Culture method of bone marrow mesenchymal stem cells

Abstract

The invention belongs to the technical field of stem cell culture, and discloses a culture method of bone marrow mesenchymal stem cells. A culture medium added with glutathione, L-ascorbic acid, L-glutamine, human serum albumin, sodium hydrosulfide, insulin, insulin-like growth factor-1, deer bone morphogenetic protein and antler stem cell exosome extract is used for culturing the bone marrow mesenchymal stem cells, and osteogenic differentiation of the bone marrow mesenchymal stem cells can be directionally induced. The culture medium does not contain animal serum, the formula is simple, the components are controllable, the cultured bone marrow mesenchymal stem cells are high in activity and good in state, the expression of marker genes of osteogenic differentiation is up-regulated, the regulation and control efficiency and accuracy of osteogenic differentiation are remarkably improved, and sufficient cell sources are provided for scientific research and clinic.

Description

Technical Field

[0001] The invention belongs to the technical field of stem cell culture, and in particular relates to a method for culturing bone marrow mesenchymal stem cells. Background Art

[0002] If a fracture occurs in a person's limbs, timely treatment will allow most patients to form callus within 2-4 weeks and then heal. However, among all fracture patients, about 5% of patients have a slow or difficult healing of their fractures. The failure of a fracture to heal is called nonunion. The causes of nonunion are complex. Clinically, the treatment of nonunion mainly relies on bone transplantation surgery, and the cure rate is also very unsatisfactory. It is the most difficult disease in orthopedics.

[0003] Bone marrow mesenchymal stem cells (BMSCs) are a type of non-hematopoietic stem cells found in the bone marrow, skeletal muscle, periosteum and trabecular bone. They have the general characteristics of mesenchymal stem cells and have the ability to differentiate into osteoblasts, chondrocytes, stromal cells and other cells under appropriate in vivo or in vitro environments. They have great potential and application value in curing fractures, nonunions and other diseases. How to induce BMSCs to differentiate into osteoblasts and obtain sufficient and active osteoblasts derived from mesenchymal stem cells for scientific research or clinical application is the first step in using BMSCs for the treatment of bone diseases. Therefore, whether it is possible to provide a culture medium and cell culture method that promotes the osteogenic differentiation of bone marrow mesenchymal stem cells is an urgent problem that technicians in this field need to solve.

[0004] The widely used BMSCs in vitro culture medium generally contains 5% to 20% animal serum. The cost of obtaining serum is high, and the quality of serum varies between different batches, making it difficult to achieve large-scale standardized cell culture. At the same time, the complex cytokines in serum are not conducive to artificially controlling the differentiation direction of mesenchymal stem cells, resulting in a large number of useless cells or even tumorigenic cells in the cultured cells. Serum-free culture can largely avoid the disadvantages brought by serum, and can achieve the directional induced differentiation of BMSCs by adding specific signal substances, nutrients, cytokines or growth factors.

[0005] Deer antler is the young antler of male deer (Cervus nippon Temminck or Cervus elaphus Linnaeus) that is not ossified and densely covered with velvet. It has been used as a medicinal material for the treatment of osteoporosis since ancient times. It takes only 2 months for deer antler to grow from the beginning to the full growth. Its bone formation rate is about 108 times that of humans. The growth, development and calcification of deer antler are similar to the calcification process of long bones. Therefore, the tissue basis and regulatory mechanism of deer antler bone calcification can be used to prepare a medium for inducing osteogenic differentiation of BMSCs. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a culture medium for promoting bone marrow mesenchymal stem cells, which does not require the addition of animal serum, has a simple and controllable formula, can achieve directional induction of osteogenic differentiation of BMSCs, and provide a stable cell source for the treatment of osteoporosis.

[0007] A second object of the present invention is to provide a method for culturing bone marrow mesenchymal stem cells.

[0008] One of the purposes of the present invention is achieved by the following technical solution:

[0009] A culture medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells comprises a basic culture medium and the following components added to the basic culture medium: glutathione, L-ascorbic acid, L-glutamine, human serum albumin, sodium hydrosulfide, insulin, insulin-like growth factor-1, deer bone morphogenetic protein, and deer antler stem cell exosome extract.

[0010] Furthermore, the content of each added ingredient is as follows: 60-100 μg / mL glutathione, 100-300 μg / mL L-ascorbic acid, 1% L-glutamine, 1% human serum albumin, 2 ng / L sodium hydrosulfide, 2-10 μg / mL insulin, 5-30 μg / mL insulin-like growth factor-1, 80-240 μg / mL recombinant deer bone morphogenetic protein, and 10-40 μg / mL deer antler stem cell exosome extract.

[0011] Optimally, the final concentration of each component is 80 μg / mL glutathione, 150 μg / mL L-ascorbic acid, 1% L-glutamine, 1% human serum albumin, 2 ng / L sodium bisulfide, 50 μg / mL insulin, 10 μg / mL insulin-like growth factor-1, 200 μg / mL recombinant deer bone morphogenetic protein, and 20 ng / L deer antler stem cell exosome extract.

[0012] Furthermore, the nucleotide sequence encoding the deer bone morphogenetic protein is shown in SEQ ID NO.1.

[0013] Furthermore, the deer bone morphogenetic protein is obtained by inducing expression of genetically engineered Escherichia coli transformed with a recombinant BMP plasmid and then isolating and purifying it.

[0014] Furthermore, the method for preparing the antler stem cell exosome extract is as follows: the supernatant of the antler stem cell crushing liquid and the supernatant of the culture medium are mixed, the supernatant is obtained by centrifugal filtration, the precipitate is obtained by a second centrifugation, the precipitate is resuspended with PBS buffer, filtered for sterilization, and freeze-dried and low-temperature crushed.

[0015] Furthermore, the first centrifugal treatment was performed at a rotation speed of 4000 rpm for 20 min, and the second centrifugal treatment was performed at a rotation speed of 10000 rpm for 20 min.

[0016] The second object of the present invention is achieved by adopting the following technical solution:

[0017] A method for culturing bone marrow mesenchymal stem cells comprises the following steps: during cell passage, gradually replacing the bone marrow mesenchymal stem cell culture medium with the culture medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells at a ratio of 25%, 50%, 75%, 90%, and 100%.

[0018] Furthermore, the culture method can induce osteogenic differentiation of bone marrow mesenchymal stem cells.

[0019] Furthermore, the bone marrow mesenchymal stem cells are human bone marrow mesenchymal stem cells.

[0020] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0021] The present invention discloses a method for culturing bone marrow mesenchymal stem cells. The culture medium used does not contain animal serum, has a simple formula, and has controllable ingredients. The cultured bone marrow mesenchymal stem cells have high activity and good condition, and the marker genes of osteogenic differentiation are upregulated, which significantly improves the regulation efficiency and accuracy of osteogenic differentiation and provides sufficient cell sources for scientific research and clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The electrophoresis diagram of the BMP protein extracted and purified from Escherichia coli in Example 1; wherein lane 0 is a 180 kDa Marker, lane 1 is a product expressed by an engineering bacterium transfected with an empty plasmid pGEX-4T-1 before purification, lane 2 is a product expressed by an engineering bacterium transfected with a recombinant plasmid pGEX-4T-1 / BMP before purification, lane 3 is a product expressed by an engineering bacterium transfected with an empty plasmid pGEX-4T-1 after purification, and lane 4 is a product expressed by an engineering bacterium transfected with a recombinant plasmid pGEX-4T-1 / BMP after purification;

[0023] Figure 2 The invention discloses a culture medium and a culture method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, and provides a percentage of cell calcification area after culturing bone marrow mesenchymal stem cells for 12 days. DETAILED DESCRIPTION

[0024] The technical scheme of the present invention is further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0025] In the examples, the second generation human BMSCs (Cat. No. CP-H166), human BMSCs culture medium (Cat. No. CM-H166), human bone marrow mesenchymal stem cell osteogenic differentiation medium (Cat. No. PD-014) and DMEM culture medium (Cat. No. PM150210) were purchased from Wuhan Pronosai Life Science Technology Co., Ltd.; the vector plasmid pGEX_4T_l (Cat. No. HG-VYA0221) was purchased from Changsha Abiwei Biotechnology Co., Ltd.; competent Escherichia coli BL21 (Cat. No. EC0114) was purchased from Thermo Scientific; RNA extraction kit (Cat. No. RC102-01), reverse transcription cDNA kit (Cat. No. R433-01), and qPCR kit (Cat. No. R333-01) were purchased from Nanjing Novozyme Biotechnology Co., Ltd.; PCR and qPCR primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0026] Example 1

[0027] Obtaining Deer Osteogenic Protein:

[0028] The mRNA sequence encoding the osteogenic protein of sika deer (Cervus nippon) was obtained from the NCBI database (Accession HQ877675.1, SEQ ID NO.1), and the following primers were designed to obtain the deer osteogenic protein gene:

[0029] BMP_F1: 5'-CCTGGTAACCGAATGCTGATGGT-3' (SEQ ID NO. 2);

[0030] BMP_R1: 5'-TCAGCGGCACCCACATCCCTCCACTA-3' (SEQ ID NO.3);

[0031] BMP_F2: 5'-TTCCGCGTggatccCCTGGTAACCGAATGCTGA-3' (SEQ ID NO.4);

[0032] BMP_R2: 5'-CGGCCGCTCctcgagACCCACATCCCTCCACTA-3' (SEQ ID NO. 5).

[0033] Using cDNA from antler stem cells as a template, primers BMP_F1 and BMP_R1 were used to amplify the deer osteogenic protein gene, and primers BMP_F2 and BMP_R2 were used to perform PCR amplification to obtain the deer osteogenic protein gene with BamHI and XhoI restriction sites at both ends. The DNA fragment was double-digested with BamHI and XhoI, and then ligated with the plasmid pGEX_4T_1 that was also double-digested with BamHI and XhoI to obtain the recombinant plasmid pGEX_4T_1 / BMP.

[0034] The empty plasmid pGEX-4T-l and the recombinant plasmid pGEX-4T-l / BMP were transformed into the Escherichia coli expression strain BL21, and the Escherichia coli genetically engineered bacteria with high expression of deer osteogenic protein were obtained after culture and screening. The engineered bacteria were inoculated into a 100 mL Erlenmeyer flask containing 10 mL LB medium, and cultured on a shaker at 37°C and 220 rpm for 12 hours, and then inoculated into a fermenter containing LB medium at a ratio of 1:100 for amplification culture, and cultured at 37°C and 220 rpm for 3 hours until the OD600 was about 0.6, and then 0.5 mM IPTG was added, and induced culture was carried out at 16°C for 20 hours.

[0035] The cells were collected by centrifugation at 8000rpm for 10min, washed once with washing buffer, suspended with breaking buffer, broken by ultrasonic wave, and collected by centrifugation at 10000rpm for 10min. The protein expression was detected by SDS-PAGE, and the protein was purified by glutathione-Sepharose4B column, and the protein purification was detected by SDS-PAGE. Figure 1 The induced expression product of Escherichia coli transfected with pGEX_4T_l / BMP showed an obvious protein band at 46kDa, which was consistent with the molecular weight of the recombinant protein deer osteogenic protein. Except for the main band at 46kDa, there were almost no other miscellaneous bands, indicating that the recombinant protein deer osteogenic protein was successfully purified.

[0036] Example 2

[0037] Extraction of exosomes from antler stem cells:

[0038] Deer antler stem cells are isolated from the periosteum of the antler shank of sika deer. Deer antler stem cells are inoculated into a culture bottle containing DMEM basal medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and incubated in a 37°C, 5% CO2 incubator. Cells are passaged when they grow to more than 80% confluence.

[0039] The culture medium supernatant of the 5th generation of antler stem cells was collected, and the antler stem cells were ultrasonically broken, centrifuged at 1000rpm for 20min, and the supernatant was filtered to obtain the supernatant. The treated cell disruption liquid supernatant and the culture medium supernatant were mixed to obtain a crude extract of antler stem cell exosomes. The crude exosome extract was centrifuged at 4000rpm for 20min, and the supernatant was filtered using a filter with a pore size of 70μm. The filtrate was centrifuged for a second time, and the speed was 10000rpm for 20min to obtain the precipitate, which was the antler stem cell exosomes. The precipitate was resuspended in PBS buffer, filtered using a 0.22μm bacterial sieve to obtain a antler stem cell exosome suspension, placed in a vacuum freeze dryer for drying, and low-temperature crushed after drying to obtain a freeze-dried powder of antler stem cell exosome extract, which was sealed and stored at room temperature in a dry place with a humidity not exceeding 50%.

[0040] Example 3

[0041] Bone marrow mesenchymal stem cell culture:

[0042] The medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells was prepared using DMEM basal medium, so that the final concentration of each component was 80 μg / mL glutathione, 150 μg / mL L-ascorbic acid, 1% L-glutamine, 1% human serum albumin, 2 ng / L sodium hydrosulfide, 50 μg / mL insulin, 10 μg / mL insulin-like growth factor-1, 200 μg / mL recombinant deer bone morphogenetic protein, and 20 ng / L deer antler stem cell exosome extract.

[0043] Resuspend the cells in a special medium for bone marrow mesenchymal stem cells containing 10% FBS and adjust the cell density to 2×10 5 / mL, inoculated in a culture flask with a bottom area of ​​25cm and placed in a 37℃, 5% CO2 incubator for culture. The culture medium was changed for the first time after 48h, and then every 1d. When the cell confluence reached 80%, it was digested with 0.25% trypsin and then subcultured at a ratio of 1:3. The subculture medium is:

[0044] Table 1: Subculture medium components in the culture method of the present invention for promoting osteogenic differentiation of bone marrow mesenchymal stem cells

[0045]

[0046]

[0047] Example 4

[0048] Example 4 provides a culture medium and a cell culture method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells. The other features are the same as those of Example 3, except that: the culture medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells contains: 60 μg / mL glutathione, 300 μg / mL L-ascorbic acid, 2 μg / mL insulin, 5 μg / mL insulin-like growth factor-1, 80 μg / mL recombinant deer bone morphogenetic protein, and 40 μg / mL deer antler stem cell exosome extract.

[0049] Example 5

[0050] Example 5 provides a culture medium and a cell culture method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells. The other features are the same as those of Example 3, except that: the culture medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells contains: 100 μg / mL glutathione, 100 μg / mL L-ascorbic acid, 1% L-glutamine, 1% human serum albumin, 2 ng / L sodium hydrosulfide, 10 μg / mL insulin, 30 μg / mL insulin-like growth factor-1, 240 μg / mL recombinant deer bone morphogenetic protein, and 10 μg / mL deer antler stem cell exosome extract.

[0051] Comparative Example 1

[0052] Comparative Example 1 provides a culture medium and a cell culture method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Other features are the same as those of Example 3, except that: deer bone morphogenetic protein is not added to the culture medium, and 200 μg / mL human serum albumin is additionally added instead.

[0053] Comparative Example 2

[0054] Comparative Example 2 provides a culture medium and a cell culture method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells. The other features are the same as those of Example 3, except that: no antler stem cell exosome extract is added to the culture medium, and double (400 μg / mL) deer bone morphogenetic protein is added.

[0055] Comparative Example 3

[0056] Comparative Example 3 provides a culture medium and a cell culture method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Other features are the same as those of Example 3, except that: no antler stem cell exosome extract is added to the culture medium, and 220 μg / mL deer bone morphogenetic protein is added.

[0057] Comparative Example 4

[0058] Comparative Example 4 provides a culture medium and a cell culture method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Other features are the same as those of Example 3, except that: no antler stem cell exosome extract and no deer bone morphogenetic protein are added to the culture medium.

[0059] Comparative Example 5

[0060] Control group: resuspend the cells in a special medium for bone marrow mesenchymal stem cells containing 10% FBS and adjust the cell density to 2×10 5 The cells were inoculated into a 25 cm culture flask and placed in a 37°C, 5% CO2 incubator for culture. The medium was changed for the first time after 48 hours, and then every 1 day. When the cell confluence reached 80%, the cells were digested with 0.25% trypsin and passaged at a ratio of 1:3. The passage medium was a special medium for bone marrow mesenchymal stem cells.

[0061] Test Example 1

[0062] MTT assay to detect cell survival:

[0063] The cell survival rate of the bone marrow mesenchymal stem cells cultured in the above-mentioned Examples 3-4 and Comparative Examples 1-5 was determined at the 5th, 10th, 15th and 20th generations, respectively. The cells were digested to prepare a single cell suspension, and the cells were counted under a microscope. The bone marrow mesenchymal stem cells were inoculated on a 96-well plate with 3000 cells per well, and 5 replicates were made for each example. The culture medium was supplemented to 200 μL per well, respectively, and placed in an incubator at 37°C and containing 5% CO2 for 48 hours. MTT was weighed and dissolved in sterile PBS (NaCl 8g, KCl 0.2g, Na2HPO4 1.44g, KH2PO40.24g, pH adjusted to 7.4, constant volume 1L) to form a solution with a concentration of 5 mg / mL, 20 μL of MTT solution was added to each well of the 96-well plate, and incubated in an incubator for 4 hours. Place the 96-well plate in a plate centrifuge and centrifuge at 2000r for 5 minutes, remove the supernatant, add 150μL DMSO to each well, place on a shaker and shake at low speed for 10 minutes to fully dissolve the crystals. Use an ELISA reader to detect the light absorption value of each well at a wavelength of 490nm and record the results.

[0064] The wells added with culture medium, MTT and dimethyl sulfoxide were used as blank wells, and the survival rates of bone marrow mesenchymal stem cells in different culture media and different passage numbers were calculated: survival rate = [(experimental well - blank well) / (control well - blank well)] × 100%.

[0065] Table 2 Survival rate of bone marrow mesenchymal stem cells

[0066]

[0067]

[0068] The results are shown in Table 2. When the number of passages of bone marrow mesenchymal stem cells is less than 10 generations, the cell survival rate of all serum-free medium groups (Examples 3-4, Comparative Examples 1-4) is not much different from that of the control group (Comparative Example 5). When the number of passages exceeds 10 generations, the concentration of each component in the culture medium affects the activity of bone marrow mesenchymal stem cells. When the final concentration of each component is 80μg / mL glutathione, 150μg / mL L-ascorbic acid, 1% L-glutamine, 1% human serum albumin, 2ng / L sodium hydrosulfide, 50μg / mL insulin, 10μg / mL insulin-like growth factor-1, 200μg / mL recombinant deer bone morphogenetic protein, 20ng / L deer antler stem cell exosome extract, the cell viability of bone marrow mesenchymal stem cells is optimal; arbitrarily adjusting the concentration of the component or deleting the component will reduce the efficacy of the culture medium. When passaged for 20 generations, the survival rates of bone marrow mesenchymal stem cells in Examples 3-5 were all over 85%, which was 20% higher than that in Comparative Examples 1-4 with omitted components. It can be seen that adding deer bone morphogenetic protein and antler stem cell exosome extract to the culture medium can effectively improve the cell survival rate of bone marrow mesenchymal stem cells, allowing them to maintain a high cell viability during high-frequency continuous passages.

[0069] Test Example 2

[0070] Take the bone marrow mesenchymal stem cells cultured for 12 days with the culture medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells in P7 substitute in Examples 3-4 and Comparative Examples 1-4, discard the culture medium, wash with 6°C pre-cooled PBS, fix with 4% paraformaldehyde at room temperature for 10 minutes, discard the paraformaldehyde, wash three times with PBS, stain with 0.5% alizarin red for 10 minutes, discard the staining solution, wash with water three times, osteoblasts will calcify, and the calcified osteoblasts will be stained red by alizarin red. Observe the staining situation under a microscope, and the percentage of calcification area can be calculated based on the area of ​​the stained cells.

[0071] The results are as follows Figure 2 As shown, it is shown that the culture medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells prepared by the present invention can induce differentiation to obtain more osteoblasts, and the optimal concentration for inducing osteogenic differentiation is 80μg / mL glutathione, 150μg / mL L-ascorbic acid, 1% L-glutamine, 1% human serum albumin, 2ng / L sodium hydrosulfide, 50μg / mL insulin, 10μg / mL insulin-like growth factor-1, 200μg / mL recombinant deer bone morphogenetic protein, and 20ng / L deer antler stem cell exosome extract.

[0072] Test Example 3

[0073] qPCR detection of important indicator gene expression of osteogenic differentiation:

[0074] Bone marrow mesenchymal stem cells were cultured for 5 days using the medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells in P7 surrogate in Examples 3-4 and Comparative Examples 1-4, respectively, and their RNA was extracted. The gene expression of osteogenic-related markers (Runx2, OPN, OCN) was detected using a qPCR kit. Three replicate wells were made for each test sample.

[0075] Primer sequences used:

[0076] RUNX2_F: 5'-CTTCAAGGTGGTAGCCCTCG-3' (SEQ ID NO. 6);

[0077] RUNX2_R: 5'-TAACAGCAGAGGCATTCCGG-3' (SEQ ID NO.7);

[0078] OPN_F: 5'-AACAGAATAAATAAGCTCAA-3' (SEQ ID NO.8);

[0079] OPN_R: 5'-CCTTGTCCCTGGACTTACAT-3' (SEQ ID NO.9);

[0080] OCN_F: 5'-TGAAGAGACCCAGGCGCTA-3' (SEQ ID NO. 10);

[0081] OCN_R: 5'-CACAGTCCGGATTGAGCTCA-3' (SEQ ID NO. 11);

[0082] GAPDH_F: 5'-GTCTCCTCTGACTTCAACAGCG-3' (SEQ ID NO.12)

[0083] GAPDH_R: 5'-ACCACCCTGTTGCTGTAGCCAA-3' (SEQ ID NO.13)

[0084] Amplification parameters: 94℃ 5min; 94℃ 30sec, 55℃ 30sec, 72℃ 60sec, 30 cycles; 72℃ 10min. GAPDH was used as the housekeeping gene and 2 -△△CT Statistical data: △Ct = Ct target gene - Ct internal reference, then calculate the average △Ct of the control group, recorded as △Ct control average, △△Ct = △Ct sample - △Ct control average.

[0085] Table 3 Gene expression of osteogenesis-related markers

[0086] Group Runx2 OPN OCN Example 3 4.179±0.047** 3.117±0.055** 5.637±0.164** Example 4 4.113±0.056** 3.025±0.159** 5.596±0.133** Example 5 4.022±0.113** 2.875±0.127** 5.315±0.176** Comparative Example 1 2.508±0.048** 2.884±0.124** 3.824±0.074** Comparative Example 2 2.866±0.058** 1.742±0.102** 4.201±0.129** Comparative Example 3 2.375±0.094** 1.980±0.079** 4.098±0.144** Comparative Example 4 1.536±0.061* 1.211±0.042* 1.792±0.095* Comparative Example 5 1.000±0.036 1.000±0.053 1.000±0.061

[0087] As can be seen from Table 3, the culture medium and cell culture method for promoting osteogenic differentiation of bone marrow mesenchymal stem cells in the present invention can significantly promote the relative expression levels of RUNX2, OPN and OCN.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. A medium for promoting bone marrow mesenchymal stem cells, characterized in that: The invention comprises a basal medium and the following components added to the basal medium: glutathione, L-ascorbic acid, L-glutamine, human serum albumin, sodium hydrosulfide, insulin, insulin-like growth factor-1, deer bone morphogenetic protein, and deer antler stem cell exosome extract.

2. The culture medium according to claim 1, characterized in that The content of each added ingredient is as follows: 60-100 μg / mL glutathione, 100-300 μg / mL L-ascorbic acid, 1% L-glutamine, 1% human serum albumin, 2 ng / L sodium hydrosulfide, 2-10 μg / mL insulin, 5-30 μg / mL insulin-like growth factor-1, 80-240 μg / mL recombinant deer bone morphogenetic protein, and 10-40 μg / mL deer antler stem cell exosome extract.

3. The culture medium according to claim 1 or 2, characterized in that The nucleotide sequence encoding the deer bone morphogenetic protein is shown in SEQ ID NO.

1.

4. The culture medium according to claim 3, characterized in that The deer bone morphogenetic protein is prepared by inducing expression of genetic engineering bacteria of Escherichia coli transformed with a recombinant BMP plasmid and then separating and purifying it.

5. The culture medium according to claim 1, characterized in that The preparation method of the antler stem cell exosome extract is as follows: the supernatant of the antler stem cell crushing liquid and the supernatant of the culture medium are mixed, the supernatant is obtained by centrifugation, the precipitate is obtained by a second centrifugation, the precipitate is resuspended with PBS buffer, the precipitate is filtered and sterilized, and the precipitate is freeze-dried and low-temperature crushed.

6. A method for culturing bone marrow mesenchymal stem cells, characterized in that: During the cell passage process, the medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells as claimed in claim 1 or 2 is gradually replaced with the medium for bone marrow mesenchymal stem cells at a ratio of 25%, 50%, 75%, 90% and 100%.

7. The method for culturing bone marrow mesenchymal stem cells according to claim 6, characterized in that: It can induce bone marrow mesenchymal stem cells to differentiate into osteoblasts.

8. The method for culturing bone marrow mesenchymal stem cells according to claim 6 or 7, characterized in that: The bone marrow mesenchymal stem cells are human bone marrow mesenchymal stem cells.

9. A method for inducing osteogenic differentiation of bone marrow mesenchymal stem cells, characterized in that: Bone marrow mesenchymal stem cells are cultured using the culture medium according to claim 1 and the culture method according to claim 6.

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