CN118995592B

CN118995592B - Acanthopanax exosome-like nano vesicle osteogenesis induction medium, preparation method thereof, osteogenesis induction differentiation method and application

Info

Publication number: CN118995592B
Application number: CN202411330539.3A
Authority: CN
Inventors: 楚佳奇; 钟焯岚; 李鹏; 赵梓全
Original assignee: Affiliated Hospital of Guangdong Medical University
Current assignee: Affiliated Hospital of Guangdong Medical University
Priority date: 2024-09-24
Filing date: 2024-09-24
Publication date: 2025-07-01
Anticipated expiration: 2044-09-24
Also published as: CN118995592A

Abstract

An osteogenesis inducing culture medium for acanthopanax exosome-like nano vesicles, a preparation method of the osteogenesis inducing culture medium for the acanthopanax exosome-like nano vesicles, an osteogenesis inducing differentiation method, application of the acanthopanax exosome-like nano vesicles in preparing the osteogenesis inducing culture medium and application of the acanthopanax exosome-like nano vesicles in preparing osteogenesis inducing medicines. The invention takes the acanthopanax bark exosome-like nano vesicle solution as an addition component of the osteogenesis inducing culture medium, can greatly improve the osteogenesis differentiation efficiency of mesenchymal stem cells, has obvious osteogenesis differentiation effect, and has good cell compatibility. The acanthopanax exosome-like nano vesicle can be effectively taken up by human bone marrow mesenchymal stem cells, so that the osteogenesis induction effect of the acanthopanax exosome-like nano vesicle is enhanced. The acanthopanax exosome-like nano vesicle osteogenesis inducing medium can be prepared into osteogenesis inducing medicaments for preventing and treating osteoporosis, bone defect, bone nonunion or bone delayed healing.

Description

Acanthopanax exosome-like nano vesicle osteogenesis induction medium, preparation method thereof, osteogenesis induction differentiation method and application

Technical Field

The invention relates to the technical field of stem cells of biomedical technology, in particular to an osteogenesis induction culture medium for acanthopanax bark exosome-like nano vesicles, a preparation method of the osteogenesis induction culture medium for the acanthopanax bark exosome-like nano vesicles, an osteogenesis induction differentiation method, application of the acanthopanax bark exosome-like nano vesicles in preparation of the osteogenesis induction culture medium and application of the acanthopanax bark exosome-like nano vesicles in preparation of osteogenesis induction medicines.

Background

Osteoporosis is a major health problem faced by middle-aged and elderly people, and epidemiological data shows that the prevalence of osteoporosis in people over 40 years old in our country is as high as 25.6%, while it is rising to 32.0% in people over 65 years old. Osteoporosis is a systemic skeletal disease caused by an imbalance between osteoclasts and osteoblasts, and is mainly characterized by reduced bone mass and reduced mineral density. This pathological state can lead to systemic bone loss and a significant increase in bone fragility. The reduction in bone density not only exacerbates the deterioration of bone microstructure, but also promotes degradation of matrix proteins, thereby significantly increasing the risk of fracture. Currently, therapeutic options for osteoporosis include bisphosphate, hormone Replacement Therapy (HRT), selective Estrogen Receptor Modulator (SERM) and diels, but these methods have limited efficacy and can cause serious side effects over long periods of time.

Human bone marrow mesenchymal stem cells (hBM-MSCs) have been attracting attention for their multipotent differentiation potential and are capable of differentiating into various tissue cells such as bone, cartilage, muscle, tendon and ligament. At present, research at home and abroad has widely proved the osteogenic differentiation capability of hBM-MSCs, so that the hBM-MSCs become ideal cells in bone tissue regeneration and repair engineering. The discovery provides a new strategy and thought for treating bone defects, bone nonunions, delayed bone healing and other refractory bone diseases.

The current osteogenesis inducing culture medium has various formulas, common components comprise basal culture medium (such as alpha-MEM, low sugar DMEM and the like), fetal bovine serum, antibiotics (penicillin and streptomycin), dexamethasone, ascorbic acid, beta-glycerophosphate sodium salt and the like, but the content of specific components is different. However, there are still many problems in the osteogenesis inducing process, such as low induction efficiency, unstable effect, long induction time, etc., which limit the application of hBM-MSCs in the clinical prevention and treatment of osteoporosis. At present, research and development of a safe and efficient osteogenic differentiation method are urgently needed to promote research of osteogenic differentiation mechanisms of hBM-MSCs and promote clinical application of the hBM-MSCs in preventing and treating diseases such as osteoporosis.

Therefore, aiming at the defects of the prior art, an acanthopanax bark exosome-like nano vesicle osteogenesis induction culture medium, a preparation method of the acanthopanax bark exosome-like nano vesicle osteogenesis induction culture medium, an osteogenesis induction differentiation method, application of the acanthopanax bark exosome-like nano vesicles in preparing the osteogenesis induction culture medium and application of the acanthopanax bark exosome-like nano vesicle osteogenesis induction culture medium in preparing osteogenesis induction medicines are provided to solve the defects of the prior art.

Disclosure of Invention

One of the purposes of the invention is to provide an acanthopanax exosome-like nano vesicle osteogenesis induction medium which avoids the defects of the prior art. The acanthopanax bark exosome-like nano vesicle osteogenesis induction medium can improve osteogenesis differentiation effect.

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

provides an osteogenesis induction culture medium for acanthopanax exosome-like nano vesicles, which comprises acanthopanax exosome-like nano vesicle solution, dexamethasone, L-ascorbic acid, beta-sodium glycerophosphate and a complete culture medium.

The preparation method of the acanthopanax bark exosome-like nano vesicle solution comprises the steps of soaking acanthopanax bark in PBS solution to obtain soaking solution, filtering and centrifuging the soaking solution for multiple times to obtain supernatant, performing first time resuspension precipitation operation on the supernatant to obtain a resuspended solution, performing sucrose gradient density centrifugation and purification on the resuspended solution to obtain purified solution, performing dilution precipitation operation on the purified solution by the PBS solution to obtain precipitate, and performing second time resuspension precipitation operation on the precipitate to obtain the acanthopanax bark exosome-like nano vesicle solution.

The acanthopanax exosome-like nano vesicle osteogenesis induction medium comprises the following components:

The acanthopanax exosome-like nano vesicle solution is 1-40 mug/mL;

dexamethasone 80nM to 120nM;

l-ascorbic acid 40-60 mu M;

8 mM-12 mM of beta-sodium glycerophosphate;

the complete culture medium is a low-sugar DMEM culture medium containing 5-15% of Vol.

Further, the acanthopanax exosome-like nano vesicle osteogenesis induction medium comprises the following components:

the acanthopanax exosome-like nano vesicle solution is 2.5 mug/mL-5.0 mug/mL;

Dexamethasone 100nM;

L-ascorbic acid 50. Mu.M;

10mM sodium beta-glycerophosphate;

The complete medium is a low-sugar DMEM medium containing 10% vol. Fetal bovine serum, 1% vol. Green streptomycin.

Preferably, the acanthopanax exosome-like nano vesicle solution is prepared by the following steps:

s1, cutting cortex acanthopanacis into pieces, and entering S2;

s2, soaking the acanthopanax bark cut into pieces in the S1 in a PBS solution for 8-12 hours to obtain a soaking solution;

S3, filtering the soaking solution obtained in the step S2 by using medical gauze to obtain crude filtrate;

s4, centrifuging the rough filtrate obtained in the step S3 for 15-30 min under the condition that the centrifugal force is 1000-2000 g to obtain a first supernatant, and then filtering the first supernatant by using a filter screen with the aperture of 70-40 mu m to obtain a first filtrate;

S5, centrifuging the first filtrate obtained in the S4 for 30-60 min under the condition that the centrifugal force is 3000-5000 g to obtain a second supernatant, and filtering the second supernatant by using a filter screen with the aperture of 0.7-0.4 mu m to obtain a second filtrate;

S6, centrifuging the second filtrate obtained in the S5 for 30-60 min under the condition that the centrifugal force is 8000-12000 g to obtain a third supernatant, and filtering the third supernatant by using a filter screen with the aperture of 0.7-0.4 mu m to obtain a third filtrate;

S7, centrifuging the third filtrate obtained in the S6 for 50-100 min under the condition that the centrifugal force is 80000-120000 g to obtain a first precipitate, and then re-suspending the first precipitate by using a sterile PBS solution to obtain a re-suspended solution;

s8, sequentially adding sucrose solutions with different mass concentrations into a centrifuge tube from low to high, adding the resuspended solution obtained in the step S7, centrifuging for 100-150 min under the centrifugal force of 100000-150 g, and collecting a separation layer between the intermediate sucrose solution layers to obtain a purified solution;

S9, diluting the purified solution obtained in the S8 by using a sterile PBS solution, centrifuging for 100-150 min under the condition that the centrifugal force is 100000-150000 g, and collecting a precipitate to obtain a second precipitate;

s10, re-suspending the second precipitate obtained in the step S9 by using a sterile PBS solution, and filtering the re-suspended solution by using a 0.22 mu m filter to obtain a solution which is the acanthopanax bark exosome-like nano vesicle solution.

Further preferably, the acanthopanax exosome-like nano vesicle solution is prepared by the following steps:

s1, cutting root bark parts of cortex acanthopanacis into cortex acanthopanacis small pieces with the cross section of 0.2cm ²～1.0cm², and entering S2;

s2, soaking the acanthopanax bark small blocks obtained in the S1 for 10 hours according to the weight-volume ratio (g/mL) of 1:10-20 of the PBS solution to obtain a soaking solution;

S4, centrifuging the rough filtrate obtained in the step S3 for 30min under the condition that the centrifugal force is 1500g to obtain a first supernatant, filtering the first supernatant by using a filter screen with the aperture of 70 mu m, and filtering the filtered filtrate by using a filter screen with the aperture of 40 mu m to obtain the first filtrate;

S5, centrifuging the first filtrate obtained in the step S4 for 30-30 min under the condition that the centrifugal force is 4000g to obtain a second supernatant, and filtering the second supernatant by using a filter screen with the aperture of 0.7 mu m to obtain the second filtrate;

S6, centrifuging the second filtrate obtained in the step S5 for 60min under the centrifugal force of 10000g to obtain a third supernatant, and filtering the third supernatant by using a filter screen with the aperture of 0.45 mu m to obtain a third filtrate;

S7, centrifuging the third filtrate obtained in the S6 under the vacuum condition of 4 ℃ and the centrifugal force of 100000g for 70min to obtain a first precipitate, and then re-suspending the first precipitate by using a sterile PBS solution to obtain a re-suspended solution;

S8, sequentially adding 2mL of sucrose solution with mass concentration of 15%, 30%, 45% and 60% into a centrifuge tube, adding the resuspended solution obtained in the step S7, centrifuging for 120min under the vacuum condition of 4 ℃ and the centrifugal force of 120000g, and collecting a separation layer between sucrose solution layers with the concentration of 30% and 45% to obtain a purified solution;

s9, diluting the purified solution obtained in the step S8 by using a sterile PBS solution, centrifuging for 150min under the vacuum condition of 4 ℃ and the centrifugal force of 120000g, and collecting a precipitate to obtain a second precipitate;

A second object of the present invention is to provide a method for preparing an osteoinductive medium, which avoids the disadvantages of the prior art. The osteogenesis inducing culture medium can be prepared by a simple method, and the osteogenesis inducing culture medium can improve osteogenesis differentiation effect.

the preparation method of the osteogenesis inducing culture medium comprises the following steps:

step (1), dissolving dexamethasone in absolute ethyl alcohol to obtain a dexamethasone solution;

dissolving L-ascorbic acid in PBS buffer solution to obtain L-ascorbic acid solution;

beta-sodium glycerophosphate is put into PBS buffer solution to obtain beta-sodium glycerophosphate solution;

soaking cortex Acanthopancis in PBS solution to obtain soaking solution, filtering and centrifuging the soaking solution for multiple times to obtain supernatant, performing first time resuspension precipitation operation on the supernatant to obtain solution after resuspension, performing gradient density centrifugation and purification on the solution after resuspension to obtain purified solution, performing dilution precipitation operation on the purified solution by PBS solution to obtain precipitate, and performing second time resuspension precipitation operation on the precipitate to obtain the cortex Acanthopancis exosome-like nano vesicle solution;

And (2) adding fetal bovine serum, penicillin and streptomycin into low-sugar DMEM to obtain a complete culture medium, and then adding the dexamethasone solution, the L-ascorbic acid solution, the beta-sodium glycerophosphate solution and the acanthopanax bark exosome-like nano vesicle solution into the complete culture medium, and uniformly mixing to obtain the osteogenic induction culture medium.

A third object of the present invention is to provide a method for osteoinductive differentiation that avoids the disadvantages of the prior art. The osteogenic induction differentiation method is based on an osteogenic induction culture medium of acanthopanax exosome-like nano vesicles, and can improve the osteogenic differentiation effect of mesenchymal stem cells (hBM-MSCs).

Provides an osteogenic induction differentiation method, which cultures mesenchymal stem cells (hBM-MSCs) by adopting the acanthopanax bark exosome-like nano vesicle osteogenic induction culture medium.

The fourth object of the present invention is to avoid the defects of the prior art and provide an application of acanthopanax exosome-like nano vesicles in preparing osteoinduction culture medium. The acanthopanax bark exosome-like nano vesicle promotes osteogenic induction differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs), and the addition of the acanthopanax bark exosome-like nano vesicle in an osteogenic induction culture medium has a good osteogenic differentiation effect.

Provides the application of acanthopanax exosome-like nano vesicles in preparing osteoinduction culture medium. The acanthopanax exosome-like nano vesicle promotes osteogenic induction differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs).

The fifth purpose of the invention is to avoid the defects of the prior art and provide the application of the acanthopanax exosome-like nano vesicle osteogenesis induction medium in preparing the osteogenesis induction medicine. The acanthopanax exosome-like nano vesicle osteogenesis induction medium can improve osteogenesis differentiation effect, so that the acanthopanax exosome-like nano vesicle osteogenesis induction medium can be used in osteogenesis induction medicines.

The fifth purpose of the invention is to avoid the defects of the prior art and provide the application of the acanthopanax exosome-like nano vesicle osteogenesis induction medium in preparing the osteogenesis induction medicine. The osteogenesis inducing culture medium of the acanthopanax bark exosome-like nano vesicle can improve the osteogenesis differentiation effect, and the osteogenesis inducing medicine prepared from the osteogenesis inducing culture medium of the acanthopanax bark exosome-like nano vesicle has the osteogenesis differentiation effect.

provides the application of the acanthopanax exosome-like nano vesicle osteogenesis induction medium in preparing osteogenesis induction medicines.

The acanthopanax exosome-like nano vesicle osteogenesis induction medium promotes osteogenesis induction differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs).

The application of the acanthopanax exosome-like nano vesicle osteogenesis inducing medium in preparing the medicines for preventing and treating at least one bone disease of osteoporosis, bone defect, bone nonunion or bone delayed healing.

The invention relates to an acanthopanax exosome-like nano vesicle osteogenesis induction culture medium, a preparation method of the acanthopanax exosome-like nano vesicle osteogenesis induction culture medium, an osteogenesis induction differentiation method, application of the acanthopanax exosome-like nano vesicles in preparing the osteogenesis induction culture medium and application of the acanthopanax exosome-like nano vesicle osteogenesis induction culture medium in preparing osteogenesis induction medicines, wherein the acanthopanax exosome-like nano vesicle osteogenesis induction culture medium contains an acanthopanax exosome-like nano vesicle solution, dexamethasone, L-ascorbic acid, beta-sodium glycerophosphate and a complete culture medium. The preparation method of the acanthopanax bark exosome-like nano vesicle solution comprises the steps of immersing the acanthopanax bark in a PBS solution to obtain an immersion liquid, filtering and centrifuging the immersion liquid for multiple times to obtain a supernatant, carrying out first time resuspension precipitation operation on the supernatant to obtain a resuspension solution, carrying out gradient density centrifugation and purification on the solution after the resuspension to obtain a purified solution, carrying out dilution precipitation operation on the purified solution by the PBS solution to obtain a precipitate, and carrying out second time resuspension precipitation operation on the precipitate to finally obtain the acanthopanax bark exosome-like nano vesicle solution. The invention takes the acanthopanax bark exosome-like nano vesicle solution as an addition component of the osteogenic induction medium, can greatly improve the osteogenic differentiation efficiency of mesenchymal stem cells, has obvious osteogenic differentiation effect, has no obvious inhibition effect on proliferation of human bone marrow mesenchymal stem cells (hBM-MSCs), and has good cell compatibility. The acanthopanax exosome-like nano vesicle can be effectively absorbed by human bone marrow mesenchymal stem cells (hBM-MSCs), so that the osteogenesis induction effect is enhanced. The acanthopanax exosome-like nano vesicle osteogenesis inducing medium can be prepared into osteogenesis inducing medicaments for preventing and treating osteoporosis, bone defect, bone nonunion or bone delayed healing.

Drawings

The invention is further illustrated by the accompanying drawings, which are not to be construed as limiting the invention in any way.

Fig. 1 is a transmission electron micrograph of acanthopanax bark exosome-like nanovesicle solution.

FIG. 2 is a graph showing the particle size range and particle concentration distribution of acanthopanax bark exosome-like nanovesicle solution.

FIG. 3 is a graph showing the proliferation activity of acanthopanax exosome-like nanovesicle solution at different concentrations on human bone marrow mesenchymal stem cells (hBM-MSCs).

FIG. 4 is a photomicrograph of an acanthopanax exosome-like nanovesicle solution taken by endocytosis of human bone marrow mesenchymal stem cells (hBM-MSCs).

FIG. 5 is a photograph of osteoinductive differentiation alizarin red staining of acanthopanax bark exosome-like nanovesicle solution pairs at different concentrations.

FIG. 6 is a graph showing the detection of the expression of ALP, a related gene in osteoblastic differentiation of human mesenchymal stem cells (hBM-MSCs) by a real-time fluorescent quantitative PCR method.

FIG. 7 is a graph showing the detection of the expression of the related gene RUNX2 in the differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) by a real-time fluorescent quantitative PCR method.

FIG. 8 is a graph showing the protein expression of differentiation markers of human bone marrow mesenchymal stem cells (hBM-MSCs).

Detailed Description

The technical scheme of the invention is further described with reference to the following examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the examples described below are purchased from conventional biochemical reagent stores or pharmaceutical operators unless otherwise specified. Wherein dexamethasone was purchased from Sigma Co., USA and L-ascorbic acid was purchased from Sigma Co., USA. Sodium beta-glycerophosphate was purchased from Sigma, usa. Ficoll-Histopaque cube-1077 was purchased from Sigma Co. Sterile PBS buffer was purchased from jino corporation, china. Australian Fetal Bovine Serum (FBS) was purchased from Gibco corporation of America. 0.25% -EDTA trypsin was purchased from Gibco corporation of America. Low sugar DMEM medium is purchased from Gibco corporation of America. alpha-MEM medium was purchased from Gibco, inc. of U.S.A. Penicillin/Streptomycin (P/S) was purchased from Gibco corporation of America. BCA protein concentration assay kit (GENSTAR) was purchased from beijing kangrun, china. Cell Counting Kit-8 (CCK 8) kit was purchased from Zeta Life, USA. PKH26 fluorescent labeling dye was purchased from Sigma Co., USA. Anti-Runx2 Anti-body (AF 5186) was purchased from China philic biological company. Anti-ALP Anti (R23427) was purchased from China plus company. Anti-Osteopontin antibody (sc-21142) Santa Cruz, inc. of Santa Cruz, USA. Anti-beta-actin Anti-body (Cat No. 66009-1-Ig) was purchased from Wuhan Sanying corporation of China. NucleoZol RNA extraction reagents were purchased from MACHEREY-NAGEL, germany. Reverse transcription kits were purchased from Northenan Corp. SYBR fluorescent quantitation reagent was purchased from Norpran Corp. Human bone marrow mesenchymal stem cells were approved by the ethical committee of the affiliated hospital of the university of medical science in Guangdong, bone marrow 10 mL was aseptically harvested from the proximal femur of patients undergoing hip replacement with informed consent, monocytes were isolated using Ficoll-Histopaque (d=1.077 g/mL), resuspended in alpha-MEM complete medium (containing 10% vol. Fetal bovine serum and 1% p/S), and sub-cultured in a 37 ℃ 5% CO ₂ incubator. Cortex Acanthopancis is from Heilongjiang river.

Example 1

An osteogenesis inducing culture medium for nanometer vesicle of cortex Acanthopancis comprises nanometer vesicle solution of cortex Acanthopancis, dexamethasone, L-ascorbic acid, sodium beta-glycerophosphate and complete culture medium.

The content of which is as follows:

acanthopanax exosome-like nano vesicle solution (also called AgELNs for short) 1 [ mu ] g/mL-40 [ mu ] g/mL;

dexamethasone 80nM to 120nM;

l-ascorbic acid 40-60 mu M;

8 mM-12 mM of beta-sodium glycerophosphate.

The complete medium is a low-sugar DMEM medium containing 5% -15% of Vol. Fetal bovine serum and 0.5% -2% of Vol. Green streptomycin (also called penicillin and streptomycin).

The preparation method of the acanthopanax bark exosome-like nano vesicle solution comprises the steps of immersing the acanthopanax bark in a PBS solution to obtain an immersion liquid, filtering the immersion liquid and centrifuging for multiple times to obtain a supernatant, carrying out first time resuspension precipitation operation on the supernatant to obtain a resuspended solution, carrying out sucrose gradient density centrifugation and purification on the resuspended solution to obtain a purified solution, carrying out dilution precipitation operation on the purified solution by the PBS solution to obtain a precipitate, and carrying out second time resuspension precipitation operation on the precipitate to obtain the acanthopanax bark exosome-like nano vesicle solution. The preparation method comprises the following steps:

S1, cleaning soil on the surface of fresh Chinese medicinal cortex acanthopanacis by using double distilled water, cutting the root bark part of the cortex acanthopanacis into cortex acanthopanacis small blocks with the cross-sectional area of 0.2cm ²～1.0cm², and entering S2;

S2, soaking the acanthopanax bark small blocks obtained in the S1 with PBS solution for 8-12 hours according to the weight-volume ratio (g/mL) of 1:10-20 to obtain a soaking solution;

S7, centrifuging the third filtrate obtained in the S6 for 50-100 min under the vacuum condition of 4 ℃ and the centrifugal force of 80000 g-120000 g to obtain a first precipitate, and then re-suspending the first precipitate by using a sterile PBS solution to obtain a re-suspended solution;

s8, sequentially adding sucrose solutions with different mass concentrations into a centrifuge tube from low to high, adding the resuspended solution obtained in the step S7, centrifuging for 100-150 min under the vacuum condition of 4 ℃ and the centrifugal force of 100000-150 g, and collecting a separation layer between the intermediate sucrose solution layers to obtain a purified solution;

s9, diluting the purified solution obtained in the step S8 by using a sterile PBS solution, centrifuging for 100-150 min under the vacuum condition of 4 ℃ and the centrifugal force of 100000-150000 g, and collecting a precipitate to obtain a second precipitate;

s10, re-suspending the second precipitate obtained in the step S9 by using a sterile PBS solution, and filtering the re-suspended solution by using a 0.22 mu m filter to obtain a solution which is the acanthopanax bark exosome-like nano vesicle solution, wherein the concentration of the acanthopanax bark exosome-like nano vesicle solution is 4.31 multiplied by 1011 particles/mL, and the average particle size range is 89nm.

The preparation method of the acanthopanax exosome-like nano vesicle osteogenesis induction medium of the embodiment comprises the following steps:

step (1), dexamethasone is prepared, namely dexamethasone is prepared, absolute ethyl alcohol is used for dissolving the dexamethasone to prepare 1mM dexamethasone solution, filtering and sterilizing are carried out through a 0.22 mu m filter membrane, sub-packaging is carried out in an EP tube, and the EP tube is stored at the temperature of minus 20 ℃;

preparing L-ascorbic acid, namely dissolving the L-ascorbic acid with PBS buffer solution to prepare 50mM L-ascorbic acid solution, filtering and sterilizing with a 0.22 mu m filter membrane, subpackaging in an EP tube, and storing at-20 ℃;

the preparation of beta-sodium glycerophosphate comprises dissolving beta-sodium glycerophosphate in PBS buffer solution to obtain 1M beta-sodium glycerophosphate solution, filtering with 0.22 μm filter membrane for sterilization, packaging in EP tube, and storing at-20deg.C.

Preparing an osteogenesis inducing culture medium, namely obtaining the complete culture medium according to the addition amount of fetal calf serum, penicillin and streptomycin in the complete culture medium in low-sugar DMEM, measuring the corresponding concentration amount of dexamethasone, L-ascorbic acid, beta-sodium glycerophosphate and acanthopanax exosome-like nano vesicle solution in the osteogenesis inducing culture medium according to the invention, and uniformly mixing to obtain the required osteogenesis inducing culture medium.

The acanthopanax bark exosome-like nano vesicle osteogenesis induction medium takes the acanthopanax bark exosome-like nano vesicle solution as an osteogenesis induction medium additive component, can greatly improve the osteogenesis differentiation efficiency of mesenchymal stem cells, has obvious osteogenesis differentiation effect, has no obvious inhibition effect on proliferation of human bone marrow mesenchymal stem cells, and has good cell compatibility. The acanthopanax exosome-like nano vesicle can be effectively taken up by human bone marrow mesenchymal stem cells, so that the osteogenesis induction effect of the acanthopanax exosome-like nano vesicle is enhanced. The acanthopanax exosome-like nano vesicle osteogenesis inducing medium can be prepared into osteogenesis inducing medicaments for preventing and treating osteoporosis, bone defect, bone nonunion or bone delayed healing.

Example 2

An osteogenic induction medium for acanthopanax exosome-like nanovesicles, which is otherwise identical to example 1, except for the following contents:

acanthopanax exosome-like nano vesicle solution is 1.0 mug/mL;

Dexamethasone 80nM;

L-ascorbic acid 40. Mu.M;

beta-sodium glycerophosphate 8mM.

The complete medium was a low-sugar DMEM medium containing 5% vol. Foetal calf serum, 0.5% vol. Green streptomycin.

The acanthopanax exosome-like nanovesicle solution of this embodiment is prepared by the following steps:

s1, cleaning soil on the surface of fresh Chinese medicinal cortex acanthopanacis by using double distilled water, cutting the root bark part of the cortex acanthopanacis into cortex acanthopanacis small blocks with the cross-sectional area of 0.2cm ², and entering S2;

s2, soaking the acanthopanax bark small blocks obtained in the step S1 for 12 hours according to the weight-to-volume ratio (g/mL) of the acanthopanax bark small blocks to PBS solution of 1:10 to obtain a soaking solution;

S4, centrifuging the rough filtrate obtained in the step S3 for 30min under the condition that the centrifugal force is 1000g to obtain a first supernatant, filtering the first supernatant by using a filter screen with the aperture of 60 mu m, and filtering the filtered filtrate by using a filter screen with the aperture of 40 mu m to finally obtain a first filtrate;

S5, centrifuging the first filtrate obtained in the step S4 for 60min under the condition that the centrifugal force is 3000g to obtain a second supernatant, and then filtering the second supernatant by using a filter screen with the aperture of 0.7 mu m in sequence to obtain a second filtrate;

S6, centrifuging the second filtrate obtained in the step S5 for 60min under the centrifugal force of 8000g to obtain a third supernatant, and filtering the third supernatant by using a filter screen with the aperture of 0.6 mu m to obtain a third filtrate;

S7, centrifuging the third filtrate obtained in the S6 for 100min under the vacuum condition of 4 ℃ and the centrifugal force of 80000g to obtain a first precipitate, and then re-suspending the first precipitate by using a sterile PBS solution to obtain a re-suspended solution;

S8, sequentially adding 2mL of sucrose solution with mass concentration of 20%, 30%, 45% and 60% into a centrifuge tube from low to high, adding the resuspended solution obtained in the step S7, centrifuging for 150min under the vacuum condition of 4 ℃ and the centrifugal force of 100000g, and collecting a separation layer between sucrose solution layers with the concentration of 30% and 45% to obtain a purified solution;

s9, diluting the purified solution obtained in the step S8 by using a sterile PBS solution, centrifuging for 150min under the vacuum condition of 4 ℃ and the centrifugal force of 100000g, and collecting a precipitate to obtain a second precipitate;

S10, re-suspending the second precipitate obtained in the step S9 by using a sterile PBS solution, and filtering the re-suspended solution by using a 0.22 mu m filter to obtain a solution which is the acanthopanax exosome-like nano vesicle solution.

The preparation method of the acanthopanax exosome-like nanovesicle osteogenesis inducing medium of this example is the same as that of example 1.

Example 3

acanthopanax exosome-like nano vesicle solution of 2.5 mug/mL;

Dexamethasone 100nM;

L-ascorbic acid 50. Mu.M;

10mM sodium beta-glycerophosphate.

Wherein the complete medium is a low-sugar DMEM medium containing 10% Vol. Fetal calf serum and 1% Vol. Green streptomycin.

S1, cleaning soil on the surface of fresh Chinese medicinal cortex acanthopanacis by using double distilled water, cutting the root bark part of the cortex acanthopanacis into cortex acanthopanacis small blocks with the cross-sectional area of 0.5cm ², and entering S2;

s2, soaking the acanthopanax bark small blocks obtained in the step S1 for 8 hours according to the weight-to-volume ratio (g/mL) of 1:15 of the PBS solution to obtain a soaking solution;

S4, centrifuging the rough filtrate obtained in the step S3 for 30min under the condition that the centrifugal force is 1500g to obtain a first supernatant, filtering the first supernatant by using a filter screen with the aperture of 70 mu m, and filtering the filtered filtrate by using a filter screen with the aperture of 40 mu m to finally obtain a first filtrate;

s5, centrifuging the first filtrate obtained in the step S4 for 30min under the condition that the centrifugal force is 4000g to obtain a second supernatant, and filtering the second supernatant by using a filter screen with the aperture of 0.7 mu m to obtain a second filtrate;

S6, centrifuging the second filtrate obtained in the step S5 for 60min under the centrifugal force of 10000g to obtain a third filtrate, and filtering a third supernatant by using a filter screen with the aperture of 0.45 mu m to obtain the third filtrate;

S8, sequentially adding 2mL of sucrose solution with mass concentration of 15%, 30%, 45% and 60% into a centrifuge tube from low to high, adding the resuspended solution obtained in the step S7, centrifuging for 120min under the vacuum condition of 4 ℃ and the centrifugal force of 120000g, and collecting a separation layer between sucrose solution layers with the concentration of 30% and 45% to obtain a purified solution;

Example 4

5.0 mug/mL of acanthopanax exosome-like nano vesicle solution;

Dexamethasone 100nM;

L-ascorbic acid 50. Mu.M;

10mM sodium beta-glycerophosphate.

s2, soaking the acanthopanax bark small blocks obtained in the step S1 for 10 hours according to the weight-to-volume ratio (g/mL) of 1:15 of the PBS solution to obtain a soaking solution;

Example 5

10.0 mug/mL of acanthopanax exosome-like nano vesicle solution;

Dexamethasone 120nM;

l-ascorbic acid 60. Mu.M;

Beta-sodium glycerophosphate 12mM.

Wherein the complete medium is a low-sugar DMEM medium containing 15% Vol. Fetal calf serum and 2% Vol. Green streptomycin.

The acanthopanax exosome-like nanovesicle solution of the example was prepared by the following steps:

S2, soaking the acanthopanax bark small blocks obtained in the step S1 for 9 hours according to the weight-to-volume ratio (g/mL) of the acanthopanax bark small blocks to PBS solution of 1:20 to obtain a soaking solution;

S4, centrifuging the rough filtrate obtained in the step S3 for 15min under the condition that the centrifugal force is 2000g to obtain a first supernatant, filtering the first supernatant by using a filter screen with the aperture of 60 mu m, and filtering the filtered filtrate by using a filter screen with the aperture of 40 mu m to finally obtain a first filtrate;

S5, centrifuging the first filtrate obtained in the step S4 for 30min under the condition that the centrifugal force is 5000g to obtain a second supernatant, and filtering the second supernatant by using a filter screen with the aperture of 0.6 mu m to obtain a second filtrate;

S6, centrifuging the second filtrate obtained in the step S5 for 30min under the condition that the centrifugal force is 12000g to obtain third filtrate, and filtering the third supernatant by using a filter screen with the aperture of 0.5 mu m to obtain third filtrate;

S7, centrifuging the third filtrate obtained in the S6 under the vacuum condition of 4 ℃ and the centrifugal force of 120000g for 50min to obtain a first precipitate, and then re-suspending the first precipitate by using a sterile PBS solution to obtain a re-suspended solution;

s8, sequentially adding 2mL of sucrose solution with mass concentration of 10%, 30%, 45% and 70% into a centrifuge tube from low to high, adding the resuspended solution obtained in the step S7, centrifuging for 100min under the vacuum condition of 4 ℃ and the centrifugal force of 150000g, and collecting a separation layer between sucrose solution layers with the concentration of 30% and 45% to obtain a purified solution;

s9, diluting the purified solution obtained in the step S8 by using a sterile PBS solution, centrifuging for 100min under the vacuum condition of 4 ℃ and the centrifugal force of 150000g, and collecting a precipitate to obtain a second precipitate;

Example 6

An osteogenic induction medium for acanthopanax exosome-like nanovesicles has the same other characteristics as in example 3, except that the acanthopanax exosome-like nanovesicle solution is 20.0 μg/mL, and the other components are the same as in example 3.

S1, cleaning soil on the surface of fresh Chinese medicinal cortex acanthopanacis by using double distilled water, cutting the root bark part of the cortex acanthopanacis into cortex acanthopanacis small blocks with the cross-sectional area of 1.0cm ², and entering S2;

s2, soaking the acanthopanax bark small blocks obtained in the step S1 for 10 hours according to the weight-to-volume ratio (g/mL) of the acanthopanax bark small blocks to PBS solution of 1:18 to obtain a soaking solution;

s4, centrifuging the rough filtrate obtained in the step S3 for 20min under the condition that the centrifugal force is 1500g to obtain a first supernatant, filtering the first supernatant by using a filter screen with the aperture of 60 mu m, and filtering the filtered filtrate by using a filter screen with the aperture of 50 mu m to finally obtain a first filtrate;

S5, centrifuging the first filtrate obtained in the step S4 for 40min under the condition that the centrifugal force is 4000g to obtain a second supernatant, and filtering the second supernatant by using a filter screen with the aperture of 0.6 mu m to obtain a second filtrate;

S6, centrifuging the second filtrate obtained in the step S5 for 40min under the condition that the centrifugal force is 11000g to obtain third filtrate, and filtering the third supernatant by using a filter screen with the aperture of 0.5 mu m to obtain third filtrate;

S7, centrifuging the third filtrate obtained in the S6 for 80 minutes under the vacuum condition of 4 ℃ and the centrifugal force of 110000g to obtain a first precipitate, and then re-suspending the first precipitate by using a sterile PBS solution to obtain a re-suspended solution;

S8, sequentially adding 2mL of sucrose solution with mass concentration of 10%, 30%, 45% and 70% into a centrifuge tube from low to high, adding the resuspended solution obtained in the step S7, centrifuging for 120min under the vacuum condition of 4 ℃ and the centrifugal force of 140000g, and collecting a separation layer between sucrose solution layers with the concentration of 30% and 45% to obtain a purified solution;

S9, diluting the purified solution obtained in the step S8 by using a sterile PBS solution, centrifuging for 120min under the vacuum condition of 4 ℃ and the centrifugal force of 140000g, and collecting a precipitate to obtain a second precipitate;

The preparation method of the acanthopanax exosome-like nanovesicle osteogenesis inducing medium of this example is the same as that of example 3.

Example 7

An osteogenic induction medium for acanthopanax exosome-like nanovesicles has the same other characteristics as in example 1, except that the acanthopanax exosome-like nanovesicle solution is 40.0 mug/mL, and the other components are the same as in example 3. The preparation method of the acanthopanax bark exosome-like nano vesicle solution and the acanthopanax bark exosome-like nano vesicle osteogenesis inducing medium of the present embodiment is the same as that of embodiment 3.

Comparative example 1

An osteogenesis inducing medium was the same as in example 1 except that the acanthopanax exosome-like nanovesicles were 0. Mu.g/mL and the other components were the same as in example 3.

Preparing L-ascorbic acid, namely dissolving the L-ascorbic acid with PBS buffer solution to prepare 50 mML-ascorbic acid solution, filtering and sterilizing the solution with a 0.22 mu m filter membrane, subpackaging the solution in an EP tube, and storing the solution at-20 ℃;

Preparing an osteogenesis inducing culture medium, namely obtaining the complete culture medium according to the addition amount of fetal calf serum, penicillin and streptomycin in the complete culture medium in low-sugar DMEM, measuring the corresponding concentration amount of dexamethasone, L-ascorbic acid and beta-sodium glycerophosphate in the osteogenesis inducing culture medium according to the embodiment, and uniformly mixing to obtain the required osteogenesis inducing culture medium.

Detection step and result analysis

1. Morphological observation under transmission electron microscope of acanthopanax exosome-like nanovesicles

The acanthopanax bark exosome-like nano vesicles are dripped on a special carrier net of a Transmission Electron Microscope (TEM) and are naturally adsorbed for 5min. Then, uranium acetate was used for negative dyeing for 5min. After the dyeing is completed, the dyeing liquid is removed, and the carrying net is placed in a drying oven to be dried for 20min. After drying, the morphology of the sample was observed using a transmission electron microscope, as shown in fig. 1.

As shown in fig. 1, the acanthopanax bark-derived exosome-like nanoparticle solution (AgELNs) of the present invention shows a surface depression and a disc-like vesicle structure under a transmission electron microscope.

2. Particle size range and concentration detection of acanthopanax bark exosome-like nano vesicles

The acanthopanax exosome-like nanovesicle solution (AgELNs) of the present invention was diluted 100-fold with PBS solution. Single particle size analysis was performed using a Nano Coulter nanoparticle analyzer to obtain particle size range and particle concentration data for the samples, as shown in fig. 2.

As can be seen from fig. 2, the concentration of acanthopanax bark-derived exosome-like nano-particles in the acanthopanax bark exosome-like nano-vesicle solution (AgELNs) is 4.31×1011 particles/mL, and the average particle diameter range is 89 nm.

3. Protein concentration determination of acanthopanax exosome-like nano vesicle

The obtained acanthopanax bark-derived exosome-like nanoparticle solution (AgELNs) is quantified by a BCA protein detection kit, and the specific steps are as follows:

(1) Preparation of standards protein standards (2 mg/mL) were diluted with sterile PBS buffer to final concentrations of 0mg/mL, 0.025mg/mL, 0.05mg/mL, 0.1mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, respectively. Standard for each concentration 20 μl was added to the standard wells of a 96-well plate to prepare a standard protein concentration profile.

(2) Sample preparation the acanthopanax exosome-like nanovesicle solution (AgELNs) of the invention is dissolved in sterile PBS buffer, and 20 μl of the sample is added into the sample well of the 96-well plate.

(3) And (3) reacting the BCA working solution, namely adding 200 mu L of BCA working solution into each hole, and incubating for 20-30 min at 37 ℃.

(4) Absorbance determination absorbance at 562 nm was determined using a microplate reader.

(5) Protein concentration calculation, namely calculating the protein concentration in the sample according to a standard protein concentration curve.

The BCA quantitative protein concentration is used for measuring and calculating the using amount of the acanthopanax bark derived exosome-like nano particle solution (AgELNs) in the subsequent dosing treatment experiment, and finally the protein concentration of the acanthopanax bark exosome-like nano vesicle solution extracted by the differential ultracentrifugation method is measured to be 0.547mg/mL.

4. Effect of acanthopanax exosome-like nanovesicle solution (AgELNs) on proliferation activity of human bone marrow mesenchymal stem cells (hBM-MSCs)

(1) And adding the acanthopanax bark exosome-like nano vesicles with the concentration of 0 mug/mL, 2.5 mug/mL, 5 mug/mL, 10 mug/mL, 20 mug/mL and 40 mug/mL into the alpha-MEM complete culture medium respectively to obtain the proliferation culture medium with the corresponding concentration of the acanthopanax bark exosome-like nano vesicles. And simultaneously taking alpha-MEM complete culture medium as a blank control.

(2) Cell seeding 3000 hBM-MSCs per well in 96-well plates. After cells are grown for 12 hours in an adherence way, the osteogenesis culture medium with different acanthopanax exosome-like nano vesicle concentrations is used, and the complete culture medium in the osteogenesis culture medium contains 10% Vol. Fetal calf serum and 1% Vol. Green streptomycin, and is cultured under the conditions of 37 ℃ and 5% CO ₂. The effect of acanthopanax exosome-like nanovesicle solution (AgELNs) on proliferation activity of human bone marrow mesenchymal stem cells (hBM-MSCs) was detected using CCK8 kit at 72 hours after treatment, respectively, to obtain fig. 3.

The proliferation activity of acanthopanax bark exosome-like nano vesicle concentration (AgELNs) on human bone marrow mesenchymal stem cells (hBM-MSCs) is observed through a CCK-8 kit, and then the absorbance value of cells at 450 nm is detected through an enzyme-labeled instrument, so that the number of living cells can be indirectly reflected, and whether the proliferation activity of the acanthopanax bark exosome-like nano vesicle concentration extracted through a differential ultracentrifugation method on the human bone marrow mesenchymal stem cells is influenced or not is verified, so that the safety of the acanthopanax bark exosome-like nano vesicle on the cells is evaluated. The CCK8 method in FIG. 3 shows that even when the maximum concentration of the acanthopanax exosome-like nano vesicle solution (AgELNs) is 40 mug/mL, the proliferation of human bone marrow mesenchymal stem cells (hBM-MSCs) is not obviously inhibited.

5. Uptake internalization experiment of acanthopanax exosome-like nanovesicle solution (AgELNs)

The ingestion internalization experiment is carried out according to the requirements of a reagent instruction book, and comprises the following steps:

(1) And (3) preparing a staining working solution, namely uniformly mixing 4 mu L of PKH26 fluorescent labeling dye with 200 mu L of Diluent C, uniformly mixing 100 mu L of PBS diluted AgELNs with 200 mu L of Diluent C, adding the working solution, uniformly mixing into a 500 mu L system, and incubating for 15min at normal temperature in a dark place.

(2) The staining was stopped and washed, and 500. Mu.L of exosome-free serum was added to stop the reaction, and diluted well with PBS solution. Unbound dye was removed by ultracentrifugation at 100,000 g for 70min under vacuum at 4 ℃ and AgELNs (PKH 26-AgELNs) labeled with PKH26 fluorescent labeling dye was collected.

(3) Cell processing, namely adding PKH26-AgELNs into a prepared complete culture medium to act on hBM-MSCs. After 6 hours, the culture supernatant was aspirated, the cells were washed 2 times with sterile PBS, fixed with 4% paraformaldehyde for 20min, washed 5 min/times X3 times with PBS, and the fixative was washed thoroughly.

(4) And (3) staining, namely adding DAPI solution to stain the cell nuclei, incubating for 30min at room temperature in a dark place, and fully washing the staining solution with PBS.

(5) Microscopic observations in a slightly moist state, the intracellular PKH26-AgELNs was observed and photographed under an inverted fluorescence microscope to give FIG. 4.

PKH26 marked acanthopanax exosome-like nano vesicle solution is dark gray, DAPI marked human bone marrow mesenchymal stem cells are bright gray, merge is positioned by using DAPI as cells, and whether the acanthopanax exosome-like nano vesicle solution marked by PKH26 is ingested by the cells is observed. The PKH26 fluorescent dye labeling experiment result in FIG. 4 shows that acanthopanax exosome-like nano vesicle solution (AgELNs) can be smoothly taken in into cells by human bone marrow mesenchymal stem cells (hBM-MSCs).

6. Effect of acanthopanax exosome-like nano vesicle solution (AgELNs) on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs)

(1) And evaluating the effect of osteogenic differentiation, namely taking 5 th generation hBM-MSCs with good in-vitro expansion growth, inoculating the hBM-MSCs into a 12-well plate according to the cell quantity of 5 multiplied by 10 ^{^4} per hole, and culturing the hBM-MSCs in a cell incubator with 37 ℃ and 5% CO ₂ saturated humidity. When the cells are grown to 80% fusion by adherence, the supernatant of the original culture medium is sucked, and an osteogenic differentiation culture medium (namely, the osteogenic differentiation culture medium obtained in comparative example 1, example 3, example 4 and example 5) with the solution concentration of the acanthopanax exosome-like nano vesicle of 0 mug/mL, 2.5 mug/mL, 5 mug/mL and 10 mug/mL is added for induction culture, and the osteogenic differentiation culture medium is replaced every 3 days. After 14 days of continuous induction, cells were fixed with 75% alcohol and stained with alizarin red to identify osteogenic differentiation effects, resulting in fig. 5.

The alizarin red staining result in fig. 5 shows that calcium salt deposition after osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) induced by acanthopanax exosome-like nano vesicle solution (AgELNs) treatment is increased compared with the control group.

(2) And (3) gene expression detection:

RNA sample collection RNA samples were collected according to the instructions of the reagent manufacturers after 5 days of culture in osteogenic differentiation medium (i.e., osteogenic differentiation medium obtained in comparative example 1, example 3, example 4, example 5) containing acanthopanax bark exosome-like nanovesicle solution at a concentration of 0. Mu.g/mL, 2.5. Mu.g/mL, 5.0. Mu.g/mL, 10.0. Mu.g/mL.

B. reverse transcription, transferring the extracted RNA sample into enzyme-free EP tube, adding DEPC water, standing for 5min, centrifuging at 4deg.C with centrifugal force of 12000 g for 15min, collecting supernatant into new enzyme-free EP tube, adding equal amount of isopropanol, precipitating for 10min, and centrifuging again. The RNA was washed three times with 75% DEPC ethanol after discarding the supernatant, and the RNA concentration and purity were measured with NanoDrop100 after air-drying. 1. Mu.g of RNA was used for reverse transcription, 4 XgDNA was added to remove genomic DNA, DEPC water was added to 16. Mu.L, the reaction was carried out at 42℃for 2min, 5 XqPCR Mix was added to carry out 37℃for 15min and 85℃for 15s, and cDNA samples were produced.

C. Real-time fluorescent quantitative PCR (qRT-PCR) 20. Mu.L of reaction system was prepared according to the following conditions:

d. After the amplification cycle, the gene expression of the osteogenic related markers (ALP, RUNX 2) was examined using GAPDH as housekeeping gene and statistical data using the 2- (^△△ CT) method, as shown in FIGS. 6 and 7.

(3) Protein level detection

A. cell lysis and protein extraction 5 th generation human bone marrow mesenchymal stem cells (hBM-MSCs) were inoculated into 6 cm dishes at a density of 3X 10 ^{^5}/well and induced to culture in osteogenic induction medium of comparative example 1 (0. Mu.g/mL), example 3 (2.5. Mu.g/mL), example 4 (5.0. Mu.g/mL), example 5 (10.0. Mu.g/mL) for 12 days. Cells were collected, lysed with RIPA buffer and sonicated, centrifuged at 12000 g for 15min at 4 ℃ to remove cell debris, and protein concentration was measured.

SDS-PAGE and protein transfer about 20. Mu.g of the protein samples were prepared and subjected to SDS-PAGE gel electrophoresis, and the proteins were transferred to 0.22 μm PVDF membrane. After blocking with skimmed milk, primary antibodies (ALP, RUNX2, beta-actin) were incubated overnight at 4 ℃. Unbound primary antibody was washed away with TBST (10 min/time, 3 total), the secondary antibody was incubated at room temperature for 1 hour, and unbound secondary antibody was washed away again with TBST. The target strip was acquired using LiCOR Odyssey DLx near infrared imaging system to obtain fig. 8.

As can be seen from FIGS. 6-8, the results of verifying the osteogenic related markers by qRT-PCR (FIGS. 6-7) and Western blot (FIG. 8) show that the expression of ALP and RUNX2 in hBMSCs treated with acanthopanax exosome-like nanovesicle solution is increased and increased in concentration dependence. And as can be seen from fig. 6 and 7, the osteogenic effect of the acanthopanax exosome-like nanovesicle solution at a concentration of 10 μg/mL showed a decreasing trend compared to the osteogenic-related marker at a concentration of 5.0 μg/mL.

In conclusion, the osteogenic induction culture medium with the acanthopanax bark exosome-like nano vesicle solution as an additive component can greatly improve the osteogenic differentiation efficiency of human bone marrow mesenchymal stem cells (hBM-MSCs), has obvious osteogenic differentiation effect, has no obvious inhibition effect on proliferation of human bone marrow mesenchymal stem cells (hBM-MSCs), and has good cell compatibility. The acanthopanax exosome-like nano vesicle can be effectively absorbed by human bone marrow mesenchymal stem cells (hBM-MSCs), so that the osteogenesis induction effect is enhanced.

Example 8

An osteoinductive differentiation method, which cultures mesenchymal stem cells using an acanthopanax exosome-like nanovesicle osteoinductive medium as in any one of examples 1 to 7. The mesenchymal stem cells are human bone marrow mesenchymal stem cells (hBM-MSCs).

The osteogenic induction differentiation method is carried out by adopting the osteogenic induction culture containing the acanthopanax bark exosome-like nano vesicles, can greatly improve the osteogenic differentiation efficiency of mesenchymal stem cells, has obvious osteogenic differentiation effect, has no obvious inhibition effect on proliferation of human bone marrow mesenchymal stem cells (hBM-MSCs) by the acanthopanax bark exosome-like nano vesicles, and has good cell compatibility. The acanthopanax exosome-like nano vesicle can be effectively absorbed by human bone marrow mesenchymal stem cells (hBM-MSCs), so that the osteogenesis induction effect is enhanced.

Example 9

Application of acanthopanax exosome-like nano vesicles in preparing osteoinduction culture medium, the acanthopanax exosome-like nano vesicles promote osteogenic induction differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). Wherein the acanthopanax exosome-like nanovesicles and the osteogenic induction medium are obtained by the preparation methods of examples 1 to 7.

The acanthopanax bark exosome-like nano vesicle solution (AgELNs) is added into an osteogenesis induction culture medium through the proliferation activity data, the ingestion internalization experimental data and the osteogenesis differentiation effect result of the acanthopanax bark exosome-like nano vesicle solution on human bone marrow mesenchymal stem cells (hBM-MSCs), can greatly improve the osteogenic differentiation efficiency of mesenchymal stem cells, has obvious osteogenic differentiation effect, and the acanthopanax bark exosome-like nano vesicle has no obvious inhibition on the proliferation of human bone marrow mesenchymal stem cells (hBM-MSCs), and has good cell compatibility. The acanthopanax exosome-like nano vesicle can be effectively absorbed by human bone marrow mesenchymal stem cells (hBM-MSCs), so that the osteogenesis induction effect is enhanced.

Example 10

Use of acanthopanax exosome-like nanovesicle osteogenesis inducing medium as in examples 1 to 7 in the preparation of an osteogenesis inducing medicament. The acanthopanax exosome-like nano vesicle osteogenesis inducing culture medium promotes osteogenesis induced differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). Use of cortex Acanthopancis exosome-like nanovesicle osteogenesis inducing medium in preparing medicine for preventing and treating at least one bone disease selected from osteoporosis, bone defect, bone nonunion and delayed bone healing.

The acanthopanax bark exosome-like nano vesicle solution (AgELNs) can greatly improve the osteogenic differentiation effect of mesenchymal stem cells, can obviously improve the osteogenic differentiation effect, has no obvious inhibition effect on the proliferation of human bone marrow mesenchymal stem cells, and has good cell compatibility through the acanthopanax bark exosome-like nano vesicle osteogenic induction culture medium provided by the invention. Therefore, when the acanthopanax exosome-like nano vesicle osteogenesis induction medium is prepared into an osteogenesis inducing medicament, the acanthopanax exosome-like nano vesicle osteogenesis inducing medium also has an osteogenesis differentiation effect, and provides a new technical means and theoretical basis for preventing and treating osteoporosis, bone defect, bone nonunion or delayed bone healing.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. An osteogenic induction differentiation method is characterized in that an osteogenic induction culture medium of acanthopanax exosome-like nano vesicle is adopted to culture mesenchymal stem cells, wherein the mesenchymal stem cells are human bone marrow mesenchymal stem cells;

the acanthopanax exosome-like nano vesicle osteogenesis induction medium is an acanthopanax exosome-like nano vesicle solution, dexamethasone, L-ascorbic acid, beta-sodium glycerophosphate and a complete medium;

The content is as follows:

the acanthopanax exosome-like nano vesicle solution is 2.5 mu g/mL-10 mu g/mL;

dexamethasone 80nM to 120nM;

l-ascorbic acid 40-60 mu M;

8 mM-12 mM of beta-sodium glycerophosphate;

The complete culture medium is a low-sugar DMEM culture medium containing 5-15% of Vol. Fetal calf serum and 0.5-2% of Vol. Green streptomycin;

The preparation method of the acanthopanax bark exosome-like nano vesicle solution comprises the steps of soaking acanthopanax bark in PBS solution to obtain soaking solution, filtering the soaking solution and centrifuging for multiple times to obtain supernatant, carrying out first time resuspension precipitation operation on the supernatant to obtain a resuspended solution, carrying out sucrose gradient density centrifugation and purification on the resuspended solution to obtain purified solution, carrying out dilution precipitation operation on the purified solution by the PBS solution to obtain precipitate, and carrying out second time resuspension precipitation operation on the precipitate to obtain the acanthopanax bark exosome-like nano vesicle solution;

the concentration of the acanthopanax bark-derived exosome-like nano-particles in the acanthopanax bark exosome-like nano-vesicle solution is 4.31 multiplied by 1011 particles/mL, and the average particle size range is 89 nm.

2. The osteoinductive differentiation method according to claim 1, wherein the acanthopanax exosome-like nanovesicle solution is prepared by specifically:

s1, cutting cortex acanthopanacis into pieces, and entering S2;

3. The osteoinductive differentiation method of claim 2, wherein the acanthopanax exosome-like nanovesicle osteoinductive medium comprises the following contents:

the acanthopanax exosome-like nano vesicle solution is 2.5 mug/mL-5.0 mug/mL;

Dexamethasone 100nM;

L-ascorbic acid 50. Mu.M;

10mM sodium beta-glycerophosphate;

4. The application of the acanthopanax exosome-like nano vesicles in preparing an osteoinduction culture medium is characterized in that the acanthopanax exosome-like nano vesicles promote osteogenic induction differentiation of human bone marrow mesenchymal stem cells;

The acanthopanax exosome-like nano vesicles improve osteogenic induction differentiation of human bone marrow mesenchymal stem cells by up-regulating the expression level of ALP and RUNX2 of the human bone marrow mesenchymal stem cells;

the concentration of the acanthopanax bark-derived exosome-like nano particles in the acanthopanax bark exosome-like nano vesicle solution is 4.31 multiplied by 1011 particles/mL, and the average particle size range is 89 nm;

the content of the acanthopanax exosome-like nano vesicle solution is 2.5 mu g/mL-10 mu g/mL.