CN111849882A - Mesenchymal stem cell exosome and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a mesenchymal stem cell exosome and a preparation method and application thereof, belonging to the technical field of biomedicine. The invention separates and cultures bone marrow mesenchymal stem cells, adipose mesenchymal stem cells and skin mesenchymal stem cells in vitro, extracts exosomes in cell culture supernatant through an ultracentrifugation method, and administers the drug in a joint cavity administration mode. The exosomes derived from the three mesenchymal stem cells can obviously relieve relevant indications of the aged knee-joint worn rat, such as improvement of Mankin score of the aged knee-joint worn model mouse, inhibition of inflammatory factors and activation of RANK system.
Description
Technical Field
The invention relates to a mesenchymal stem cell exosome and a preparation method and application thereof, belonging to the technical field of biomedicine.
Background
According to the World Health Organization (WHO) statistics, there are approximately 3.55 billion osteoarthritis patients worldwide, a progressive and debilitating joint disease, which is expected to affect up to 25% of the population by 2040 years. The knee joint is the most complex joint of the human body and bears 60 percent of the weight of the human body, so the knee joint is extremely easy to wear and damage, the earliest and most major pathological changes are in articular cartilage, the cartilage has almost no regeneration capacity, and after diseases occur, the pathological changes can not be reversed along with the growth of the age, and the knee joint osteoarthritis can be seriously caused. The incidence of knee osteoarthritis is mostly old people, and according to statistics, the incidence rate of the knee osteoarthritis is 60% in people over 65 years old and 80% in people over 75 years old. This may be associated with slow blood circulation in the elderly, aging of the bone joints and poor knee activity. Knee osteoarthritis is a common chronic degenerative disease which seriously affects the life quality of old people, and is mainly manifested by knee stiffness, swelling, pain, limited movement and difficulty in walking, and even a person lying in bed for a long time can have contracture, muscular atrophy and the like of the knee joint. Knee osteoarthritis can only be improved through medicines at present, old patients with severe osteoarthritis need to be treated through operations, and artificial total knee replacement is a main means for treating various late-stage knee joint lesions at present, but complications of different degrees can still occur after the operations. At present, no satisfactory treatment strategy can completely cure the senile knee osteoarthritis. Therefore, the molecular mechanism of the occurrence and development of the senile knee osteoarthritis is further deeply researched, and the search for a novel treatment means for treating the senile knee osteoarthritis has very important scientific significance and clinical application value.
The stem cell is a kind of multipotential cell with self-replicating ability, under a certain condition, it can be differentiated into various functional cells, and has the potential function of regenerating various tissues and organs, and can repair and replace the tissues and organs with pathological changes, senility and injury, so that it is the main source and means for curing various difficult and complicated diseases in the future medical field. Mesenchymal stem cells are important members of the stem cell family, derived from the mesoderm, and are the ideal seed cells for regenerative medicine. Mesenchymal stem cells are present in various tissues such as bone marrow, placenta, umbilical cord blood, fat, skin, etc., and although mesenchymal stem cells can be isolated from placenta, umbilical cord and umbilical cord blood, their sources are limited. Bone marrow, fat and skin tissues also contain a large amount of mesenchymal stem cells, which can be derived from the self, are not limited by sources and have no rejection reaction, thereby forming a hot spot in medical research.
However, stem cell therapy has many considerable problems, such as abnormal differentiation of stem cells themselves, immune rejection, and long-term storage and transportation of stem cells. For the above reasons, it is important to find a therapeutic approach to replace stem cells. Exosomes are vesicular vesicles of lipid bilayers secreted by most cells in the body, approximately 40-150nm in diameter, whose function depends on the cell type from which they are derived. The vesicular vesicles contain various proteins (such as various enzymes, lipids, growth factors and cytokines) and some coding and non-coding RNAs (ribonucleic acids), so that a brand-new cell-cell information transmission system is formed, the physiological state of cells is influenced, and the occurrence and the progress of various diseases are closely related. Currently, more and more studies indicate that exosomes play a crucial role in the early diagnosis and treatment of disease.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a mesenchymal stem cell exosome and a preparation method and application thereof.
In order to solve the technical problems, the invention provides a preparation method of a mesenchymal stem cell exosome, which comprises the following steps: and (2) separating and culturing mesenchymal stem cells in vitro, and extracting exosomes in cell culture solution supernatant by an ultracentrifugation method, wherein the mesenchymal stem cells comprise bone marrow mesenchymal stem cells, skin epidermis mesenchymal stem cells and adipose mesenchymal stem cells.
Preferably, the method for isolating and culturing the mesenchymal stem cells comprises the following steps:
taking bilateral femurs and shinbones of rats, washing bone marrow with DMEM/F-12 culture solution containing 10% FBS, placing the bone marrow in a culture dish, and filtering; centrifuging the filtrate, discarding the supernatant, resuspending the cells in DMEM/F-12 culture solution containing 10% FBS, and culturing in a cell culture box; after 3 days, the liquid is changed for the first time, and then every other day, when the bottom of the bottle is full of cells, the ratio of the liquid to the cell is 1: 2, passage in proportion; analyzing and screening by a flow cytometer to obtain the bone marrow mesenchymal stem cells.
Preferably, the method for isolating and culturing adipose-derived mesenchymal stem cells comprises the following steps:
washing subcutaneous adipose tissues taken down in surgery with PBS solution to remove impurities, shearing the extracted adipose tissues, transferring the sheared adipose tissues into a screw injector, connecting a fat emulsifier, repeatedly injecting to break the adipose tissues, and filtering to obtain adipose mesenchymal stem cell suspension; centrifuging, removing supernatant, adding 10% FBS-containing DMEM medium for resuspension, inoculating into culture flask, standing at 37 deg.C and 5% CO2Culturing in an incubator; and (3) after 24h, replacing the culture medium, removing nonadherent cells, replacing the culture medium every other two days for 1 time, and digesting with pancreatin until the fusion degree reaches 80-90 percent and the ratio of 1: 3, carrying out passage; detecting cell phenotype by a flow cytometer to obtain the adipose-derived stem cells.
Preferably, the method for isolating and culturing the skin epidermal mesenchymal stem cells comprises the following steps:
washing and soaking fresh skin tissue taken down in operation with PBS containing double antibody under aseptic condition, separating epidermis, cutting epidermis into pieces, adding 0.05% recombinant trypsin containing 0.02% EDTA, digesting at 37 deg.C for 30min, terminating digestion with culture medium containing 10% FBS, filtering suspension cells to obtain single cell suspension; after centrifugation, suspending the cells by using a KSFM culture medium, inoculating the cells into an IV collagen-coated culture bottle for culture for 10min, sucking the upper non-adherent cells, adding the KSFM culture medium for continuous culture, changing the culture solution every other day until the fusion degree reaches 80-90%, digesting by using pancreatin, and performing the steps of 1: 3, carrying out passage; analyzing and screening by a flow cytometer to obtain the skin epidermal mesenchymal stem cells.
Preferably, the specific method for extracting exosomes in cell culture solution supernatant comprises the following steps:
filtering the stem cell culture solution, and centrifuging the filtrate at high speed and gradient, at first, centrifuging 300g for 10min at 4 ℃, and taking the supernatant; centrifuging at 2000g for 30min, and collecting supernatant; centrifuging at 10000g for 30min, and collecting supernatant; centrifuging at 140000g for 120min, and discarding the supernatant to obtain a precipitate, namely an exosome secreted by the mesenchymal stem cells; then, the obtained exosomes were resuspended and washed with precooled PBS, and centrifuged again at 140000g for 120 min; finally, the suspension was resuspended in 50. mu.l of 4 ℃ PBS and transferred to a low-adhesion tube to obtain exosomes.
The invention also provides the mesenchymal stem cell exosome prepared by the method.
The invention also provides application of the mesenchymal stem cell exosome in preparing a medicine for treating senile knee joint abrasion.
The invention also provides a medicament containing the mesenchymal stem cell exosome.
The invention achieves the following beneficial effects: the invention separates and cultures bone marrow-derived mesenchymal stem cells, skin epidermis-derived mesenchymal stem cells and fat-derived mesenchymal stem cells in vitro, extracts exosomes in cell culture supernatant by a high-speed gradient centrifugation method, and administers the medicament by a joint cavity administration mode. The exosomes derived from the three mesenchymal stem cells can obviously relieve relevant indications of the aged knee-joint worn rat, such as improvement of Mankin score of the aged knee-joint worn model mouse, inhibition of inflammatory factors and activation of RANK system.
Drawings
Figure 1 effect of exosomes of mesenchymal stem cells on the Mankin score of aged knee wear model rats.
Grouping: 1- -control group; 2-a model group of senile knee joint wear; 3- - -bone marrow mesenchymal stem cell exosome group; 4- - -adipose-derived mesenchymal stem cell exosome group; 5- -skin mesenchymal stem cell exosome group. P <0.001 compared to group 1; # P <0.01 compared to group 2.
Figure 2 effect of exosomes of mesenchymal stem cells on inflammatory factors of aged knee wear model rats.
Grouping: 1- -control group; 2-a model group of senile knee joint wear; 3- - -bone marrow mesenchymal stem cell exosome group; 4- - -adipose-derived mesenchymal stem cell exosome group; 5- -skin mesenchymal stem cell exosome group. P <0.01, P <0.001 compared to group 1; # P <0.05, # P <0.01 compared to group 2.
Figure 3 effect of exosomes of mesenchymal stem cells on RANK system activity in aged knee wear model rats.
Grouping: 1- -control group; 2-a model group of senile knee joint wear; 3- - -bone marrow mesenchymal stem cell exosome group; 4- - -adipose-derived mesenchymal stem cell exosome group; 5- -skin mesenchymal stem cell exosome group. P <0.01, P <0.001 compared to group 1; # P <0.05, # P <0.01 compared to group 2.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Example 1
The invention provides a preparation method of a mesenchymal stem cell exosome, which comprises the following steps: and (3) separating and culturing the mesenchymal stem cells in vitro, and extracting exosomes in the supernatant of the cell culture solution by adopting a high-speed gradient centrifugation method.
1. Bone marrow mesenchymal stem cell isolation and culture
After anaesthetizing, male SD rats of 2 months old are killed by breaking neck, taking thighbone and shinbone at two sides, adopting sterile physiological saline and 75% ethanol solution to wash, cutting two ends of the thighbone to expose a marrow cavity, repeatedly washing the marrow cavity by DMEM/F-12(1:100 double antibody) culture solution containing 10% FBS, collecting washing liquid, repeatedly blowing and beating to fully disperse cells, and then filtering by a 200-mesh stainless steel filter screen. Centrifuging the filtrate at 300g for 5min, discarding supernatant, resuspending the cells in DMEM/F-12(1:100 double antibody) culture medium containing 10% FBS, and culturing in cell culture box (37 deg.C, 5% CO)2). Changing the liquid for the first time after 3 days, changing the liquid every other day, observing the growth condition of the plant through a microscope until the fusion degree reaches 80-90%, digesting the plant by pancreatin, and then, changing the ratio of the raw materials according to 1: and (5) carrying out passage at a ratio of 2. The bone marrow mesenchymal stem cell surface antigen was identified by the following method. The medium in the flask was aspirated, washed 3 times with PBS solution, digested with trypsin, and then digested with 10% FBS in DMEM/F-12(1:100 double antibody) medium to stop the digestion and form a cell suspension. And transferring the cell suspension into a centrifuge tube, centrifuging for 5min at 300g, removing supernatant, washing with PBS solution, centrifuging for 3 times, and preparing the cell suspension with PBS. Will be provided with The cell suspension was divided into 3 tubes and the number of cells per tube was adjusted to 1X 105Respectively dripping PE marked CD44 and CD34 monoclonal antibodies under the condition of keeping out of the sun, dripping PBS for the rest 1 tube as a blank control, and incubating for 30min at room temperature in the absence of the sun. Centrifuging at 300g for 5 min; the supernatant was decanted, the pellet washed 3 times with PBS solution, and the cells resuspended in 1ml of PBS solution. And analyzing and screening by using a flow cytometer, and using the obtained bone marrow mesenchymal stem cells for subsequent experiments.
2. Obtaining and culturing adipose-derived mesenchymal stem cells
The subcutaneous adipose tissues removed by the operation are placed in a sterile container, washed by PBS, and the upper layer of grease and the lower layer of PBS are sucked off, and the operation is repeated for 5 times to remove impurities. Cutting adipose tissue into pieces (<1mm small block) is moved back into a screw injector, connected with a 0.8mm fat emulsifier and another screw injector, and injected repeatedly for 20 times; the fat emulsifier with the diameter of 0.6mm is replaced, and the bolus injection is repeated for 20 times. Filtering the crushed adipose tissues into a new centrifuge tube through a filter head with the diameter of 0.2mm, centrifuging for 5min at 300g, discarding supernatant, and precipitating to obtain the component rich in adipose-derived mesenchymal stem cells. Adding 10% FBS-containing DMEM medium, resuspending, filtering with 200 mesh screen, and filtering at 10000/cm2Inoculating to a new culture flask, and standing at 37 deg.C and 5% CO 2Culturing in an incubator for 24h, replacing culture medium, removing non-attached cells, replacing culture medium every two days for 1 time, until the fusion degree reaches 80-90%, washing with PBS, digesting with recombinant trypsin (0.25%), and passaging at a ratio of 1: 3. Selecting 2 nd generation adipose-derived mesenchymal stem cells, digesting with recombinant trypsin (0.25%) when the cells grow and fuse to 80% -90%, centrifuging, washing with PBS for 3 times, counting the cells, and taking 10 cells from each tube6The individual cells were resuspended in 100. mu.L PBS, flow antibodies APC-CD44, APC-CD45, FITC-CD34 were added, respectively, incubated at 4 ℃ for 30min, the unlabeled antibody was washed off with PBS, and 300. mu.L PBS was added to resuspend the cells. No antibody was added as a negative control. The Beckman flow cytometer detects cell phenotypes.
3. Obtaining and culturing of skin epidermal mesenchymal stem cells
Placing fresh skin tissue in sterile container, washing with PBS containing double antibody, soaking for 1-2 hr, and separating epidermisAnd cutting the epidermis into pieces<1mm small blocks), transferring into a small conical flask, adding 0.05% trypsin solution containing 0.02% EDTA, completely soaking tissue fragments, digesting in water bath at 37 ℃ for 30min, adding culture medium containing 10% FBS to stop digestion, repeatedly blowing tissue fragments to fully separate cells, filtering with a filter membrane with the aperture of 50 mu m to remove impurities after suspending the cells, and obtaining the single cell suspension rich in the epidermal mesenchymal stem cells. Centrifuging at 300g for 5min, discarding supernatant, and pressing the settled cells to 10 5Cells/cm2Inoculating to IV collagen coated culture bottle, culturing in cell culture box for 10min, removing non-adherent cells, adding appropriate amount of KSFM culture medium, and culturing in cell culture box (37 deg.C, 5% CO)2) The culture medium is replaced every two days, the growth condition of the culture medium is observed by a microscope, and when the fusion degree reaches 80-90%, the culture medium is digested by pancreatin. Then subculturing is carried out according to the ratio of 1: 3. Sorting the epidermal stem cells by a flow cytometer, comprising the following steps. Taking out cultured primary epidermal cells, removing culture medium by suction, adding PBS, washing for 3 times, adding 0.05% recombinant trypsin digestive juice containing 0.02% EDTA, digesting at 37 deg.C for 2min, adding 10ml digestive stop solution to stop digestion, blowing to remove cell wall, centrifuging for 5min at 300g, discarding supernatant, adding precooled PBS, suspending cells, centrifuging for 5min at 300g, adding PBS to resuspend cells, counting cells, and counting according to 10%6Cells/tube were packed, antibodies (CD71-FITC and CD49f-PE) were added, and epidermal mesenchymal stem cell cultures were screened for flow cytometry analysis for subsequent experiments.
4. Exosome preparation
And extracting exosome secreted by the mesenchymal stem cells by adopting a high-speed gradient centrifugation method. Filtering the mesenchymal stem cell culture solution by using a 0.22 mu m screen, and centrifuging the filtrate at high speed and gradient, wherein 300g of the filtrate is centrifuged for 10min at 4 ℃ to obtain a supernatant; centrifuging at 2000g for 30min, and collecting supernatant; centrifuging at 10000g for 30min, and collecting supernatant; and 140000g, centrifuging for 120min, and discarding the supernatant to obtain a precipitate, namely the exosome secreted by the mesenchymal stem cells. The resulting exosomes were then resuspended and washed with pre-cooled PBS and centrifuged again at 140000g for 120 min. Finally, the suspension was resuspended in 50. mu.l of 4 ℃ PBS and transferred to a low-adhesion tube. The obtained exosome is observed in shape and size through an electron microscope, and the marker protein of the exosome is detected by a Western method, so that the obtained precipitate is confirmed to be a cell exosome. And finally, confirming the concentration of the exosome by using a nanoparticle tracking analyzer for subsequent experiments.
Example 2
The mesenchymal stem cell exosome prepared in example 1 is treated by a joint cavity administration mode to an aged joint abrasion model rat, after 8 weeks of administration, the rat is killed after anesthesia, joint cartilage and synovial tissue are separated, and histological and molecular biological detection is carried out.
1. Preparing an aged joint wear model:
through the operation induction of the instability of the medial menisci (DMM) osteoarthritis model on the inner side of the knee joint, the pathogenesis process of the senile osteoarticular abrasion can be simulated in a short time. The specific method comprises the following steps: male SD rats of 2 months age were anesthetized and fixed on the surgical platform in supine position. The method comprises the following steps of sequentially cutting the skin of the knee joint of a rat, carrying out blunt separation on soft tissues, removing the fat tissue under the patella after searching to the patellar ligament, opening a joint capsule, positioning the medial meniscus and the ligament connected with the tibial plateau, namely the medial meniscus ligament, thoroughly shearing the ligament by an ophthalmologic scissors, moving the meniscus to ensure that the ligament is completely broken, flushing the joint cavity by using sterile normal saline, and sequentially suturing cuts. Sham operated groups served as controls, and no loss incision was made after opening the joint capsule to expose the meniscus. After eight weeks, rats in each group were taken for bilateral knee joint, fixed with 4% PFA, decalcified with 10% EDTA, embedded in paraffin, and sectioned continuously (6 μm) until anterior cruciate ligament appeared. Safranin-O staining was performed, histological changes were observed under a mirror and joint structures were scored: cartilage structure (0-5), chondrocytes (0-3), Safranin-O staining (0-5), and tide integrity (0-1). The total of the 4 scores is the Mankin score, and the higher the score is, the more serious the cartilage degeneration is. Rats with significantly increased Mankin scores compared to the control group (sham) were considered to be model rats that induced successful senile joint wear.
2. Exosome dosing:
in the embodiment, the joint cavity injection administration mode is adopted to treat senile constipationNode abrasion model rats. Senile joint abrasion model rats prepared by the above method, each rat was given 1.0x107Each nanoparticle exosome was dissolved in physiological saline at a volume of 5 μ l per administration, administered 1 time every 3 days for 4 weeks. Control rats were given equal volumes of saline. After the treatment, histological and molecular biological detection is performed.
3. Tissue morphology detection
After anesthesia, rats were sacrificed and each group of rats was bilateral knee-joint fixed with 4% PFA, decalcified with 10% EDTA, paraffin embedded, and serially sectioned (6 μm) until anterior cruciate ligament appeared. Safranin-O staining was performed, histological changes were observed under a mirror and joint structures were scored: cartilage structure (0-5), chondrocytes (0-3), Safranin-O staining (0-5), and tide integrity (0-1). The total of the 4 scores is the Mankin score, and the higher the score is, the more serious the cartilage degeneration is. The detection result is shown in fig. 1, and the Mankin score can be remarkably reduced by the exosomes of the three mesenchymal stem cells.
4. Inflammatory factor detection
The synovial tissues in the joints of each group of rats and the synovial tissues infiltrated to the cartilage surface are taken, total RNA is extracted, mRNA expression of inflammatory factors IL-6, IL-1 beta and TNF-alpha is detected through qRT-PCR, and the result is shown in figure 2, and the exosomes of the three mesenchymal stem cells can inhibit the mRNA expression of IL-6, IL-1 beta and TNF-alpha.
5. Detection of the Activity of the RANK System
The OPG-RANKL-RANK system is a cellular signaling pathway that regulates bone remodeling. The ligand of nuclear transcription factor KB Receptor Agonist (RANKL) can be secreted by osteoblasts, which can bind to nuclear factor KB receptor activator RANK on the surface of osteoclast precursor cells to promote osteoclastogenesis, while Osteoprotegerin (OPG) can competitively bind to RANKL to inhibit osteoclastogenesis. The balance of OPG and RANKL plays a key role in the pathophysiology of bone. Recent studies have found that articular subchondral bone remodeling in osteoarthritis patients can induce a degenerative change that exacerbates the cartilage adjacent to it, and the OPG-RANKL-RANK system in turn regulates this process.
Synovial tissues in joints of rats in each group and synovial tissues infiltrated to the surface of cartilage are extracted, total RNA of the synovial tissues is extracted, mRNA expression of TRAP, NFATc1, Cath-K and MMP-9 induced by RANKL is detected through qRT-PCR, and the result is shown in figure 3, and the mRNA expression of TRAP, NFATc1, Cath-K and MMP-9 can be inhibited by exosomes of three mesenchymal stem cells.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The preparation method of the mesenchymal stem cell exosome is characterized by comprising the following steps: and (2) separating and culturing mesenchymal stem cells in vitro, and extracting exosomes in cell culture solution supernatant by an ultracentrifugation method, wherein the mesenchymal stem cells comprise bone marrow mesenchymal stem cells, skin epidermis mesenchymal stem cells and adipose mesenchymal stem cells.
2. The method for preparing mesenchymal stem cell exosomes according to claim 1, wherein the method for isolating and culturing mesenchymal stem cells comprises:
taking bilateral femurs and shinbones of rats, washing bone marrow with DMEM/F-12 culture solution containing 10% FBS, placing the bone marrow in a culture dish, and filtering; centrifuging the filtrate, discarding the supernatant, resuspending the cells in DMEM/F-12 culture solution containing 10% FBS, and culturing in a cell culture box; after 3 days, the liquid is changed for the first time, and then every other day, when the bottom of the bottle is full of cells, the ratio of the liquid to the cell is 1: 2, passage in proportion; analyzing and screening by a flow cytometer to obtain the bone marrow mesenchymal stem cells.
3. The method for preparing mesenchymal stem cell exosomes according to claim 1, wherein the method for isolating and culturing adipose mesenchymal stem cells comprises the following steps:
washing subcutaneous adipose tissue with PBS solution to remove impurities, cutting the extracted adipose tissue, transferring into screw injector, and connecting with fat emulsifier Repeatedly injecting to break adipose tissues, and then filtering to obtain adipose mesenchymal stem cell suspension; centrifuging, removing supernatant, adding 10% FBS-containing DMEM medium for resuspension, inoculating into culture flask, standing at 37 deg.C and 5% CO2Culturing in an incubator; and (3) after 24h, replacing the culture medium, removing nonadherent cells, replacing the culture medium every other two days for 1 time, and digesting with pancreatin until the fusion degree reaches 80-90 percent and the ratio of 1: 3, carrying out passage; detecting cell phenotype by a flow cytometer to obtain the adipose-derived stem cells.
4. The method for preparing mesenchymal stem cell exosomes according to claim 1, wherein the method for isolating and culturing skin epidermal mesenchymal stem cells comprises the following steps:
washing and soaking fresh skin tissue taken down in operation with PBS containing double antibody under aseptic condition, separating epidermis, cutting epidermis into pieces, adding 0.05% recombinant trypsin containing 0.02% EDTA, digesting at 37 deg.C for 30min, terminating digestion with culture medium containing 10% FBS, filtering suspension cells to obtain single cell suspension; after centrifugation, suspending the cells by using a KSFM culture medium, inoculating the cells into an IV collagen-coated culture bottle for culture for 10min, sucking the upper non-adherent cells, adding the KSFM culture medium for continuous culture, changing the culture solution every other day until the fusion degree reaches 80-90%, digesting by using pancreatin, and performing the steps of 1: 3, carrying out passage; analyzing and screening by a flow cytometer to obtain the skin epidermal mesenchymal stem cells.
5. The method for preparing exosomes of mesenchymal stem cells according to claim 1, which is characterized in that the specific method for extracting exosomes in supernatant of cell culture solution comprises the following steps:
filtering the stem cell culture solution, and centrifuging the filtrate at high speed and gradient, at first, centrifuging 300g for 10min at 4 ℃, and taking the supernatant; centrifuging at 2000g for 30min, and collecting supernatant; centrifuging at 10000g for 30min, and collecting supernatant; centrifuging at 140000g for 120min, and discarding the supernatant to obtain a precipitate, namely an exosome secreted by the mesenchymal stem cells; then, the obtained exosomes were resuspended and washed with precooled PBS, and centrifuged again at 140000g for 120 min; finally, the suspension was resuspended in 50. mu.l of 4 ℃ PBS and transferred to a low-adhesion tube to obtain exosomes.
6. Mesenchymal stem cell exosomes prepared according to the method of any one of claims 1-5.
7. Use of mesenchymal stem cell exosomes according to claim 6 in the preparation of a medicament for treating senile knee wear.
8. A medicament comprising the mesenchymal stem cell exosome of claim 6.
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