Exosome containing anti-aging factors as well as preparation method and application of exosome

Abstract

The invention discloses an exosome containing anti-aging factors as well as a preparation method and application of the exosome. The preparation method comprises the following steps: transfecting an Oct4 plasmid, a Sox2 plasmid and a Klf4 plasmid into a host cell, and culturing to obtain a cell culture supernatant containing the exosome; performing extraction treatment on the cell culture supernatant to obtain an exosome containing the anti-aging factor; the mass ratio of the Oct4 plasmid to the Sox2 plasmid to the Klf4 plasmid is 3: 1: 1. The ratio of the anti-aging factors Oct4 to Sox2 to Klf4 in the exosome containing the anti-aging factors prepared by the preparation method is 3: 1: 1, and the anti-aging effect of the exosome is remarkably improved. Besides, when the host cells are bone marrow mesenchymal stem cells, the prepared exosome containing the anti-aging factors has a homologous membrane targeting effect, and can target the bone marrow mesenchymal stem cells in bone tissues to treat osteoporosis caused by bone aging.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to exosomes containing anti-aging factors and a preparation method and application thereof. Background Art

[0002] In the field of life sciences, research on aging has always been a hot topic. In March 2022, scientists at the Salk Institute introduced a method to restore aging cells to a youthful state through the "Yamananaka factors". These four factors are Oct4, Sox2, Klf4 and c-Myc, referred to as "OSKM". This research provides new ideas for delaying aging and extending lifespan, and may also bring hope for the treatment of various aging-related diseases.

[0003] Transcription factors Oct4, Sox2, KIf4, and c-Myc are classic somatic cell reprogramming-related factors. Studies have found that they can maintain the pluripotency of embryonic stem cells. The Oct4 gene works by binding to an 8bp gene expression sequence (ATGCAAAT) in the regulatory regions of multiple genes. These genes are usually involved in self-renewal and differentiation. Oct4 is expressed in the inner cell mass of early embryos. Embryos lacking Oct4 expression cannot develop to the blastocyst stage. Sox2 is a DNA binding protein that interacts with a specific gene sequence to act as a gene enhancer. Sox2 interacts with Oct4 to form a stable DNA binding domain. Sox2 deletion mutations prevent embryos from successfully developing into blastocysts. KIf4 is a zinc finger transcription factor that binds to the core sequence (CACCC) and regulates cell proliferation and differentiation. Oct4, Sox2, and KIf4 usually bind to the promoter region together. Although the detailed mechanism is still unclear, existing studies have suggested that genes encoding somatic cell regulation may be inhibited by these factors, while genes encoding self-renewal and pluripotency may be turned on by the binding of these factors.

[0004] Oct4, Sox2 and KIf4 factors have great potential in the field of life sciences, but the delivery system has restricted the further application of OSK. At present, there are three main methods to effectively deliver the three factors of OSK into cells: biotransfection, physical transfection and chemical transfection. Among them, biotransfection mainly uses viruses and other microorganisms as gene introduction vectors, with lentivirus, adenovirus and adeno-associated virus being the most common. Its infection efficiency can reach more than 90%, but there are problems such as safety. Physical transfection mainly includes microinjection, electroporation and gene gun. No matter which one requires specific equipment, it is expensive and not conducive to large-scale production. Chemical transfection method is the most commonly used transfection method in biomedical research, mainly including two categories: cationic liposomes and cationic polymers, but transfection reagents may be toxic to cells, especially at high concentrations, which may cause cell death or functional impairment.

[0005] Therefore, the existing technology still needs to be improved and developed. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an exosome containing anti-aging factors and a preparation method and application thereof, aiming to solve the problems of poor delivery method and limited therapeutic effect when Oct4, Sox2 and KIf4 factors are used.

[0007] The technical solution of the present invention is as follows:

[0008] In a first aspect, a method for preparing exosomes containing anti-aging factors is provided, the preparation method comprising the steps of:

[0009] Oct4 plasmid, Sox2 plasmid and Klf4 plasmid were transfected into host cells, and cultured to obtain cell culture supernatant containing exosomes;

[0010] extracting the cell culture supernatant to obtain exosomes containing anti-aging factors;

[0011] Wherein, the mass ratio of the Oct4 plasmid, the Sox2 plasmid and the Klf4 plasmid is 3:1:1.

[0012] In a preferred technical solution, the host cell is a bone marrow mesenchymal stem cell.

[0013] The preferred technical solution, the extraction process specifically includes the steps of:

[0014] performing a first centrifugation on the cell culture supernatant, discarding the precipitate and retaining the supernatant to obtain a first supernatant;

[0015] Performing a second centrifugation on the first supernatant, discarding the precipitate and retaining the supernatant, to obtain a second supernatant;

[0016] The second supernatant is subjected to ultrafiltration and concentration treatment using an 80-120 kD ultrafiltration tube, the lower layer of liquid is discarded and the upper layer of liquid is retained to obtain a concentrated solution;

[0017] The concentrated solution is subjected to high-speed centrifugation, the precipitate is discarded and the supernatant is retained to obtain an ultrafiltrate;

[0018] The ultrafiltrate is subjected to filtering and sterilization treatment to obtain a sterilized liquid;

[0019] The sterilization solution is subjected to ultracentrifugation, the supernatant is discarded and the precipitate is retained to obtain exosomes containing anti-aging factors.

[0020] According to a preferred technical solution, the conditions for the first centrifugal treatment are: centrifugal force 600-1000 g, and centrifugal time 8-12 min.

[0021] According to a preferred technical solution, the conditions for the second centrifugal treatment are: centrifugal force 2000-2800 g, and centrifugal time 8-12 min.

[0022] According to a preferred technical solution, the ultrafiltration concentration treatment is carried out under the following conditions: centrifugal force of 3500-4500 g and centrifugal time of 20-30 min.

[0023] According to a preferred technical solution, the conditions for the high-speed centrifugation treatment are: centrifugal force 9000-11000 g, and centrifugal time 50-70 min.

[0024] According to a preferred technical solution, the ultracentrifugation treatment conditions are: centrifugal force 90,000 to 110,000 g, and centrifugation time 100 to 150 min.

[0025] In a second aspect, an exosome containing anti-aging factors is provided, wherein the exosome is prepared by the preparation method described in the first aspect.

[0026] In a third aspect, a use of the exosomes described in the second aspect in the preparation of a drug for treating aging-related diseases is provided.

[0027] In a fourth aspect, a use of the exosomes described in the second aspect in the preparation of a drug for treating osteoporosis.

[0028] Beneficial effects: The present invention provides a method for preparing exosomes containing anti-aging factors. The exosomes containing anti-aging factors prepared by the preparation method have a ratio of 3:1:1 among the anti-aging factors Oct4, Sox2 and Klf4, and their anti-aging effects are significantly improved. In addition, when the host cells are bone marrow mesenchymal stem cells, the prepared exosomes containing anti-aging factors have a homologous membrane targeting effect, and can target bone marrow mesenchymal stem cells in bone tissue to treat osteoporosis caused by bone aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a graph showing the results of Western Blot detection of the contents of aging-related factors P53 / P21 / P16 in the Aging group, Aging+OSK111 group and Aging+OSK311 group in Example 1.

[0030] Figure 2 This is a graph showing the results of detecting the H3K9me3 content in the Young group, the Aging group, the Aging+OSK111 group, and the Aging+OSK311 group by immunofluorescence assay in Example 1.

[0031] Figure 3This is a graph showing the results of detecting the content of P21 in the Young group, the Aging group, the Aging+OSK111 group and the Aging+OSK311 group by immunofluorescence assay in Example 1.

[0032] Figure 4 This is a graph showing the results of evaluating the degree of aging of cells in the Young group, the Aging group, the Aging+OSK111 group, and the Aging+OSK311 group by the cell scratch experiment in Example 1.

[0033] Figure 5 This is a graph showing the results of evaluating the cell viability in the Young group, the Aging group, the Aging+OSK111 group, and the Aging+OSK311 group through the cell proliferation experiment in Example 1.

[0034] Figure 6 This is a transmission electron micrograph of the exosomes prepared in Example 1.

[0035] Figure 7 This is the particle size distribution diagram of the exosomes prepared in Example 1.

[0036] Figure 8 It is a graph showing the fluorescence imaging results of bone tissue after DiD-stained BMSC-exo, DiD-stained HEK293T-exo and PBS were injected into mice in Example 1.

[0037] Fig. 9 This is a diagram of the fluorescence imaging results of the heart, liver, spleen, lung and kidney after DiD-stained BMSC-exo, DiD-stained HEK293T-exo and PBS were injected into mice in Example 1.

[0038] Fig.10 This is a diagram of paraffin sections of bone tissue after DiD-stained BMSC-exo and DiD-stained HEK293T-exo were injected into mice in Example 1.

[0039] Fig.11 This is a graph showing the evaluation results of the osteoporosis treatment effect in Example 1. DETAILED DESCRIPTION

[0040] The present invention provides an exosome containing anti-aging factors and a preparation method and application thereof. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0041] Exosomes are nanoscale lipid bilayer vesicles that are actively secreted by cells, and their cavities or lipid bilayers contain a variety of biological components such as proteins, lipids, and nucleic acids. Exosomes have low immunogenicity, high physicochemical stability, high tissue penetration, and innate transport capabilities, and are therefore expected to become a new type of drug delivery carrier. At present, research on exosomes as drug delivery carriers has made gratifying progress. Exosomes can not only optimize the efficiency of old drugs for new uses by encapsulating small molecule chemical drugs, but can also become a "logistics system" for delivering protein and nucleic acid drugs.

[0042] Based on this, an embodiment of the present invention provides a method for preparing exosomes containing anti-aging factors, the preparation method comprising the steps of:

[0043] Oct4 plasmid, Sox2 plasmid and Klf4 plasmid were transfected into host cells, and cultured to obtain cell culture supernatant containing exosomes;

[0044] extracting the cell culture supernatant to obtain exosomes containing anti-aging factors;

[0045] Wherein, the mass ratio of the Oct4 plasmid, the Sox2 plasmid and the Klf4 plasmid is 3:1:1.

[0046] Specifically, in the prior art, when conducting relevant research on the anti-aging factors Oct4, Sox2, and Klf4, the transfection ratio of the Oct4 plasmid, the Sox2 plasmid, and the Klf4 plasmid is usually 1:1:1, that is, the dosage is equal, but the anti-aging effect exhibited is limited. The present invention adjusts the transfection ratio of the Oct4, Sox2, and Klf4 plasmids to significantly improve the anti-aging effect. Furthermore, the present invention designs exosomes containing Oct4, Sox2, and Klf4 factors, and the exosomes have the characteristics of good biocompatibility. Using exosomes as a delivery system can make these anti-aging factors safe and effective in treatment.

[0047] It is understood that in the art, the Oct4 plasmid refers to a plasmid containing the Oct4 gene in an expression frame, which can be transcribed into Oct4 mRNA and translated into Oct4 protein in a host cell; the Sox2 plasmid refers to a plasmid containing the Sox2 gene in an expression frame, which can be transcribed into Sox2 mRNA and translated into Sox2 protein in a host cell; the Klf4 plasmid refers to a plasmid containing the Klf4 gene in an expression frame, which can be transcribed into Klf4 mRNA and translated into Klf4 protein in a host cell.

[0048] In one embodiment, the host cell is a bone marrow mesenchymal stem cell.

[0049] Bone Marrow Mesenchymal Stem Cells (BMSC) are a type of adult stem cell that mainly exists in the bone marrow stroma. They have multiple differentiation potentials to form bone, cartilage, fat, nerve and myoblasts. Exosomes have the ability to target homologous tissues, which depends on the phenotype, composition and tissue origin of the source cells. Exosomes derived from bone marrow mesenchymal stem cells (BMSC-exos) are extracellular vesicles that can perform the functions of bone marrow mesenchymal stem cells. After intravenous injection, engineered BMSC-exos loaded with drugs can target bone marrow mesenchymal stem cells located in bone tissue in vivo, thereby achieving the effect of treating osteoporosis.

[0050] In one embodiment, the extraction process specifically comprises the steps of:

[0051] performing a first centrifugation on the cell culture supernatant, discarding the precipitate and retaining the supernatant to obtain a first supernatant;

[0052] Performing a second centrifugation on the first supernatant, discarding the precipitate and retaining the supernatant, to obtain a second supernatant;

[0053] The second supernatant is subjected to ultrafiltration and concentration treatment using an 80-120 kD ultrafiltration tube, the lower layer of liquid is discarded and the upper layer of liquid is retained to obtain a concentrated solution;

[0054] The concentrated solution is subjected to high-speed centrifugation, the precipitate is discarded and the supernatant is retained to obtain an ultrafiltrate;

[0055] The ultrafiltrate is subjected to filtering and sterilization treatment to obtain a sterilized liquid;

[0056] The sterilization solution is subjected to ultracentrifugation, the supernatant is discarded and the precipitate is retained to obtain exosomes containing anti-aging factors.

[0057] In one embodiment, the conditions of the first centrifugation treatment are: centrifugal force 600-1000g, centrifugal time 8-12min.

[0058] In one embodiment, the conditions of the second centrifugation treatment are: centrifugal force 2000-2800g, centrifugal time 8-12min.

[0059] In one embodiment, the ultrafiltration concentration treatment is carried out under the following conditions: centrifugal force of 3500-4500 g and centrifugal time of 20-30 min.

[0060] In one embodiment, the conditions of the high-speed centrifugation treatment are: centrifugal force 9000-11000g, centrifugal time 50-70min.

[0061] In one embodiment, the conditions of the ultracentrifugation treatment are: centrifugal force 90000-110000 g, centrifugation time 100-150 min.

[0062] An embodiment of the present invention provides an exosome containing anti-aging factors, wherein the exosome is prepared by the preparation method as described above.

[0063] An embodiment of the present invention provides a use of the exosomes as described above in preparing a drug for treating aging-related diseases.

[0064] An embodiment of the present invention provides a use of the exosomes as described above in preparing a drug for treating osteoporosis.

[0065] The present invention will be further described below by means of specific examples.

[0066] In the embodiment of the present invention, the Oct4 plasmid, Sox2 plasmid and Klf4 plasmid are divided into mouse and human sources, mouse cells are transfected with mouse plasmids, and human cells are transfected with human plasmids. All plasmids are customized from Origene Biotechnology Company, and are obtained by inserting the Oct4 gene, Sox2 gene and Klf4 gene into their respective expression vectors, and the restriction sites are SgfI and MluI. The relevant information is as follows:

[0067] Expressed genes Species Transcript Plasmid Part Number Expression vector part number Oct4 Human NM_002701 RG211998 PS100010 Sox2 Human NM_003106 JS0076 PS100041 Klf4 Human NM_004235 JS0077 PS100042 Oct4 Mouse NM_013633 MG205861 PS100010 Sox2 Mouse NM_011443 JS0078 PS100041 Klf4 Mouse NM_010637 JS0079 PS100042

[0068] The culture medium involved in the embodiment is as follows:

[0069] Culture medium containing 2% fetal bovine serum: 2% (v / v) fetal bovine serum + high glucose DMEM + 1% (v / v) double antibody;

[0070] Serum-free medium: high-glucose DMEM;

[0071] Complete culture medium: 10% (v / v) fetal bovine serum + high glucose DMEM + 1% (v / v) double antibody;

[0072] Exosome-free serum culture medium: 10% (v / v) exosome-free serum + high-glucose DMEM + 1% (v / v) double antibody;

[0073] Among them, fetal bovine serum was purchased from gibco, catalog number 10099-141; high glucose DMEM was purchased from gibco, catalog number C11995500BT; bispecific antibodies were purchased from gibco, catalog number 15140163; exosome-free serum was purchased from Vivacell, catalog number C38010050.

[0074] Example 1

[0075] This embodiment provides an exosome containing anti-aging factors, and the preparation method thereof is as follows:

[0076] 1. Preparation of bone marrow mesenchymal stem cells

[0077] In this example, two types of bone marrow mesenchymal stem cells were prepared:

[0078] 1. Mouse bone marrow mesenchymal stem cells (BMSC): Anesthetize C57BL / 6 mice and clean their leg hair. Use cotton balls soaked in PBS containing 10% double antibody to scrub the sampling site; kill the mice, remove the leg bones, and remove the soft tissue attached to the leg bones; remove the bulges at both ends of the femur and tibia, and use a syringe to absorb complete culture medium to flush the bone marrow cavity cells into the culture bottle. Fully mix the cells in the culture bottle, observe under a microscope, and change the culture medium regularly.

[0079] 2. Human mesenchymal stem cells (iMSC): Mesenchymal stem cells (MSC) are differentiated from human induced pluripotent stem cells (iPSC), which were purchased from Beijing Saibei Biotechnology Co., Ltd.

[0080] Experiments have found that both BMSC and iMSC can be used to prepare exosomes, and the prepared exosomes can target bone tissue in in vivo animal experiments. Among them, mouse-derived BMSCs have limited proliferation capacity, so the preparation of exosomes from them is relatively complex and limited in quantity; while iMSCs have stronger proliferation capacity, and the preparation of exosomes from them is relatively simple and easy to obtain, which is more conducive to practical applications.

[0081] 2. Determination of the ratio of anti-aging factors

[0082] In the prior art, when Oct4, Sox2, and Klf4 plasmids are transfected into cells, the ratio usually used is 1:1:1, that is, the dosage is equal, but the anti-aging effect exhibited is limited. Therefore, this example adjusts the transfection ratio of Oct4, Sox2, and Klf4 plasmids to explore the possibility of further improving the anti-aging effect.

[0083] In this example, the Young group (BMSCs from 3-month-old mice), the Aging group (BMSCs from 18-month-old mice), the Aging+OSK111 group (BMSCs from 18-month-old mice transfected with Oct4, Sox2, and Klf4 plasmids, wherein the mass ratio of the Oct4, Sox2, and Klf4 plasmids was 1:1:1, and the total final concentration of the plasmids added during the transfection was 1 μg / ml), and the Aging+OSK311 group (BMSCs from 18-month-old mice transfected with Oct4, Sox2, and Klf4 plasmids, wherein the mass ratio of the Oct4, Sox2, and Klf4 plasmids was 3:1:1, and the total final concentration of the plasmids added during the transfection was 1 μg / ml).

[0084] The levels of aging-related factors P53 / P21 / P16 in the Aging group, Aging+OSK111 group, and Aging+OSK311 group were detected by Western Blot. Figure 1 As shown. Figure 1 It can be seen that the expression of aging-related factors P53 / P21 / P16 in the Aging+OSK311 group was significantly lower than that in the Aging group, while the expression of aging-related factors P53 / P21 / P16 in the Aging+OSK111 group was not significantly lower than that in the Aging group.

[0085] The levels of H3K9me3 and P21 in the Young group, Aging group, Aging+OSK111 group, and Aging+OSK311 group were detected by immunofluorescence assay. Figure 2 and 3 As shown. Figure 2 It can be seen that the expression of H3K9me3 in the Aging+OSK111 group and the Aging+OSK311 group was higher than that in the Aging group; among them, the expression of H3K9me3 in the Aging+OSK311 group was more significantly increased, close to that in the Young group. Figure 3 It can be seen that the expression of P21 in the Aging+OSK111 group and the Aging+OSK311 group was lower than that in the Aging group; among them, the expression of P21 in the Aging+OSK311 group was more significantly reduced, which was equivalent to that in the Young group.

[0086] The cell scratch assay was used to evaluate the degree of cell senescence in the Young group, Aging group, Aging+OSK111 group, and Aging+OSK311 group. Figure 4 As shown. Figure 4It can be seen that the migration and repair abilities of the Aging+OSK111 group and the Aging+OSK311 group were improved compared with the Aging group; among them, the migration and repair abilities of the Aging+OSK311 group were more significantly improved, comparable to those of the Young group; while the migration and repair abilities of the Aging+OSK111 group were limitedly improved.

[0087] Cell proliferation assay was used to evaluate the cell viability in the Young group, Aging group, Aging+OSK111 group, and Aging+OSK311 group. Figure 5 As shown. Figure 5 It can be seen that the cell viability of the Aging+OSK311 group was significantly improved compared with the Aging group and was equivalent to that of the Young group; while the cell viability of the Aging+OSK111 group was not significantly improved compared with the Aging group.

[0088] In summary, when Oct4, Sox2, and Klf4 plasmids were transfected into aged BMSCs at a mass ratio of 3:1:1, the proportion of aged BMSCs could be significantly reduced, and the aged cells could be restored to a young state; however, when Oct4, Sox2, and Klf4 plasmids were transfected into aged BMSCs at a mass ratio of 3:1:1, the proportion of aged BMSCs could be reduced to a limited extent, and the senescent cells could only be partially restored.

[0089] 3. Transfection of Bone Marrow Mesenchymal Stem Cells

[0090] 1. Prepare bone marrow mesenchymal stem cells: 24 hours before transfection, passage the bone marrow mesenchymal stem cells to 10cm 2 In a cell culture dish, the method is as follows: take a 10 cm2 dish with a 100% confluence of bone marrow mesenchymal stem cells. 2 The cell culture dish was discarded with the original culture medium and washed 1-2 times with 2-3 ml of phosphate buffer saline (PBS), the PBS was discarded, 1 ml of 0.25% (w / v) trypsin was added to digest the cells for 2 min, and then 3 ml of complete culture medium was added to stop the digestion. The cell suspension was repeatedly blown with a gun tip to disperse the aggregated cells, and the cell suspension was transferred to a 15 ml centrifuge tube, which was placed in a centrifuge and centrifuged at 1000 r / pm for 1 min. After the centrifugation, the centrifuge tube was removed, the supernatant was aspirated, 2 ml of complete culture medium was added to resuspend the cell pellet, and 1 ml of the cell suspension was aspirated into a new 10 cm 2 Place the cells in a cell culture dish and add 8 ml of complete culture medium to the dish. Shake the dish to evenly distribute the cells and place the dish in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0091] 2. Transfection of bone marrow mesenchymal stem cells: 2 hours before transfection, discard the 10cm 2 Replace the original culture medium in the cell culture dish with 10ml of culture medium containing 2% fetal bovine serum and continue to culture; take 1ml of serum-free culture medium to a 1.5ml EP tube, take 6μg of Oct4 plasmid, 2μg of Sox2 plasmid and 2μg of Klf4 plasmid and add them to the serum-free culture medium in the EP tube, add 30μl of 0.1% PEI transfection reagent (polysciences, 24765) to the plasmid solution, blow gently to mix, let stand for 15 minutes, and obtain a "transfection reagent-plasmid" mixture; add the "transfection reagent-plasmid" mixture to the cell culture medium of the cells to be transfected, and shake gently to evenly distribute the "transfection reagent-plasmid" mixture. After mixing evenly, place the cell culture dish in a 37°C, 5% CO2 incubator for 12 to 24 hours.

[0092] 4. Extraction of Exosomes

[0093] After transfection, discard the original culture medium containing the transfection reagent, shake up and down with 5 ml of phosphate buffered saline solution, wash three times to remove exosomes from other sources in ordinary fetal bovine serum to prevent exosomes from other sources from affecting subsequent experimental analysis, continue to culture with exosome-free serum medium for 48 to 60 hours, collect the culture supernatant, and extract exosomes. The specific method of exosome extraction is as follows:

[0094] 1. Collect cell culture supernatant: The cell culture supernatant contains exosomes loaded with Oct4, Sox2, and Klf4 factors. Before collection, the culture bottle needs to be shaken to fully collect the exosomes attached to the cells and the bottom of the cell culture dish. After collecting the cell culture supernatant, rinse the culture bottle with 5 ml of PBS in turn and collect the rinse fluid, and merge it into the collected cell culture supernatant.

[0095] 2. First centrifugation: Take the cell culture supernatant and centrifuge it at 4°C, 800g for 10 minutes. After the centrifugation, discard the precipitate and keep the first supernatant. The precipitate in this step contains a large number of dead cells.

[0096] 3. Second centrifugation: Take the first supernatant and centrifuge it at 4°C, 2500g for 10 minutes. After the centrifugation, discard the precipitate and keep the second supernatant. The precipitate in this step contains a large amount of cell debris.

[0097] 4. Ultrafiltration concentration: Take the second supernatant, place it in a 15ml 100kD ultrafiltration tube, and centrifuge it at 4℃, 4000g for 25min to concentrate the second supernatant to about 5ml (note that the inclined surface of the filter cartridge is perpendicular to the shaft, and the maximum amount of filtrate that can be added by the parallel rotor and the angled rotor is different). After the centrifugation, discard the lower layer of liquid and retain the upper layer of concentrated liquid;

[0098] 5. High-speed centrifugation: Take the concentrated liquid and centrifuge it at 4°C and 10,000g for 1 hour. After centrifugation, discard the precipitate and retain the ultrafiltrate. The precipitate in this step contains a large amount of impurities.

[0099] 6. Filtration and sterilization: Filter the ultrafiltrate obtained by high-speed centrifugation through a 0.22 μm filter to sterilize it and obtain a sterilized solution.

[0100] 7. First ultracentrifugation: Take the sterilized solution, place it in an 8.9 ml ultracentrifuge tube, and ultracentrifuge it at 100,000 g and 4°C for 2 hours. After the centrifugation, discard the supernatant and retain the precipitate. The precipitate obtained in this step contains a large amount of exosomes, which is the crude exosome product.

[0101] 8. Second ultracentrifugation: Resuspend the crude exosome product obtained from the first ultracentrifugation to 8.9 ml with PBS, and ultracentrifuge again at 100,000 g, 4°C for 2 h. After the centrifugation, gently discard the supernatant with a 1 ml long pipette tip. The resulting precipitate is the exosome containing anti-aging factors. Resuspend the exosomes with 200 μl PBS and store at -80°C after collection.

[0102] 4. Exosome Characterization

[0103] The prepared exosomes containing anti-aging factors were characterized. The morphology of the exosomes was observed by transmission electron microscopy. Figure 6 The particle size distribution of exosomes was detected by Malvern laser particle size analyzer. The results are shown in Figure 7 shown.

[0104] V. Effect Evaluation

[0105] 1. Evaluation of bone targeting effect

[0106] The exosomes containing anti-aging factors prepared from BMSC are recorded as BMSC-exo; the exosomes containing anti-aging factors prepared from HEK293T are recorded as HEK293T-exo. The difference between HEK293T-exo and BMSC-exo is only the different cell sources for preparing exosomes, and the other preparation steps are the same.

[0107] BMSC-exo and HEK293T-exo were stained with DiD fluorescent dye and injected into mice through the tail vein (dose 2 mg / kg). An equal volume of PBS was used as a blank control. Samples were collected 24 hours after injection and the fluorescence intensity was observed on a small animal in vivo imaging instrument. The results of bone tissue fluorescence imaging are shown in Figure 2. Figure 8 As shown, the results of fluorescence imaging of the heart, liver, spleen, lungs and kidneys are as follows Fig. 9 The stronger the fluorescence intensity, the more Exo accumulated in that part of the surface.

[0108] Figure 8 In the experiment, the group injected with PBS was the blank control group, without any fluorescence; the group injected with HEK293T-exo was the control group (no bone targeting), the bone tissue had a certain fluorescence but it was weaker than that injected with BMSC-exo; the group injected with BMSC-exo was the experimental group, the fluorescence intensity of the bone tissue was high, indicating that the exosomes accumulated more in this part and BMSC-exo had a bone targeting effect.

[0109] Fig. 9 In the experiment, the group injected with PBS was the blank control group, without any fluorescence; the group injected with HEK293T-exo was the control group (no bone targeting), because HEK293T-exo had no bone targeting effect, so when the injection volume was the same, the fluorescence intensity of major organs such as heart, liver, spleen, lung, and kidney was stronger than that injected with BMSC-exo; the group injected with BMSC-exo was the experimental group, because BMSC-exo had bone targeting effect, so when the injection volume was the same, BMSC-exo would have a certain amount of targeted aggregation to bone tissue, and relatively speaking, the fluorescence intensity in major organs such as heart, liver, spleen, lung, and kidney was weaker than that injected with HEK293T-exo, further indicating that BMSC-exo had bone targeting effect.

[0110] BMSC-exo and HEK293T-exo were stained with DiD fluorescent dye and injected into mice through the tail vein (dosage 2 mg / kg). Samples were collected 24 hours after injection and paraffin sections were performed. The results are shown in Fig.10 shown.

[0111] Fig.10 In the experiment, a large amount of red fluorescence was observed in the bone marrow of mice injected with BMSC-exo, while less fluorescence was observed in the bone marrow of mice injected with HEK293T-exo, further indicating that BMSC-exo has a bone-targeting effect.

[0112] 2. Evaluation of osteoporosis treatment effect

[0113] In this embodiment, an ovariectomized group, a natural aging group, and a blank control group were set up. The ovariectomized group was obtained by removing bilateral ovaries of 3-month-old C57BL / 6 mice and then modeling for 3 months; the natural aging group was 18-month-old C57BL / 6 mice; and the blank control group was obtained by raising 3-month-old C57BL / 6 mice without treatment for 3 months, i.e., 6-month-old mice.

[0114] The exosomes containing anti-aging factors prepared from BMSCs were injected into the tail vein of mice in the ovariectomized group and the naturally aged group (dosage 2 mg / kg), and micro-CT images of the mice were taken after 1 month and 3 months to observe the changes in cancellous bone mass. The experimental results are as follows: Fig.11 The higher the trabecular density, the better the effect of osteoporosis treatment.

[0115] Fig.11 A is the blank control group, B is the ovariectomized group without treatment, C is the ovariectomized group after 1 month of treatment with exosomes containing anti-aging factors, D is the ovariectomized group after 3 months of treatment with exosomes containing anti-aging factors, E is the natural aging group without treatment, F is the natural aging group after 1 month of treatment with exosomes containing anti-aging factors, and D is the natural aging group after 3 months of treatment with exosomes containing anti-aging factors. Fig.11 It can be seen that the bone mass of 6-month-old mice was used as the blank control. The ovaries of the mice in the ovariectomized group were removed at 3 months of age, and the trabeculae basically disappeared after 3 months. The trabeculae in the mice in the natural aging group basically disappeared at 18 months, proving that both groups had osteoporosis. After 1 month of continuous drug treatment, the number of trabeculae in the ovariectomized group and the natural aging group increased slightly. After 3 months of continuous drug treatment, the number of trabeculae increased significantly and basically returned to the 6-month-old level, indicating that continuous drug treatment for 3 months can significantly increase trabecular density and improve osteoporosis.

[0116] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing exosomes containing anti-aging factors, characterized in that: The preparation method comprises the steps of: Oct4 plasmid, Sox2 plasmid and Klf4 plasmid were transfected into host cells, and cultured to obtain cell culture supernatant containing exosomes; extracting the cell culture supernatant to obtain exosomes containing anti-aging factors; Wherein, the mass ratio of the Oct4 plasmid, the Sox2 plasmid and the Klf4 plasmid is 3:1:

1.

2. The preparation method according to claim 1, characterized in that: The host cells are bone marrow mesenchymal stem cells.

3. The preparation method according to claim 1, characterized in that: The extraction process specifically comprises the steps of: performing a first centrifugation on the cell culture supernatant, discarding the precipitate and retaining the supernatant to obtain a first supernatant; Performing a second centrifugation on the first supernatant, discarding the precipitate and retaining the supernatant, to obtain a second supernatant; The second supernatant is subjected to ultrafiltration and concentration treatment using an 80-120 kD ultrafiltration tube, the lower layer of liquid is discarded and the upper layer of liquid is retained to obtain a concentrated solution; The concentrated solution is subjected to high-speed centrifugation, the precipitate is discarded and the supernatant is retained to obtain an ultrafiltrate; The ultrafiltrate is subjected to filtering and sterilization treatment to obtain a sterilized liquid; The sterilization solution is subjected to ultracentrifugation, the supernatant is discarded and the precipitate is retained to obtain exosomes containing anti-aging factors.

4. The preparation method according to claim 3, characterized in that: The conditions of the first centrifugal treatment are: centrifugal force 600-1000g, centrifugal time 8-12min; And / or, the conditions of the second centrifugal treatment are: centrifugal force 2000-2800g, centrifugal time 8-12min.

5. The preparation method according to claim 3, characterized in that: The conditions of the ultrafiltration concentration treatment are: centrifugal force 3500-4500g, centrifugal time 20-30min.

6. The preparation method according to claim 3, characterized in that: The conditions of the high-speed centrifugation treatment are: centrifugal force 9000-11000g, centrifugal time 50-70min.

7. The preparation method according to claim 3, characterized in that: The conditions of the ultracentrifugation treatment are: centrifugal force 90000-110000g, centrifugal time 100-150min.

8. An exosome containing anti-aging factors, characterized in that: The exosomes are prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the exosomes according to claim 8 in the preparation of a drug for treating aging-related diseases.

10. Use of the exosomes according to claim 8 in preparing a drug for treating osteoporosis.

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