Preparation method and application of low-oxygen pretreated pilose antler stem cell exosome preparation

Abstract

The invention discloses a preparation method of a hypoxia pretreated cornu cervi pantotrichum stem cell exosome preparation, which comprises the following steps: S1, performing isolated culture on cornu cervi pantotrichum stem cells ABPCs to obtain the cornu cervi pantotrichum stem cells ABPCs subjected to isolated culture for later use; s2, performing low-oxygen pretreatment on the cornu cervi pantotrichum stem cells ABPCs obtained in the step S1 to obtain primary cornu cervi pantotrichum stem cell exosomes ABPCs-EXOs; and S3, extracting and purifying the primary cornu cervi pantotrichum stem cell exosome ABPCs-EXOs obtained in the step S2 to obtain a hypoxia-pretreated cornu cervi pantotrichum stem cell exosome preparation, and performing hypoxia-pretreated cornu cervi pantotrichum stem cell exosome preparation to promote osteogenic differentiation of bone marrow mesenchymal stem cells.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing a hypoxia-pretreated deer antler stem cell exosome preparation and its application. Background Technology

[0002] Oral and maxillofacial bone defects caused by trauma, congenital malformations, tumors, and other reasons not only affect patients' chewing and speech functions but also negatively impact their appearance and overall well-being. Researchers have attempted to repair defects using autologous bone and various bone substitutes; however, autologous bone transplantation is often limited by donor site size and is accompanied by complications such as pain, infection, and nerve damage. Meanwhile, single bone substitutes generally suffer from poor shapeability and slow bone formation.

[0003] Cellular tissue engineering has emerged as a promising strategy for tissue repair, with stem cells, being pluripotent cells capable of self-replication, forming a core component of regenerative medicine. However, stem cell therapy faces several challenges in clinical practice, such as immune rejection, tumorigenicity, and the survival and viability of stem cells after allogeneic cell implantation. Furthermore, autologous stem cell transplantation is limited by technological limitations and the quality of the patient's own stem cells. In fact, transplanted cells rapidly undergo apoptosis, necrosis, or other death mechanisms induced by physical and biochemical factors. Therefore, it is necessary to find a suitable cell-free therapy.

[0004] Studies have shown that the therapeutic effect of mesenchymal stem cells (MSCs) does not appear to depend on the physical proximity of transplanted cells within the tissue; rather, its therapeutic effect is primarily due to the release of paracrine factors. The synergistic effect of small molecules secreted by MSCs can reduce cell damage and enhance tissue repair capabilities. Exosomes, a major component of paracrine factors, are tiny vesicles secreted by cells, ranging in size from 50-150 nm and enclosed in a lipid bilayer. They contain various proteins, miRNAs, lncRNAs, and liposomes. Due to their unique structure, they can enter cells via endocytosis, playing a crucial role in intercellular communication and possessing enormous therapeutic potential.

[0005] Furthermore, mesenchymal stem cells actually exist in the hypoxic microenvironment (1.5% O2) in vivo, while the normal oxygen concentration in the existing culture environment (around 20% O2) is higher than their original survival environment. Therefore, the difference in oxygen concentration in the in vitro culture environment will affect the biological activity of stem cells, including cell proliferation, cell differentiation, and anti-inflammatory cell responses, thereby affecting their ability to repair damaged tissues. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a hypoxia-pretreated antler blastema progenitor cell exosome preparation and its application. The preparation is a hypoxia-pretreated antler blastema progenitor cell (ABPCs) exosome preparation to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), thereby solving the problems of low exosome yield and unstable osteogenic activity in the prior art.

[0007] The objective of this invention is achieved as follows:

[0008] A method for preparing a hypoxia-pretreated deer antler stem cell exosome preparation includes the following steps:

[0009] S1. Isolation and culture of deer antler stem cells ABPCs to obtain isolated and cultured deer antler stem cells ABPCs for later use;

[0010] S2. The deer antler stem cell ABPCs obtained in S1 were subjected to hypoxia pretreatment to obtain primary deer antler stem cell exosomes ABPCs-EXOs; specifically as follows: S2.1. The deer antler stem cell ABPCs were counted and divided into 1×10⁻⁶ cells. 7 Inoculate at a density of / dish into 10cm dishes;

[0011] S2.2 When the confluence of antler stem cell ABPCs reaches 70%-80%, discard the complete culture medium, wash the cells three times with 1×PBS, replace the culture medium with exosome-free serum medium, place the cell culture dish in a hypoxic incubator, and culture in a constant temperature incubator. Collect the supernatant, which is the primary antler stem cell exosome ABPCs-EXOs. The primary antler stem cell exosome ABPCs-EXOs should be stored at -80 ℃ for subsequent use. The oxygen concentration in the hypoxic incubator is 1.5%, and the carbon dioxide concentration is 5%.

[0012] S3. After extracting and purifying the primary deer antler stem cell exosomes ABPCs-EXOs obtained in S2, a hypoxia-pretreated deer antler stem cell exosome preparation was obtained.

[0013] In S2.2, the exosome-free serum culture medium is made by adding 10% (v / v) exosome-free FBS and 1% (v / v) penicillin (final concentration 100 U / mL penicillin + 100 μg / mL streptomycin) to a low-glucose (1 g / L glucose concentration) DMEM medium.

[0014] In step S2.2, the temperature of the incubator is 37 ℃ and the incubation time is 48 h.

[0015] The specific operation of S1 is as follows: S1.1, collect deer antler mesenchymal tissue that has grown for 5-6 days, wash it 3 times with PBS solution containing 1% (v / v) double antibiotics, slice the deer antler tissue longitudinally, and carefully locate and separate the mesenchymal layer;

[0016] S1.2 Cut the tissue into 0.7 mm thin slices, and then digest them in 200 U / mL type I collagenase for 30 min. Under a microscope, rough edges can be seen in the tissue. The tissue is then cultured as primary cells in DMEM complete medium. When the cells crawl out from the periphery of the adherent tissue block, the culture medium is replaced. The DMEM complete medium is DMEM medium containing 10 wt% fetal bovine serum.

[0017] S1.3 When the cell density reaches 70%, digest with 0.25% (w / v) trypsin for 1 min, centrifuge to collect the cells, and then passage them in DMEM complete medium to obtain P1 cells; repeat the above method to continue passage to obtain P3 cells.

[0018] The specific operation of S3 is as follows: S3.1, Place the collected supernatant in a 50 mL centrifuge tube, centrifuge for 10 min to remove cell pellet, collect the supernatant, transfer it to an ultrafiltration centrifuge tube, centrifuge at 4 ℃ for 10 min, repeat centrifugation until the supernatant is concentrated to 20 mL, and collect the supernatant.

[0019] S3.2 Add ESC reagent to the supernatant obtained in S3.1, wherein the ESC reagent dosage ratio is: 5 mL of ESC reagent per 20 mL of cell culture supernatant;

[0020] S3.3 After adding ESC reagent, tighten the centrifuge tube, mix it with a vortex mixer for 1 min, and then place it at 4 ℃ for at least 2 h to obtain the mixture.

[0021] S3.4. Take out the centrifuge tube containing the mixture and centrifuge at 10000g for 50 min at 4 ℃. Discard the supernatant and obtain the centrifuged precipitate, which is rich in exosome particles.

[0022] S3.5 Take 1× PBS and beat the centrifuged precipitate obtained in S3.4 evenly until it dissolves to obtain a resuspension. Transfer the resuspension to a new 1.5 mL centrifuge tube. The PBS ratio is: 5 mL PBS solution is added to every 20 mL of cell culture supernatant.

[0023] S3.6 Centrifuge a 1.5 mL centrifuge tube containing the resuspension at 4 ℃ and 12000g for 2 min, and retain the supernatant. The supernatant is rich in exosome particles, which is the crude exosome particle product.

[0024] S3.7 Transfer the crude exosome particles obtained in S3.6 into the EPF column chamber and centrifuge at 3000g for 10 min at 4 ℃. After centrifugation, collect the liquid at the bottom of the EPF column tube. This liquid is the hypoxia pretreated deer antler stem cell exosome preparation. After aliquoting, store in a -80 ℃ low temperature freezer.

[0025] The application of a hypoxia-pretreated deer antler stem cell exosome preparation to promote osteogenic differentiation of bone marrow mesenchymal stem cells.

[0026] The beneficial effects of this invention are as follows: 1. Compared with commonly used bone marrow mesenchymal stem cells and umbilical cord mesenchymal stem cells, it has the following advantages: ① Under the same culture conditions, compared with human mesenchymal stem cells, deer antler stem cells show a higher proliferation capacity (8.6~11.7 times), consistent with the trend of in vivo research results; ② Deer antlers can produce up to 30 kg of bone tissue (up to 2.0 cm / d) within a few months at a rapid growth rate. The rapid growth of deer antlers provides a rare model for studying tissue regeneration, in which the rapid proliferation of cells is precisely regulated and does not become cancerous; ③ The periodic regeneration of deer antlers not only provides a valuable opportunity for humans to understand how nature endows mammalian organs with complete regenerative capabilities, but also provides a source of deer antler stem cells; ④ They are easy to obtain, isolate, and culture. These cell populations not only function as tissue-specific stem cells in adult male deer, but also maintain the characteristics of embryonic origin throughout the animal's life. Therefore, hypoxia-pretreated deer antler stem cells are promising seed cells in bone tissue regeneration engineering.

[0027] 2. The paracrine pathway is a crucial pathway for mesenchymal stem cells to function, and exosomes (EXOs) have broad research and application prospects. ① They promote cell proliferation and transmit intercellular signals; ② They can exist stably in tissues for a long time; ③ They possess unique properties such as natural cell targeting; ④ They are cellular-free, requiring no matching recipients or donors, thus eliminating the risks and obstacles associated with allogeneic or xenotransplantation, resulting in lower immunogenicity and tumorigenicity, and avoiding ethical issues; ⑤ Exosome extraction technology is becoming increasingly mature, with higher purity and quality levels, and they are simple and readily available for storage. Therefore, exosomes have broad research prospects and application potential in tissue regenerative medicine.

[0028] 3. Hypoxia pretreatment of stem cells can significantly promote the release of their exosomes, providing a new approach for bone tissue engineering regeneration; oxygen concentration is crucial for the biological behavior of stem cells. The physiological oxygen concentration in vivo is lower than the conventional oxygen concentration used in cell culture. Therefore, compared with exosomes produced under physiological conditions, hypoxia pretreatment can promote the release of exosomes, and these exosomes can significantly enhance osteogenic differentiation activity (such as BMP2 activity, calcium nodule formation, and other indicators). Attached Figure Description

[0029] Figure 1 A is a culture diagram of deer antler stem cells; A and B are diagrams of the tip of deer antler; C is a diagram of primary deer antler stem cells; D is a diagram of deer antler stem cells after passage and purification.

[0030] Figure 2 Osteogenic differentiation identification diagram of deer antler stem cells (A is alizarin red staining diagram) and immunofluorescence identification diagram (B is vimentin diagram; C is keratin diagram; D is immunofluorescence result diagram of CD73, CD90, and CD105).

[0031] Figure 3 Image A shows the culture of bone marrow mesenchymal stem cells; image B shows primary bone marrow mesenchymal stem cells; image C shows bone marrow mesenchymal stem cells after passage and purification; image C shows their identification by flow cytometry.

[0032] Figure 4 Image of exosome morphology under a transmission electron microscope;

[0033] Figure 5 Figure 1 shows the experimental results of CCK-8 in exosomes derived from deer antler stem cells at different concentrations.

[0034] Figure 6 To compare the osteogenic differentiation capacity of exosomes derived from ABPCs pretreated with conventional osteogenic induction solution, normoxic and hypoxic solutions, the following diagrams are used: A is an alkaline phosphatase staining diagram; B is an alizarin red staining diagram; C is a qPCR experiment diagram; D is a Western blot (WB) experiment diagram; and E is an immunofluorescence experiment diagram. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0036] Isolation and culture of deer antler stem cells (ABPCs)

[0037] S1.1. Collect mesenchymal tissue from deer antlers that have grown for approximately 5 days. Wash the tissue three times with PBS containing 1% penicillin and antibiotics. Slice the antler tissue longitudinally, carefully locate and separate the mesenchymal layer, as follows: Figure 1 As shown in AB;

[0038] S1.2. Cut the tissue into 0.7 mm thin slices, then digest with type I collagenase at 200 U / mL for 30 min. Under a microscope, rough edges will be visible in the tissue. Culture the tissue in DMEM complete medium (containing 10% fetal bovine serum) for primary cell culture (37 ℃, 5% CO2). When cells begin to migrate out from the periphery of the adherent tissue block, replace the culture medium. Figure 1 As shown in C;

[0039] S1.3. When the cell density reaches 70%, digest with 0.25% trypsin for 1 min, then centrifuge to collect the cells (1000 r / min, 5 min), and then passage in DMEM complete medium to obtain P1 ( Figure 1 D) P3 cells were obtained by passage using the same method. P3 cells were used in all experiments.

[0040] The deer antlers mentioned were provided by Huamao Deer Industry in Gulin County, Luzhou City, Sichuan Province. Healthy male sika deer aged approximately 2-4 years were selected.

[0041] The complete culture medium is DMEM medium containing 10 wt% fetal bovine serum.

[0042] Immunofluorescence and osteogenic differentiation identification of deer antler stem cells (ABPCs)

[0043] 1. Osteogenic Differentiation Identification Experiment

[0044] (1) Third-generation ABPCs were cultured using osteogenic induction medium;

[0045] (2) After 21 days of induction culture, the osteogenic differentiation effect of ABPCs was detected using alizarin red staining.

[0046] The osteogenic induction medium consisted of 50 mL of complete medium with 500 μL of glutamine, 500 μL of sodium β-glycerophosphate, 100 μL of ascorbic acid, and 5 μL of dexamethasone.

[0047] The results are as follows Figure 2 As shown, calcium nodules were observed. Figure 2 A) indicates that ABPCs can be induced to differentiate into osteopathic forms, proving their stem nature.

[0048] 2. Immunofluorescence experiment

[0049] (1) Place cell crawling sheets into a six-well plate, seed P3 generation ABPCs and culture for 24 hours. After washing with PBS, add 2 mL of 4% paraformaldehyde for fixation for 40 minutes. After fixation, wash three times with PBS and treat with 3% hydrogen peroxide for 15 minutes.

[0050] (2) At room temperature, add 0.5% Triton to the cell slides and incubate for 10 min. Wash three times with PBS, 5 min each time. Block the cells with 15% goat serum at 37 ℃ for 30 min, and wash with PBS. Add CD73, CD90, CD105 and vimentin and keratin primary antibodies respectively, and incubate overnight at 4 ℃.

[0051] (3) The next day, after washing with PBS, add the secondary antibody corresponding to the primary antibody species, incubate at room temperature in the dark for 1 h, wash with PBS 3 times, 5 min each time. Add ready-to-use DAPI working solution, incubate at 37 ℃ in the dark for 15 min, wash with PBS 3 times, 5 min each time. Mount with 10% glycerol, observe under an upright fluorescence microscope, and detect the expression of CD73, CD90, CD105, vimentin, and keratin in ABPCs by immunofluorescence.

[0052] The results are as follows Figure 2 As shown in B, vimentin expression was positive ( Figure 2 B); Keratin expression negative ( Figure 2 C); CD73 and CD90 expression is positive, CD105 expression is negative (C) Figure 2 D), indicating that ABPCs are mesenchymal stem cells.

[0053] Isolation and culture of bone marrow mesenchymal stem cells (BMSCs)

[0054] (1) Take the femur and tibia of a rat, cut off both ends of the bone, flush out the bone marrow tissue with a syringe, and rinse repeatedly until the bone turns white;

[0055] (2) After isolation and purification, primary culture was carried out, with the medium changed every 3 days during the process. Figure 3 A); after reaching 80% growth, they are passaged to obtain P1 (A); Figure 3 B).

[0056] The rats were provided by the Experimental Animal Center of Southwest Medical University and were SPF-grade male SD rats aged 2-4 weeks.

[0057] Flow cytometry of bone marrow mesenchymal stem cells (BMSCs)

[0058] (1) After BMSCs were transferred to P3, they were digested into single-cell suspensions, centrifuged at 1000 rpm / 5 min, the original culture medium was removed, and the cells were resuspended in PBS to adjust the cell density of each sample to 1×10⁻⁶. 5 100 uL;

[0059] (2) Add 5 μL of antibodies corresponding to CD45, CD34 and CD29 respectively, mix thoroughly, and incubate on ice in the dark for 30 min; centrifuge at 1000 r / min for 5 min, and discard the supernatant;

[0060] (3) Add 1 mL of PBS buffer to wash the cells twice, resuspend the cells in 300 μL of PBS, replace with flow cytometry tubes, and perform instrumental analysis;

[0061] The results are as follows Figure 3As shown in C, flow cytometry was used to identify the negative expression of the cell surface antibodies CD45 and CD34, and the positive expression of the specific antibody CD29, thus determining the cell origin.

[0062] Deer antler stem cells were pretreated with normoxic and hypoxic methods respectively.

[0063] S2.1 Count the deer antler stem cells (ABPCs) and count them at 1 × 10⁻⁶. 7 Inoculate at a density of / dish into 10 cm dishes;

[0064] S2.2 When the confluence of deer antler stem cells (ABPCs) reaches 70%~80%, discard the complete culture medium, wash the cells three times with 1×PBS, and replace the culture medium with exosome-free serum medium (10% exosome-free FBS, DMEM, penicillin / streptomycin). Place the cell culture dishes in normal oxygen (20% O2, 5% CO2) and hypoxia incubators (1.5% O2, 5% CO2) respectively, and culture them in a constant temperature incubator at 37 ℃ for 48 h. Collect the supernatant and store it at -80℃ for subsequent use.

[0065] Extraction and purification of exosomes derived from deer antler stem cells under normoxic and hypooxic conditions—Deer antler stem cell exosomes ABPCs-EXOs

[0066] S3.1 Place the collected supernatant in a 50 mL centrifuge tube, centrifuge at 3000 rpm for 10 min to remove cell pellet, collect the supernatant, transfer it to an ultrafiltration centrifuge tube, centrifuge at 4 ℃ and 3000 rpm for 10 min, repeat centrifugation until the supernatant is concentrated to 20 mL (the concentration ratio is generally 150~200 mL to 20 mL), and collect the supernatant;

[0067] S3.2 Add Exosome Concentration Solution (ESC reagent) to the centrifuged supernatant after removing impurities. The ESC reagent dosage ratio is 5 mL per 20 mL of cell culture supernatant.

[0068] S3.3 After adding ESC reagent, tighten the centrifuge tube, mix it with a vortex mixer for 1 min, and then place it at 4℃ for at least 2 h (increasing the standing time can improve the exosome yield, but the standing time should not exceed 24 h).

[0069] S3.4. Take out the centrifuge tube containing the mixture and centrifuge at 10000g for 50 min at 4 ℃. Discard the supernatant. The precipitate is rich in exosome particles, which is the centrifuged precipitate.

[0070] S3.5. Take 1× PBS and beat the centrifuged precipitate evenly until it dissolves. Then transfer the resuspension to a new 1.5 mL centrifuge tube. The ratio of PBS used is 5 mL of PBS solution to every 20 mL of cell culture supernatant.

[0071] S3.6 Centrifuge a 1.5 mL centrifuge tube containing the resuspension at 4 ℃ and 12000g for 2 min, and retain the supernatant. The supernatant is rich in exosome particles, which is the crude exosome particle product.

[0072] S3.7 Transfer the harvested crude exosome particles into the upper chamber of an Exosome Purification Filter (EPF column) and centrifuge at 3000g for 10 min at 4 ℃. After centrifugation, collect the liquid at the bottom of the EPF column. This liquid is the hypoxia pretreated deer antler stem cell exosome preparation. After aliquoting, store in a -80℃ freezer.

[0073] Observation of exosome morphology using transmission electron microscopy

[0074] The results are as follows Figure 4 As shown, exosomes are round or elliptical vesicles with a typical double membrane structure. After negative staining, the edges appear as bright white rings, while the central region appears gray or dark due to the different electron densities of the contents, proving that exosomes were successfully extracted in this study.

[0075] Screening of deer antler stem cell exosomes ABPCs-EXOs concentration

[0076] (1) Isolate and culture BMSCs in vitro until the cells reach 80% confluence. Digest the cells and seed them into 96-well plates with 3000 cells per well. Dilute ABPCs-EXOs with serum-free DMEM medium at concentrations of 0, 5, 10, 20, and 40 μg / mL. Then use the above culture medium for BMSCs and culture at 37 ℃ with 5% CO2.

[0077] (2) After incubation for 0, 12, 24, 48 and 72 hours respectively, a CCK-8 detection solution containing 10% was prepared in serum-free DMEM medium. 100 μl of CCK-8 detection solution was added to each well and the OD value was detected at a wavelength of 450 nm using a UV spectrophotometer after 2 hours.

[0078] (3) Statistical analysis was performed on the OD values ​​of different concentrations to screen out the optimal concentration of deer antler stem cell exosomes ABPCs-EXOs.

[0079] The results are as follows Figure 5As shown in the CCK-8 results, the cell viability of the 20 μg / mL and 40 μg / mL groups was similar after 24 h of treatment with bone marrow mesenchymal stem cells. At 48 h, the 20 μg / mL group was significantly superior to the 40 μg / mL group. Further increasing the CM concentration had little effect on cell viability. Therefore, this study ultimately selected the 20 μg / mL CM concentration as the final experimental concentration.

[0080] Comparison of osteogenic differentiation capacity of BMSCs by pretreatment of ABPC-derived exosomes with normoxic and hypoxic conditions.

[0081] Bone marrow mesenchymal stem cells (BMSCs) were routinely digested, counted, and seeded into 6-well plates, divided into three groups: ① ABPCs-EXOs group (ABPCs-derived exosomes pretreated with normoxic conditions were added to osteogenic induction medium at 20 μg / mL); ② Hypo-ABPCs-EXOs group (ABPCs-derived exosomes pretreated with hypooxic conditions were added to osteogenic induction medium at 20 μg / mL); ③ control group (cultured in conventional osteogenic induction medium).

[0082] 1. Alkaline phosphatase staining

[0083] (1) Third-generation ABPCs were cultured using osteogenic induction medium;

[0084] (2) After 7 days of culture, staining was performed using the BCIP / NBT alkaline phosphatase kit.

[0085] The results are as follows Figure 6 As shown in Figure A, the alkaline phosphatase staining in the two exosome treatment groups was significantly better than that in the control group, with the Hypo-ABPCs-EXOs group showing a more pronounced staining than the ABPCs-EXOs group.

[0086] 2. Alizarin Red staining

[0087] The ABPCs were seeded and grouped as described above. After 21 days of induction culture, the osteogenic differentiation effect of ABPCs was detected using alizarin red stain.

[0088] The results are as follows Figure 6 As shown in B, the density of calcium nodules in the two exosome treatment groups was significantly higher than that in the control group, with the Hypo-ABPCs-EXOs group showing a greater increase than the ABPCs-EXOs group.

[0089] 3. Quantitative Polymerase Chain Reaction (qPCR)

[0090] (1) The cells were seeded and grouped as described above. After culturing for 72 h, the cells in the six-well plates were collected, total RNA was extracted, and the concentration of RNA in each sample was measured. Then, cDNA was synthesized using Primescript RT Master Mix reagent. The amplification and detection were performed on a real-time quantitative PCR instrument to detect the expression levels of Runt-related transcription factor-2 (Runx2) and bone morphogenetic protein-2 (BMP-2) genes, with GAPDH as the internal reference gene.

[0091] (2) Gene expression levels were recorded using the cycle threshold (Ct). All experiments were repeated three times, with three sub-wells for each sample each time. The data were analyzed using 2-ΔΔCT.

[0092] The results are as follows Figure 6 As shown in Figure C, the bone-related genes (Runx2 and BMP-2) in the two exosome treatment groups were significantly better than those in the control group. The Hypo-ABPCs-EXOs group showed more significant expression than the ABPCs-EXOs group, indicating that hypoxia pretreatment of exosomes derived from deer antler stem cells can significantly promote osteogenic differentiation of bone marrow mesenchymal stem cells compared to normoxic conditions.

[0093] 4. Western blot (WB) assay

[0094] (1) Seed cells and group cells induction as described above. After culturing for 72 h, collect cells from the six-well plates and lyse them on ice for 30 min with RIPA lysis buffer.

[0095] (2) Using a sterile plastic cell scraper, scrape cells from each culture plate, centrifuge, and remove cell debris;

[0096] (3) Then collect the supernatant and determine the total protein concentration using the BCA protein quantification kit. Add 1 / 5 of the total volume of protein loading buffer and denature at 100 °C for 10 min, then perform SDS-PAGE electrophoresis on the protein sample;

[0097] (4) Transfer the protein to a 0.45 μm PVDF membrane and block it in a protein-free rapid blocking solution for 10 min. Then incubate it with primary antibodies at 4 °C overnight. The primary antibodies involved are as follows: RUNX2, BMP2, GAPDH.

[0098] (5) After washing 3 times, incubate with the secondary antibody on a shaker at room temperature for 1 h. Use WB ultrasensitive imaging solution to visualize protein bands and use ImageJ software to analyze the gray values ​​of the bands to determine the protein expression level;

[0099] The results are as follows Figure 6 As shown in Figure D, the expression of osteogenic-related proteins (Runx2 and BMP-2) in the two exosome treatment groups was significantly higher than that in the control group, with the Hypo-ABPCs-EXOs group showing more significant expression than the ABPCs-EXOs group.

[0100] 5. Immunofluorescence experiment

[0101] (1) Seed cells and induce cell division as described above. After culturing for 72 h, prepare and fix cell smears as described in Experiment Example 2.3. Add 0.5% Triton to the cell smears at room temperature and incubate for 10 min. Wash three times with PBS, 5 min each time. Block the cells with 15% goat serum at 37 ℃ for 30 min and aspirate. Add primary antibodies (RUNX2, BMP2) respectively and incubate overnight at 4 ℃.

[0102] (2) The next day, after washing with PBS, add rhodamine-labeled secondary antibody (red), incubate at room temperature in the dark for 1 h, and wash 3 times with PBS, 5 min each time. Add FITC-labeled phalloidin (green, concentration 1:200), incubate at room temperature in the dark for 30 min, wash 3 times with PBS, add ready-to-use DAPI working solution (blue), incubate at 37 ℃ in the dark for 15 min, and wash 3 times with PBS, 5 min each time. Mount with anti-fluorescence quenching mounting medium, observe under an upright fluorescence microscope, and compare the expression of RUNX2 and BMP2 by fluorescence intensity detection.

[0103] The results are as follows Figure 6 As shown, the osteogenic-associated protein RUNX2 (RUNX2) in the two exosome treatment groups Figure 6 E), BMP2 ( Figure 6 F) expression was significantly higher than that in the control group, with the Hypo-ABPCs-EXOs group showing more significant expression than the ABPCs-EXOs group.

Claims

1. A method for preparing a hypoxia-pretreated deer antler stem cell exosome preparation, characterized in that, Includes the following steps: S1. Isolation and culture of deer antler stem cells ABPCs to obtain isolated and cultured deer antler stem cells ABPCs for later use; S2. The deer antler stem cell ABPCs obtained in S1 were subjected to hypoxia pretreatment to obtain primary deer antler stem cell exosomes ABPCs-EXOs; specifically as follows: S2.

1. The deer antler stem cell ABPCs were counted and divided into 1×10⁻⁶ cells. 7 / dish density inoculated into 10 cm dishes; S2.2 When the confluence of antler stem cell ABPCs reaches 70%-80%, discard the complete culture medium, wash the cells three times with 1×PBS, replace the culture medium with exosome-free serum medium, place the cell culture dish in a hypoxic incubator, and culture in a constant temperature incubator. Collect the supernatant, which is the primary antler stem cell exosome ABPCs-EXOs. The primary antler stem cell exosome ABPCs-EXOs should be stored at -80℃ for subsequent use. The oxygen concentration in the hypoxic incubator is approximately 1.5%, and the carbon dioxide concentration is approximately 5%. S3. After extracting and purifying the primary deer antler stem cell exosomes ABPCs-EXOs obtained in S2, a hypoxia-pretreated deer antler stem cell exosome preparation was obtained.

2. The method for preparing the deer antler stem cell exosome preparation according to claim 1, characterized in that, In S2.2, the exosome-free serum culture medium is made by adding 10% (v / v) exosome-free serum, 1% (v / v) penicillin and streptomycin to a low-glucose 1 g / L LDMEM medium, with a final concentration of 100 U / mL penicillin and 100 μg / mL streptomycin.

3. The method for preparing the deer antler stem cell exosome preparation according to claim 1, characterized in that, In step S2.2, the temperature of the incubator is 37°C, and the incubation time is 48 h.

4. The method for preparing the deer antler stem cell exosome preparation according to claim 1, characterized in that, The specific operation of S1 is as follows: S1.1, collect deer antler mesenchymal tissue that has grown for 5-6 days, wash it 3 times with PBS solution containing 1% (v / v) double antibiotics, slice the deer antler tissue longitudinally, and carefully locate and separate the mesenchymal layer; S1.

2. Cut the tissue into 0.7 mm thin slices, and then digest them in 200 U / mL type I collagenase for 30 min. Under a microscope, rough edges can be seen in the tissue. The tissue is then cultured as primary cells in DMEM complete medium. When the cells crawl out from the periphery of the adherent tissue block, the culture medium is replaced. The DMEM complete medium is DMEM medium containing 10 wt% fetal bovine serum. S1.3 When the cell density reaches 70%, digest with 0.25% (w / v) trypsin for 1 min, centrifuge to collect the cells, and then passage them in DMEM complete medium to obtain P1 cells; repeat the above method to continue passage to obtain P3 cells.

5. The method for preparing the deer antler stem cell exosome preparation according to claim 1, characterized in that, The specific operation of S3 is as follows: S3.1, Place the collected supernatant in a 50 mL centrifuge tube, centrifuge for 10 min to remove cell pellet, collect the supernatant, transfer it to an ultrafiltration centrifuge tube, centrifuge at 4℃ for 10 min, repeat centrifugation until the supernatant is concentrated to 20 mL, and collect the supernatant. S3.2 Add ESC reagent to the supernatant obtained in S3.1, wherein the ESC reagent dosage ratio is: 5 mL ESC reagent per 20 mL cell culture supernatant; S3.3 After adding ESC reagent, tighten the centrifuge tube, mix it with a vortex mixer for 1 min, and then let it stand at 4 ℃ for at least 2 h to obtain the mixture; S3.

4. Take out the centrifuge tube containing the mixture and centrifuge at 10000g for 50 min at 4 ℃. Discard the supernatant and obtain the centrifuged precipitate, which is rich in exosome particles. S3.5 Take 1× PBS and beat the centrifuged precipitate obtained in S3.4 evenly until it dissolves to obtain a resuspension. Transfer the resuspension to a new 1.5 mL centrifuge tube. The PBS ratio is: 5 mL PBS is added to every 20 mL of cell culture supernatant. S3.6 Centrifuge a 1.5 mL centrifuge tube containing the resuspension at 4℃ and 12000g for 2 min, and retain the supernatant. The supernatant is rich in exosome particles, which is the crude exosome particle product. S3.7 Transfer the crude exosome particles obtained in S3.6 into the EPF column chamber and centrifuge at 3000g for 10 min at 4℃. After centrifugation, collect the liquid at the bottom of the EPF column. This liquid is the hypoxia pretreated deer antler stem cell exosome preparation. After aliquoting, store it in a -80℃ low temperature freezer.

6. The application of a hypoxia-pretreated deer antler stem cell exosome preparation, characterized in that, It is used to promote osteogenic differentiation of bone marrow mesenchymal stem cells.

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