for 40 min to obtain a second supernatant; filter the second supernatant with a filter mesh with a pore size of 0.6 μm to obtain a second filtrate;

[0186] S6. Centrifuge the second filtrate obtained in S5 at a centrifugal force of 11000 g for 40 min to obtain a third filtrate, and filter the third supernatant with a filter mesh with a pore size of 0.5 μm to obtain a third filtrate;

[0187] S7. Centrifuge the third filtrate obtained in S6 at 4°C under vacuum conditions and at a centrifugal force of 110000 g for 80 min to obtain a first precipitate, and then resuspend the first precipitate with sterile PBS solution to obtain a resuspended solution;

[0188] S8. Sequentially add 2 mL of sucrose solutions with mass concentrations of 10%, 30%, 45%, and 70% from low to high into a centrifuge tube, and then add the resuspended solution obtained in S7. Centrifuge at 140,000 g under vacuum conditions at 4°C for 120 min, and collect the separation layer between the 30% and 45% sucrose solution layers to obtain a purified solution;

[0189] S9. Dilute the purified solution obtained in S8 with a sterile PBS solution, centrifuge at 140,000 g under vacuum conditions at 4°C for 120 min, and collect the precipitate to obtain a second precipitate;

[0190] S10. Resuspend the second precipitate obtained in S9 with a sterile PBS solution, and then filter the resuspended solution through a 0.22 μm filter. The resulting solution is the Acanthopanax cortex exosome-like nanovesicle solution.

[0191] The preparation method of the Acanthopanax cortex exosome-like nanovesicle osteogenic induction medium in this example is the same as that in Example 3.

[0192] Example 7

[0193] An Acanthopanax cortex exosome-like nanovesicle osteogenic induction medium, with other features the same as those in Example 1, except that: the Acanthopanax cortex exosome-like nanovesicle solution is 40.0 μg / mL, and other components are the same as those in Example 3. The preparation methods of the Acanthopanax cortex exosome-like nanovesicle solution and the Acanthopanax cortex exosome-like nanovesicle osteogenic induction medium in this example are the same as those in Example 3.

[0194] Comparative Example 1

[0195] An osteogenic induction medium, with other features the same as those in Example 1, except that: the Acanthopanax cortex exosome-like nanovesicles are 0 μg / mL, and other components are the same as those in Example 3.

[0196] The preparation method of the Acanthopanax cortex exosome-like nanovesicle osteogenic induction medium in this example is as follows:

[0197] Step (1). Preparation of dexamethasone: Take dexamethasone, dissolve it with absolute ethanol to prepare a 1 mM dexamethasone solution, filter and sterilize it through a 0.22 μm filter membrane, aliquot it into EP tubes, and store it at -20°C;

[0198] Preparation of L-ascorbic acid: Take L-ascorbic acid, dissolve it with PBS buffer to prepare a 50 mM L-ascorbic acid solution, filter and sterilize it through a 0.22 μm filter membrane, aliquot it into EP tubes, and store it at -20°C;

[0199] Preparation of sodium β - glycerophosphate: Take sodium β - glycerophosphate, dissolve it with PBS buffer solution to prepare a 1 M sodium β - glycerophosphate solution, filter and sterilize it through a 0.22 μm filter membrane, dispense it into EP tubes, and store it at - 20 °C.

[0200] Step (2), preparation of osteogenic induction medium: In low - glucose DMEM, according to the addition amounts of fetal bovine serum, penicillin, and streptomycin in the complete medium of the present invention, obtain the complete medium; then measure the dosages of dexamethasone, L - ascorbic acid, and sodium β - glycerophosphate in the osteogenic induction medium of this example according to the corresponding concentrations, and then mix them evenly to obtain the required osteogenic induction medium.

[0201] Detection steps and result analysis

[0202] 1. Observation of the morphology of exosome - like nanovesicles from Acanthopanax cortex under transmission electron microscope

[0203] Drop the exosome - like nanovesicles of Acanthopanax cortex of the present invention onto a special grid for transmission electron microscope (TEM), and let it adsorb naturally for 5 min. Then use uranyl acetate for negative staining, and the staining time is 5 min. After staining, remove the staining solution, and place the grid in a drying oven to dry for 20 min. After drying, use a transmission electron microscope to observe the morphology of the sample, as Figure 1 shown.

[0204] As Figure 1 shown, the exosome - like nanoparticle solution (AgELNs) derived from Acanthopanax cortex of the present invention shows a vesicle structure with surface depressions and a disc - like shape under a transmission electron microscope.

[0205] 2. Detection of the particle size range and concentration of exosome - like nanovesicles from Acanthopanax cortex

[0206] Dilute the exosome - like nanovesicle solution (AgELNs) of Acanthopanax cortex of the present invention 100 times with PBS solution. Use a Nano Coulter nanoparticle size analyzer to perform single - particle size analysis to obtain the particle size range and particle concentration data of the sample, as Figure 2 shown.

[0207] From Figure 2 it can be seen that the concentration of exosome - like nanoparticles derived from Acanthopanax cortex in the exosome - like nanovesicle solution (AgELNs) of Acanthopanax cortex is 4.31×10¹¹ particles / mL, and the average particle size range is 89 nm.

[0208] 3. Determination of the protein concentration of exosome - like nanovesicles from Acanthopanax cortex

[0209] Quantify the obtained exosome - like nanoparticle solution (AgELNs) derived from Acanthopanax cortex by using a BCA protein detection kit. The specific steps are as follows:

[0210] (1) Standard preparation: Dilute the protein standard (2 mg / mL) with sterile PBS buffer to final concentrations of 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL respectively. Take 20 μL of each concentration of the standard and add it to the standard wells of a 96-well plate to prepare a standard protein concentration curve.

[0211] (2) Sample preparation: Dissolve the exosome-like nanovesicle solution of Acanthopanax cortex (AgELNs) of the present invention in sterile PBS buffer, and take 20 μL of the sample and add it to the sample wells of a 96-well plate.

[0212] (3) BCA working solution reaction: Add 200 μL of BCA working solution to each well and incubate at 37 °C for 20 min to 30 min.

[0213] (4) Absorbance measurement: Use a microplate reader to measure the absorbance at 562 nm.

[0214] (5) Protein concentration calculation: Calculate the protein concentration in the sample according to the standard protein concentration curve.

[0215] Use the BCA quantitative protein concentration to measure and calculate the usage amount of the exosome-like nanoparticle solution of Acanthopanax cortex (AgELNs) in the subsequent drug addition treatment experiment. Finally, the protein concentration of the exosome-like nanovesicle solution of Acanthopanax cortex extracted by the differential ultracentrifugation method in the present invention is measured to be 0.547 mg / mL.

[0216] 4. Effects of exosome-like nanovesicle solution of Acanthopanax cortex (AgELNs) on the proliferation activity of human bone marrow mesenchymal stem cells (hBM-MSCs)

[0217] (1) Add exosome-like nanovesicles of Acanthopanax cortex at concentrations of 0 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL to α-MEM complete medium respectively to obtain proliferation media with corresponding concentrations of exosome-like nanovesicles of Acanthopanax cortex. At the same time, use α-MEM complete medium as a blank control.

[0218] (2) Cell seeding: 3000 hBM-MSCs were seeded in each well of a 96-well plate. After the cells were attached and grown for 12 hours, the osteogenic induction medium with different concentrations of the above-mentioned Acanthopanax cortex exosome-like nanovesicles was used. The complete culture medium in the osteogenic induction medium contained 10% Vol. fetal bovine serum and 1% Vol. penicillin-streptomycin, and cultured at 37°C and 5% CO2. After treatment, the CCK8 kit was used to detect the effect of the Acanthopanax cortex exosome-like nanovesicle solution (AgELNs) on the proliferation activity of human bone marrow mesenchymal stem cells (hBM-MSCs) at 72 hours. Figure 3 .

[0219] The effect of the concentration of Acanthopanax cortex exosome-like nanovesicles (AgELNs) on the proliferation activity of human bone marrow mesenchymal stem cells (hBM-MSCs) was observed by using a CCK-8 kit, and then the absorbance of the cells at 450 nm was detected by an ELISA instrument for 72 hours, which can indirectly reflect the number of living cells, thereby verifying whether the concentration of Acanthopanax cortex exosome-like nanovesicles extracted by differential ultracentrifugation in the present invention has an effect on the proliferation activity of human bone marrow mesenchymal stem cells, thereby evaluating the safety of Acanthopanax cortex exosome-like nanovesicles on cells. Figure 3 The CCK8 assay showed that even at the maximum concentration of 40 μg / mL, the exosome-like nanovesicle solution of Acanthopanax cortex (AgELNs) had no significant inhibitory effect on the proliferation of human bone marrow mesenchymal stem cells (hBM-MSCs).

[0220] 5. Uptake and internalization experiment of Acanthopanax cortex exosome-like nanovesicle solution (AgELNs)

[0221] This uptake and internalization experiment was carried out according to the requirements of the reagent instructions, including the following steps:

[0222] (1) Preparation of the staining working solution: Mix 4 μL of PKH26 fluorescent labeling dye with 200 μL of Diluent C, then mix 100 μL of AgELNs diluted with PBS with 200 μL of Diluent C, add the working solution and mix to make a 500 μL system, and incubate at room temperature for 15 min in a dark place.

[0223] (2) Staining termination and washing: Add 500 μL of exosome-free serum to terminate the reaction and dilute it with PBS solution. Ultracentrifuge at 100,000 g for 70 min at 4°C under vacuum to remove unbound dye and collect AgELNs labeled with PKH26 fluorescent dye (PKH26-AgELNs).

[0224] (3) Cell treatment: Add PKH26-AgELNs to the prepared complete medium and apply it to hBM-MSCs. After 6 hours, aspirate and discard the supernatant of the medium. Wash the cells twice with sterile PBS, then fix them with 4% paraformaldehyde for 20 min, and wash with PBS for 5 min / time × 3 times to thoroughly wash away the fixative.

[0225] (4) Staining: Add DAPI solution to stain the cell nuclei, incubate at room temperature in the dark for 30 min, and thoroughly wash away the staining solution with PBS.

[0226] (5) Microscopic observation: Under slightly moist conditions, observe and photograph the PKH26-AgELNs internalized by cells using an inverted fluorescence microscope to obtain Figure 4 .

[0227] The solution of exosome-like nanovesicles of Acanthopanax cortex labeled with PKH26 is dark gray, the cell nuclei of human bone marrow mesenchymal stem cells labeled with DAPI are bright gray, and Merge uses DAPI as cell localization to observe whether the solution of exosome-like nanovesicles of Acanthopanax cortex labeled with PKH26 is taken up by cells. Through Figure 4 the experimental results of PKH26 fluorescent dye labeling show that exosome-like nanovesicles of Acanthopanax cortex solution (AgELNs) can be successfully taken up by human bone marrow mesenchymal stem cells (hBM-MSCs) and enter the cells.

[0228] 6. Effect of exosome-like nanovesicles of Acanthopanax cortex solution (AgELNs) on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs)

[0229] (1) Evaluation of the effect on osteogenic differentiation: Take the 5th generation of hBM-MSCs with good in vitro amplified growth, inoculate them in a 12-well plate at a cell density of 5×10 ^4 / well, and culture them in a cell incubator at 37 °C and 5% CO2 saturated humidity. When the cells adhere and grow to 80% confluence, aspirate and discard the supernatant of the original medium, and add osteogenic differentiation medium containing exosome-like nanovesicles of Acanthopanax cortex solution at concentrations of 0 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL (i.e., in the osteogenic differentiation media obtained in Comparative Example 1, Example 3, Example 4, and Example 5) for induction culture, and change the osteogenic differentiation medium every 3 days. After continuous induction for 14 days, fix the cells with 75% alcohol, and then perform alizarin red staining to identify the osteogenic differentiation effect to obtain Figure 5 .

[0230] Through Figure 5 the alizarin red staining results show that the calcium salt deposition after osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) induced by exosome-like nanovesicles of Acanthopanax cortex solution (AgELNs) is increased compared with the control group.

[0231] (2) Gene expression detection:

[0232] a. RNA sample collection: After culturing for 5 days in osteogenic differentiation media containing acanthopanax cortex exosome-like nanovesicles at concentrations of 0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, and 10.0 μg / mL (i.e., the osteogenic differentiation media obtained in Comparative Example 1, Example 3, Example 4, and Example 5), collect RNA samples according to the requirements of the reagent manufacturer's instructions.

[0233] b. Reverse transcription: Transfer the extracted RNA samples to enzyme-free EP tubes, add DEPC water and let stand for 5 min. Centrifuge at 12,000 g for 15 min at 4°C, transfer the supernatant to a new enzyme-free EP tube, add an equal volume of isopropanol to precipitate for 10 min, and centrifuge again. After discarding the supernatant, wash the RNA three times with 75% DEPC ethanol, air dry, and detect the RNA concentration and purity using NanoDrop100. Take 1 μg of RNA for reverse transcription, add 4×gDNA to remove genomic DNA, supplement DEPC water to 16 μL, react at 42°C for 2 min, add 5×qPCR Mix and react at 37°C for 15 min, and react at 85°C for 15 s to generate cDNA samples.

[0234] c. Real-time fluorescence quantitative PCR (qRT-PCR): Prepare a 20 μL reaction system according to the following conditions:

[0235]

[0236] d. After performing the amplification cycle, using GAPDH as the housekeeping gene, use the 2-( △△ CT) method to statistically analyze the data and detect the gene expression of osteogenesis-related markers (ALP, RUNX2), as Figure 6 and Figure 7 .

[0237] (3) Protein level detection

[0238] a. Cell lysis and protein extraction: Seed the 5th passage of human bone marrow mesenchymal stem cells (hBM-MSCs) at a density of 3×10 ^5 / well in 6 cm dishes, and induce and culture in the osteogenic induction media of Comparative Example 1 (0 μg / mL), Example 3 (2.5 μg / mL), Example 4 (5.0 μg / mL), and Example 5 (10.0 μg / mL) for 12 days. Collect the cells, lyse them with RIPA buffer and perform ultrasonic fragmentation, centrifuge at 12,000 g at 4°C for 15 min to remove cell debris, and measure the protein concentration.

[0239] b. SDS-PAGE and protein blotting: After preparing the protein samples, approximately 20 μg of the samples were subjected to SDS-PAGE gel electrophoresis, and the proteins were transferred onto a 0.22 μm PVDF membrane. After blocking with skim milk, the primary antibodies (ALP, RUNX2, β-actin) were incubated overnight at 4°C. The unbound primary antibodies were washed away with TBST (10 min / time, 3 times in total), the secondary antibody was incubated for 1 hour at room temperature, and the unbound secondary antibody was washed away with TBST again. The target bands were collected using a LiCOR Odyssey DLx near-infrared imaging system to obtain Figure 8 .

[0240] It can be seen through Figures 6 - 8 that osteogenesis-related markers were verified by qRT-PCR ( Figures 6 - 7 ) and Western blot ( Figure 8 ). The results showed that the expressions of ALP and RUNX2 in hBMSCs treated with the exosome-like nanovesicle solution of Acanthopanax cortex increased and showed a concentration-dependent increase. Moreover, it can be seen through Figure 6 and Figure 7 that when the concentration of the exosome-like nanovesicle solution of Acanthopanax cortex was 10 μg / mL, the osteogenic effect showed a downward trend compared with that at a concentration of 5.0 μg / mL for osteogenesis-related markers.

[0241] In summary, the osteogenic induction medium with the exosome-like nanovesicle solution of Acanthopanax cortex as an additive component in the present invention can significantly improve the osteogenic differentiation efficiency of human bone marrow mesenchymal stem cells (hBM-MSCs), has a significant osteogenic differentiation effect, and the exosome-like nanovesicles of Acanthopanax cortex have no obvious inhibitory effect on the proliferation of human bone marrow mesenchymal stem cells (hBM-MSCs), and have good cell compatibility. The exosome-like nanovesicles of Acanthopanax cortex can also be effectively taken up by human bone marrow mesenchymal stem cells (hBM-MSCs), thereby enhancing their osteogenic induction effect.

[0242] Example 8

[0243] An osteogenic induction and differentiation method, in which mesenchymal stem cells are cultured using the osteogenic induction medium of the exosome-like nanovesicles of Acanthopanax cortex as described in any one of Examples 1 to 7. The mesenchymal stem cells are human bone marrow mesenchymal stem cells (hBM-MSCs).

[0244] This osteogenic induction and differentiation method is carried out by osteogenic induction culture containing exosome-like nanovesicles of Acanthopanax cortex, which can greatly improve the osteogenic differentiation efficiency of mesenchymal stem cells, has a significant osteogenic differentiation effect, and the exosome-like nanovesicles of Acanthopanax cortex have no obvious inhibitory effect on the proliferation of human bone marrow mesenchymal stem cells (hBM-MSCs), and have good cell compatibility. The exosome-like nanovesicles of Acanthopanax cortex can also be effectively taken up by human bone marrow mesenchymal stem cells (hBM-MSCs), thereby enhancing its osteogenic induction effect.

[0245] Example 9

[0246] Use of exosome-like nanovesicles of Acanthopanax cortex in the preparation of an osteogenic induction medium, and the exosome-like nanovesicles of Acanthopanax cortex promote osteogenic induction and differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). The exosome-like nanovesicles of Acanthopanax cortex and the osteogenic induction medium are obtained by the preparation methods of Examples 1 to 7.

[0247] Based on the data of the proliferation activity, uptake and internalization experiment, and osteogenic differentiation effect of human bone marrow mesenchymal stem cells (hBM-MSCs) by the above exosome-like nanovesicle solution of Acanthopanax cortex (AgELNs), adding exosome-like nanovesicles of Acanthopanax cortex to the osteogenic induction medium can greatly improve the osteogenic differentiation efficiency of mesenchymal stem cells, has a significant osteogenic differentiation effect, and the exosome-like nanovesicles of Acanthopanax cortex have no obvious inhibitory effect on the proliferation of human bone marrow mesenchymal stem cells (hBM-MSCs), and have good cell compatibility. The exosome-like nanovesicles of Acanthopanax cortex can also be effectively taken up by human bone marrow (hBM-MSCs) mesenchymal stem cells, thereby enhancing its osteogenic induction effect.

[0248] Example 10

[0249] Use of an osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex as in Examples 1 to 7 in the preparation of an osteogenic induction drug. The osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex promotes osteogenic induction and differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). Use of the osteogenic induction medium of exosome-like nanovesicles of Acanthopanax cortex in the preparation of at least one bone disease such as osteoporosis, bone defect, nonunion, or delayed bone healing.

[0250] Based on the data of the proliferative activity of human bone marrow mesenchymal stem cells (hBM-MSCs), the uptake and internalization experiment data, and the results of osteogenic differentiation by the above-mentioned Acanthopanax cortex exosome-like nanovesicle solution (AgELNs), it can be seen that the osteogenic induction medium of Acanthopanax cortex exosome-like nanovesicles of the present invention can significantly improve the osteogenic differentiation effect of mesenchymal stem cells, and can significantly enhance the osteogenic differentiation effect. Moreover, the Acanthopanax cortex exosome-like nanovesicles have no obvious inhibitory effect on the proliferation of human bone marrow mesenchymal stem cells and have good cell compatibility. Therefore, when the osteogenic induction medium of Acanthopanax cortex exosome-like nanovesicles is prepared into an osteogenic induction drug, it also has an osteogenic differentiation effect, providing a new technical means and theoretical basis for the prevention and treatment of osteoporosis, bone defect, nonunion or delayed union of bone.

[0251] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for osteogenic differentiation, characterized in that: Mesenchymal stem cells are cultured using an osteogenic induction medium containing exosome-like nanovesicles from Acanthopanax cortex; the mesenchymal stem cells are human bone marrow mesenchymal stem cells; The acanthopanax cortex exosome-like nanovesicle osteogenic induction medium comprises acanthopanax cortex exosome-like nanovesicle solution, dexamethasone, L-ascorbic acid, sodium β-glycerophosphate and a complete culture medium; The content is as follows: Acanthopanax cortex exosome-like nanovesicle solution: 2.5 μg / mL~10 μg / mL; Dexamethasone: 80nM~120nM; L-ascorbic acid: 40 μM to 60 μM; β-glycerophosphate sodium: 8mM~12mM; The complete culture medium is a low-glucose DMEM culture medium containing 5% Vol. to 15% Vol. fetal bovine serum and 0.5% Vol. to 2% Vol. penicillin-streptomycin; The preparation method of the Acanthopanax Cortex exosome-like nanovesicle solution is as follows: soaking the Acanthopanax Cortex in a PBS solution to obtain an immersion solution; filtering the immersion solution and centrifuging it multiple times to obtain a supernatant; The supernatant is subjected to a first resuspending precipitation operation to obtain a resuspended solution; the resuspended solution is purified by sucrose gradient density centrifugation to obtain a purified solution; the purified solution is then diluted and precipitated by a PBS solution to obtain a precipitate; the precipitate is subjected to a second resuspending precipitation operation to obtain the Acanthopanax cortex exosome-like nanovesicle solution; The concentration of the Acanthopanax cortex-derived exosome-like nanoparticles in the Acanthopanax cortex exosome-like nanovesicle solution is 4.31×10¹¹ particles / mL, and the average particle size range is 89 nm.

2. The osteogenic differentiation method according to claim 1, characterized in that: The Acanthopanax acanthopanax exosome-like nanovesicle solution is specifically prepared by the following steps: S1, cut the Acanthopanax acanthopanax bark into pieces and enter S2; S2, soaking the Acanthopanax acanthopanax cortex cut into pieces in S1 in a PBS solution for 8 h to 12 h to obtain an immersion solution; S3, filtering the soaking liquid obtained in S2 using medical gauze to obtain a coarse filtrate; S4, centrifuging the coarse filtrate obtained in S3 at a centrifugal force of 1000 g to 2000 g for 15 min to 30 min to obtain a first supernatant; then filtering the first supernatant using a filter with a pore size of 70 μm to 40 μm to obtain a first filtrate; S5, centrifuging the first filtrate obtained in S4 at a centrifugal force of 3000 g to 5000 g for 30 min to 60 min to obtain a second supernatant; then filtering the second supernatant using a filter with a pore size of 0.7 μm to 0.4 μm to obtain a second filtrate; S6, centrifuging the second filtrate obtained in S5 at a centrifugal force of 8000 g to 12000 g for 30 min to 60 min to obtain a third supernatant; then filtering the third supernatant using a filter with a pore size of 0.7 μm to 0.4 μm to obtain a third filtrate; S7, centrifuging the third filtrate obtained in S6 at a centrifugal force of 80000 g to 120000 g for 50 min to 100 min to obtain a first precipitate, and then resuspending the first precipitate with a sterile PBS solution to obtain a resuspended solution; S8, adding sucrose solutions of different mass concentrations in a centrifuge tube in order from low to high, and then adding the resuspended solution obtained in S7, centrifuging at a centrifugal force of 100000g to 150000g for 100min to 150min, collecting the separation layer between the middle sucrose solution layers, and obtaining a purified solution; S9, diluting the purified solution obtained in S8 with a sterile PBS solution, centrifuging at a centrifugal force of 100,000 g to 150,000 g for 100 min to 150 min, collecting the precipitate to obtain a second precipitate; S10, resuspending the second precipitate obtained in S9 with a sterile PBS solution, and then filtering the resuspended solution with a 0.22 μm filter, and the obtained solution is the Acanthopanax Acanthopanax Cortex exosome-like nanovesicle solution.

3. The osteogenic differentiation method according to claim 2, characterized in that: The content of the Acanthopanax cortex exosome-like nanovesicle osteogenic induction medium is as follows: Acanthopanax cortex exosome-like nanovesicle solution: 2.5 μg / mL~5.0 μg / mL; Dexamethasone: 100 nM; L-ascorbic acid: 50 μM; β-glycerophosphate sodium: 10 mM; The complete culture medium is a low-glucose DMEM culture medium containing 10% Vol. fetal bovine serum and 1% Vol. penicillin-streptomycin.

4. Use of Acanthopanax Cortex Exosome-like Nanovesicles in the Preparation of Osteogenic Induction Medium, characterized in that: The Acanthopanax cortex exosome-like nanovesicles promote osteogenic differentiation of human bone marrow mesenchymal stem cells; The Acanthopanax cortex exosome-like nanovesicles improve the osteogenic differentiation of human bone marrow mesenchymal stem cells by upregulating the expression levels of ALP and RUNX2 in human bone marrow mesenchymal stem cells; The preparation method of the Acanthopanax Cortex exosome-like nanovesicle solution is as follows: soaking the Acanthopanax Cortex in a PBS solution to obtain an immersion solution; filtering the immersion solution and centrifuging it multiple times to obtain a supernatant; The supernatant is subjected to a first resuspending precipitation operation to obtain a resuspended solution; the resuspended solution is purified by sucrose gradient density centrifugation to obtain a purified solution; the purified solution is then diluted and precipitated by a PBS solution to obtain a precipitate; the precipitate is subjected to a second resuspending precipitation operation to obtain the Acanthopanax cortex exosome-like nanovesicle solution; The concentration of the Acanthopanax cortex-derived exosome-like nanoparticles in the Acanthopanax cortex exosome-like nanovesicle solution is 4.31×10¹¹ particles / mL, and the average particle size range is 89 nm; The content of the Acanthopanax acanthopanax exosome-like nanovesicle solution is 2.5 μg / mL to 10 μg / mL.

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