A method for preparing stem cells having enhanced osteogenic differentiation ability

Abstract

This invention provides a method for preparing stem cells with enhanced osteogenic differentiation capacity, belonging to the field of biological technology. This invention significantly improves the osteogenic differentiation capacity of bone marrow mesenchymal stem cells (BMSCs) by combining deer antler polypeptide with rhodioloside. The osteogenic induction culture medium prepared in this invention includes DMEM / F12 medium, 10 mM β-glycerophosphate sodium, 50 μg / ml ascorbic acid, 100 nM dexamethasone, 20 μg / ml deer antler polypeptide, and 20 μg / ml rhodioloside. This optimized culture medium achieves a significant osteogenic promoting effect at lower concentrations, demonstrating the synergistic effect of deer antler polypeptide and rhodioloside. Simultaneously, the synergistic effect reduces the amount of components used, lowers the cost of using deer antler polypeptide, and reduces the side effects associated with high doses.

Description

Technical Field

[0001] This invention belongs to the field of biological technology, and in particular relates to a method for preparing stem cells with enhanced osteogenic differentiation capacity. Background Technology

[0002] Osteogenic differentiation is a crucial process in bone tissue formation and repair, and is of great significance for the treatment of bone-related diseases such as fractures and osteoporosis. Bone marrow mesenchymal stem cells (BMSCs) are widely used in bone tissue engineering and regenerative medicine research due to their multipotent differentiation potential, especially their ability to differentiate into osteoblasts. BMSCs can not only differentiate into osteoblasts under in vitro conditions, but also respond to bone injury signals in the in vivo microenvironment, participating in bone tissue repair and regeneration. Effectively promoting osteogenic differentiation of BMSCs has significant clinical application value for improving the efficiency of bone repair and regeneration and improving patient prognosis.

[0003] Patent application number 201710569852.6 discloses a deer antler polypeptide with the amino acid sequence VLSATDKTNVLAAWGKVGGNAPAFGAEALERM. The disclosure indicates that this deer antler polypeptide can increase the expression of osteogenic genes and promote osteoblast differentiation by regulating the TGF-β / Smad signaling pathway. However, despite showing some osteogenic promoting effects, its overall effect remains limited. Furthermore, existing literature and research data indicate that using deer antler polypeptide alone often requires high concentrations to achieve significant osteogenic effects, which may lead to dose-dependent side effects and cost issues in practical applications. Therefore, further optimization is needed to improve the application efficacy of deer antler polypeptide. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing stem cells with enhanced osteogenic differentiation capacity, thereby achieving efficient transformation of stem cells into osteoblasts.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] Firstly, this invention provides a method for preparing stem cells with enhanced osteogenic differentiation capacity, the method comprising the following steps:

[0007] (1) Prepare a basic high-efficiency osteogenic induction medium, which includes ordinary osteogenic induction medium, deer antler polypeptide and rhodioloside;

[0008] (2) The basic high-efficiency osteogenic induction culture medium was filtered and sterilized using a filter membrane;

[0009] (3) Supplement with fetal bovine serum and antibiotics to obtain a complete and efficient osteogenic induction culture medium;

[0010] (4) Seed bone marrow mesenchymal stem cells into a culture plate and replace it with a complete and efficient osteogenic induction medium after the cells reach a suitable degree of fusion.

[0011] (5) Place the culture medium in a cell culture incubator and incubate for 7-21 days.

[0012] Preferably, in the basic high-efficiency osteogenic induction culture medium, the concentration of the deer antler polypeptide is 20 μg / ml-80 μg / ml, and the concentration of the rhodioloside is 20 μg / ml-40 μg / ml.

[0013] In the selected location, the concentration of the deer antler polypeptide is 20 μg / ml, and the concentration of the rhodioloside is 20 μg / ml.

[0014] Preferably, the amino acid sequence of the deer antler polypeptide is VLSATDKTNVLAAWGKVGGNAPAFGAEALERM.

[0015] Preferably, the conventional osteogenic induction medium consists of DMEM / F12 medium, 10 mM sodium β-glycerophosphate, 50 μg / ml ascorbic acid, and 100 nM dexamethasone.

[0016] Preferably, the stem cells are bone marrow mesenchymal stem cells.

[0017] Preferably, the amount of fetal bovine serum added is 10% of the volume of the complete and efficient osteogenic induction medium, the amount of antibiotic added is 1% of the volume of the complete and efficient osteogenic induction medium, and the antibiotic is penicillin-streptomycin.

[0018] The appropriate degree of fusion is a fusion of 80% or higher.

[0019] Secondly, the present invention provides a culture medium for improving the osteogenic differentiation ability of stem cells, wherein the culture medium is composed of ordinary induction culture medium, deer antler polypeptide, rhodioloside, fetal bovine serum and antibiotics.

[0020] Preferably, the concentration of the deer antler polypeptide is 20 μg / ml-80 μg / ml, and the concentration of the rhodioloside is 20 μg / ml-40 μg / ml;

[0021] The amino acid sequence of the deer antler polypeptide is VLSATDKTNVLAAWGKVGGNAPAFGAEALERM.

[0022] Preferably, the concentration of the deer antler polypeptide is 20 μg / ml, and the concentration of the rhodioloside is 20 μg / ml.

[0023] Preferably, the conventional osteogenic induction medium consists of DMEM / F12 medium, 10 mM sodium β-glycerophosphate, 50 μg / ml ascorbic acid, and 100 nM dexamethasone;

[0024] The concentration of the fetal bovine serum is 10%, the concentration of the antibiotic is 1%, and the antibiotic is penicillin-streptomycin.

[0025] Thirdly, the present invention provides the use of a combination of deer antler polypeptide and rhodioloside in the preparation of a culture medium for promoting the mineralization of bone marrow mesenchymal stem cells, characterized in that the composition contains 20-80 μg / ml deer antler polypeptide and 20-40 μg / ml rhodioloside.

[0026] The amino acid sequence of the deer antler polypeptide is VLSATDKTNVLAAWGKVGGNAPAFGAEALERM.

[0027] Preferably, the culture medium further includes: DMEM / F12 medium, 10 mM β-glycerophosphate sodium, 50 μg / ml ascorbic acid, 100 nM dexamethasone, 10% fetal bovine serum and 1% penicillin-streptomycin solution.

[0028] Preferably, the concentration of the deer antler polypeptide is 20 μg / ml, and the concentration of the rhodioloside is 20 μg / ml.

[0029] Fourth, the present invention provides the use of a combination of deer antler polypeptide and rhodioloside in the preparation of a culture medium for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, characterized in that the composition contains 20-80 μg / ml deer antler polypeptide and 20-40 μg / ml rhodioloside.

[0030] The amino acid sequence of the deer antler polypeptide is VLSATDKTNVLAAWGKVGGNAPAFGAEALERM.

[0031] Preferably, the culture medium further includes: DMEM / F12 medium, 10 mM β-glycerophosphate sodium, 50 μg / ml ascorbic acid, 100 nM dexamethasone, 10% fetal bovine serum and 1% penicillin-streptomycin solution.

[0032] Preferably, the composition promotes osteogenic differentiation by increasing the alkaline phosphatase activity and cellular mineralization level of bone marrow mesenchymal stem cells.

[0033] Fifth, the present invention provides the use of a combination of deer antler polypeptide and rhodioloside in the preparation of a drug that accelerates bone tissue repair, characterized in that the composition contains 20-80 μg / ml deer antler polypeptide and 20-40 μg / ml rhodioloside.

[0034] The amino acid sequence of the deer antler polypeptide is VLSATDKTNVLAAWGKVGGNAPAFGAEALERM.

[0035] The drug accelerates bone tissue repair by speeding up the differentiation of osteocytes.

[0036] The beneficial effects of this invention are as follows:

[0037] This invention provides an innovative method for preparing stem cells with osteogenic differentiation capacity. The key to this method is the combined application of natural deer antler polypeptide and rhodioloside, which significantly enhances the ability of bone marrow mesenchymal stem cells to differentiate into osteoblasts.

[0038] Traditional osteogenic induction methods typically involve adding only a single inducing agent, resulting in limited effectiveness. However, this invention utilizes the combined application of deer antler polypeptide and rhodioloside, which produce a significant synergistic effect, greatly enhancing the osteogenic differentiation capacity of bone marrow mesenchymal stem cells. Experimental results show that the combined osteogenic promoting effect far exceeds the effect of using either component alone.

[0039] More importantly, because deer antler polypeptide and rhodioloside produce a synergistic effect when used in combination, the concentration of both can be significantly reduced, thereby lowering the cost of using deer antler polypeptide. At the same time, the lower concentration also helps reduce potential dose-dependent side effects, enhancing the practical application value of this method.

[0040] In summary, this invention provides a novel method for preparing highly efficient osteogenic differentiated stem cells for bone tissue engineering and regenerative medicine, which is expected to accelerate the repair process of related orthopedic diseases and has broad application prospects. At the same time, this method also opens up new avenues for the combined application of natural bioactive molecules. Attached Figure Description

[0041] Figure 1 The effects of different treatments on alkaline phosphatase (ALP) activity in bone marrow mesenchymal stem cells.

[0042] Figure 2 The effects of different treatments on cell mineralization levels in bone marrow mesenchymal stem cells;

[0043] Where A represents the staining detection results and B represents the statistical results. Detailed Implementation

[0044] Different embodiments of the present invention will be described in detail below. These detailed descriptions are not intended to limit the present invention, but should be considered as further explanations of certain aspects, features, and implementation schemes of the present invention. The terminology used in the description of the present invention is intended to depict specific implementations and is not intended to limit the present invention.

[0045] The numerical ranges mentioned in this invention should be understood to include every intermediate value within that range. This invention covers any specified value or intermediate value within a numerical range, as well as any other specified value or intermediate value within a numerical range. The upper and lower limits of these intermediate values ​​may be independently included or excluded from the scope of this invention.

[0046] Example 1

[0047] (1) Prepare conventional osteogenic induction culture medium

[0048] 1) Prepare the basic osteogenic induction medium according to the following formula: DMEM / F12 medium (1000ml), 10mM β-glycerophosphate sodium, 50μg / ml ascorbic acid, 100nM dexamethasone;

[0049] 2) The basic osteogenic induction culture medium was filtered and sterilized using a 0.22-micron filter membrane;

[0050] 3) Add 10% fetal bovine serum and 1% penicillin-streptomycin solution to obtain the basic osteogenic induction medium.

[0051] (2) Inoculation with bone marrow mesenchymal stem cells

[0052] Bone marrow mesenchymal stem cells were seeded into 6-well culture plates, 1 × 10⁶ cells per well. 5 Cells are cultured until the cell count reaches more than 80% for osteogenic induction.

[0053] (3) Osteogenic induction culture

[0054] Remove the original culture medium and replace it with conventional osteogenic induction medium. Incubate the cells at 37°C in a 5% CO2 cell culture incubator and change the culture medium every 2-3 days.

[0055] Example 2

[0056] (1) Prepare a high-efficiency osteogenic induction culture medium

[0057] 1) Prepare the high-efficiency osteogenic induction medium according to the following formula: DMEM / F12 medium (1000ml), 10mM β-glycerophosphate sodium, 50μg / ml ascorbic acid, 100nM dexamethasone, 20μg / ml deer antler polypeptide (amino acid sequence is VLSATDKTNVLAAWGKVGGNAPAFGAEALERM);

[0058] 2) Use a 0.22-micron filter membrane to filter and sterilize the high-efficiency osteogenic induction culture medium;

[0059] 3) Add 10% fetal bovine serum and 1% penicillin-streptomycin solution to obtain a highly efficient osteogenic induction culture medium.

[0060] (2) Inoculation with bone marrow mesenchymal stem cells

[0061] Bone marrow mesenchymal stem cells were seeded into 6-well culture plates, 1 × 10⁶ cells per well. 5 Cells are cultured until the cell count reaches more than 80% for osteogenic induction.

[0062] (3) Osteogenic induction culture

[0063] Remove the original culture medium and replace it with a high-efficiency osteogenic induction medium. Incubate the cells at 37°C in a 5% CO2 cell culture incubator and change the culture medium every 2-3 days.

[0064] Example 3

[0065] (1) Prepare a high-efficiency osteogenic induction culture medium

[0066] 1) Prepare the high-efficiency osteogenic induction medium according to the following formula: DMEM / F12 medium (1000ml), 10mM β-glycerophosphate sodium, 50μg / ml ascorbic acid, 100nM dexamethasone, 20μg / ml rhodioloside (CAS No.: 10338-51-9);

[0067] 2) The basic osteogenic induction culture medium was filtered and sterilized using a 0.22-micron filter membrane;

[0068] 3) Add 10% fetal bovine serum and 1% penicillin-streptomycin solution to obtain a highly efficient osteogenic induction culture medium.

[0069] (2) Inoculation with bone marrow mesenchymal stem cells

[0070] Bone marrow mesenchymal stem cells were seeded into 6-well culture plates, 1 × 10⁶ cells per well. 5 Cells are cultured until the cell count reaches more than 80% for osteogenic induction.

[0071] (3) Osteogenic induction culture

[0072] Remove the original culture medium and replace it with a high-efficiency osteogenic induction medium. Incubate the cells at 37°C in a 5% CO2 cell culture incubator and change the culture medium every 2-3 days.

[0073] Example 4

[0074] (1) Prepare a high-efficiency osteogenic induction culture medium

[0075] 1) Prepare the high-efficiency osteogenic induction medium according to the following formula: DMEM / F12 medium (1000ml), 10mM β-glycerophosphate sodium, 50μg / ml ascorbic acid, 100nM dexamethasone, 10μg / ml icariin (CAS No.: 489-32-7);

[0076] 2) Use a 0.22-micron filter membrane to filter and sterilize the high-efficiency osteogenic induction culture medium;

[0077] 3) Add 10% fetal bovine serum and 1% penicillin-streptomycin solution to obtain a highly efficient osteogenic induction culture medium.

[0078] (2) Inoculation with bone marrow mesenchymal stem cells

[0079] Bone marrow mesenchymal stem cells were seeded into 6-well culture plates, 1 × 10⁶ cells per well. 5 Cells are cultured until the cell count reaches more than 80% for osteogenic induction.

[0080] (3) Osteogenic induction culture

[0081] Remove the original culture medium and replace it with a high-efficiency osteogenic induction medium. Incubate the cells at 37°C in a 5% CO2 cell culture incubator and change the culture medium every 2-3 days.

[0082] Example 5

[0083] (1) Prepare a high-efficiency osteogenic induction culture medium

[0084] 1) Prepare the high-efficiency osteogenic induction medium according to the following formula: DMEM / F12 medium (1000ml), 10mM β-glycerophosphate sodium, 50μg / ml ascorbic acid, 100nM dexamethasone, 20μg / ml deer antler polypeptide, 20μg / ml rhodioloside.

[0085] 2) Use a 0.22-micron filter membrane to filter and sterilize the high-efficiency osteogenic induction culture medium;

[0086] 3) Add 10% fetal bovine serum and 1% penicillin-streptomycin solution to obtain a highly efficient osteogenic induction culture medium.

[0087] (2) Inoculation with bone marrow mesenchymal stem cells

[0088] Bone marrow mesenchymal stem cells were seeded into 6-well culture plates, 1 × 10⁶ cells per well. 5 Cells are cultured until the cell count reaches more than 80% for osteogenic induction.

[0089] (3) Osteogenic induction culture

[0090] Remove the original culture medium and replace it with a high-efficiency osteogenic induction medium. Incubate the cells at 37°C in a 5% CO2 cell culture incubator and change the culture medium every 2-3 days.

[0091] Example 6

[0092] (1) Prepare a high-efficiency osteogenic induction culture medium

[0093] 1) Prepare the high-efficiency osteogenic induction medium according to the following formula: DMEM / F12 medium (1000ml), 10mM β-glycerophosphate sodium, 50μg / ml ascorbic acid, 100nM dexamethasone, 20μg / ml deer antler polypeptide, 10μg / ml icariin;

[0094] 2) Use a 0.22-micron filter membrane to filter and sterilize the high-efficiency osteogenic induction culture medium;

[0095] 3) Add 10% fetal bovine serum and 1% penicillin-streptomycin solution to obtain a highly efficient osteogenic induction culture medium.

[0096] (2) Inoculation with bone marrow mesenchymal stem cells

[0097] Bone marrow mesenchymal stem cells were seeded into 6-well culture plates, 1 × 10⁶ cells per well. 5 Cells are cultured until the cell count reaches more than 80% for osteogenic induction.

[0098] (3) Osteogenic induction culture

[0099] Remove the original culture medium and replace it with a high-efficiency osteogenic induction medium. Incubate the cells at 37°C in a 5% CO2 cell culture incubator and change the culture medium every 2-3 days.

[0100] Example 7

[0101] (1) Take cells induced and cultured for 7 days according to the methods in Examples 1-6, remove the culture medium, and wash the cells with PBS;

[0102] (2) After fixing the cells with 4% paraformaldehyde, the cells were treated according to the instructions of the alkaline phosphatase detection kit (Beyotime P0321S);

[0103] (4) Add 100 μl of reaction stop solution to each well to stop the reaction, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 405 nm. Calculate the relative ALP enzyme activity of each group based on the control group.

[0104] from Figure 1The results show that, compared with bone marrow mesenchymal stem cells cultured using ordinary osteogenic induction medium, the addition of deer antler polypeptide, rhodioloside, and icariin alone can effectively improve the osteogenic differentiation capacity of bone marrow mesenchymal stem cells, which is consistent with existing technologies. Specifically, deer antler polypeptide increased osteogenic differentiation capacity by 27.4%, rhodioloside by 46.9%, and icariin by 58.2%.

[0105] Further analysis shows that the combined addition of deer antler polypeptide and rhodioloside increased the effect by 103.8% compared to the control group, while the combined addition of deer antler polypeptide and icariin only increased it by 79.3% compared to the control group. This indicates that the combined use of deer antler polypeptide and rhodioloside has a more significant effect on promoting bone growth.

[0106] To further test the effect of the combined use, this invention calculates the q-values ​​of the combined use of deer antler polypeptide and rhodioloside, as well as the q-values ​​of the combined use of deer antler polypeptide and icariin.

[0107] The results showed that the q value of the combination of deer antler polypeptide and rhodioloside was 1.038 / 0.274+0.469-(0.274×0.469)=1.69. It can be seen that the obtained q value is greater than 1.15, indicating that the combination of deer antler polypeptide and rhodioloside has a synergistic effect.

[0108] However, the q value of deer antler polypeptide and icariin is 0.793 / 0.274+0.582-(0.274×0.582)=1.14. It can be seen that the obtained q value is less than 1.15, indicating that when deer antler polypeptide and icariin are used together, the two have an additive effect.

[0109] The reason for these results may be that rhodioloside can promote osteoblast differentiation through multiple signaling pathways and has antioxidant and anti-inflammatory effects. When used in combination with deer antler polypeptide, it produces a complementary effect, thereby synergistically enhancing the osteoblast differentiation effect. Icariin, on the other hand, mainly promotes osteoblast differentiation through one or a few signaling pathways, and its pathway overlaps to some extent with that of deer antler polypeptide. Therefore, the combined effect of icariin and deer antler polypeptide is more of an additive effect than a synergistic effect.

[0110] Example 8

[0111] ALP enzyme activity is a marker of early osteoblast differentiation, while cellular mineralization is the final stage of osteoblast differentiation and an important indicator of osteoblast maturation and bone matrix formation. Although ALP enzyme activity can serve as an indicator of early osteoblast differentiation, high ALP activity does not always directly translate into high mineralization capacity. Therefore, to comprehensively evaluate the entire process of osteoblast differentiation and ensure that deer antler peptides and rhodioloside can effectively improve osteoblast differentiation at different stages, this invention investigates whether the combined use of deer antler peptides and rhodioloside can regulate cellular mineralization.

[0112] (1) Take cells cultured for 14 days according to Example 1, Example 2, Example 3 and Example 5, remove the culture medium, and wash the cells with PBS;

[0113] (2) After fixing the cells with 4% paraformaldehyde, add 1% alizarin red S staining solution and incubate at 37°C for 30 min.

[0114] (3) After rinsing with PBS, the cells were photographed under a microscope;

[0115] (4) Add 1 ml of 10% hexadecyl chloride to each well, incubate at room temperature for 1 hour, then transfer the reagent to a 96-well plate and measure the OD value at 562 nm using an ELISA reader. Calculate the relative cell mineralization level using the control group as a benchmark.

[0116] The results obtained are as follows Figure 2 As shown, from Figure 2 The results show that, compared with the control group, the use of deer antler polypeptide alone can improve the cell mineralization level to a certain extent, but the effect is very limited; the treatment with rhodioloside can significantly improve the cell mineralization level, but there is a significant difference in the cell mineralization level compared with the combined group.

[0117] Among them, the deer antler polypeptide group showed a 15.1% increase compared to the control group, the rhodioloside group showed a 39.0% increase compared to the control group, and the combined group showed an 87.7% increase compared to the control group. The calculated q value was q = 0.877 / 0.151 + 0.390 - (0.151 × 0.390) = 1.82. The q value results show that the combined use of deer antler polypeptide and rhodioloside effectively improves cell mineralization levels. Therefore, based on the results of Examples 7 and 8, we can conclude that the combined use of deer antler polypeptide and rhodioloside can effectively improve the osteogenic transformation ability of bone marrow mesenchymal stem cells, thereby preparing stem cells with osteogenic differentiation capacity.

[0118] Example 9

[0119] The above examples have demonstrated that the combined use of deer antler polypeptide and rhodioloside can effectively improve the osteogenic differentiation capacity of bone marrow mesenchymal stem cells. In order to verify whether the combined use of deer antler polypeptide and rhodioloside in other proportions or concentrations also has the same effect, this invention uses ALP enzyme activity for detection.

[0120] (1) Cell treatment:

[0121] Category A treatment: Single treatment with 20 μg / ml deer antler polypeptide and 40 μg / ml rhodioloside; combined treatment with 20 μg / ml deer antler polypeptide and 40 μg / ml rhodioloside.

[0122] Category B treatment: Single treatment with 40 μg / ml deer antler polypeptide and 20 μg / ml rhodioloside; combined treatment with 40 μg / ml deer antler polypeptide and 20 μg / ml rhodioloside.

[0123] Category C treatment: Single treatment with 40 μg / ml deer antler polypeptide and 40 μg / ml rhodioloside; combined treatment with 40 μg / ml deer antler polypeptide and 40 μg / ml rhodioloside.

[0124] Category D treatment: Single treatment with 80 μg / ml deer antler polypeptide and 20 μg / ml rhodioloside, or combined treatment with 80 μg / ml deer antler polypeptide and 20 μg / ml rhodioloside.

[0125] The basic osteogenic induction medium for all treatments was the same as in Example 1, and the culture method was also the same as in Example 1. The control group was cultured using the osteogenic induction medium of Example 1.

[0126] (2) Take cells from various treatments, remove the culture medium, and wash the cells with PBS;

[0127] (3) After fixing the cells with 4% paraformaldehyde, the cells were treated according to the instructions of the alkaline phosphatase detection kit.

[0128] (4) Add 100 μl of reaction stop solution to each well to stop the reaction, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 405 nm. Calculate the relative ALP enzyme activity of each group based on the control group.

[0129] For treatment A: the effect of 20 μg / ml deer antler polypeptide was 27.4% as shown in Example 7, the effect of 40 μg / ml rhodioloside was 54.6%, and the effect of the combined group was 112.4%. The calculated q value was 1.68.

[0130] For treatment type B: the efficacy of 40 μg / ml deer antler polypeptide was 36.5%, the efficacy of 20 μg / ml rhodioloside was 46.9%, and the efficacy of the combined group was 136.7%. The calculated q value was 2.06.

[0131] For treatment type C: the efficacy of 40 μg / ml deer antler polypeptide was 36.5%, the efficacy of 40 μg / ml rhodioloside was 54.6%, and the efficacy of the combined group was 138.3%. The calculated q value was 1.94.

[0132] For treatment type D, the efficacy of 80 μg / ml deer antler polypeptide was 37.1%, the efficacy of 20 μg / ml rhodioloside was 46.9%, and the efficacy of the combined group was 135.5%, with a calculated q value of 2.03.

[0133] The results above show that when deer antler polypeptide and rhodioloside are combined at different concentrations and ratios provided by the present invention, they can all produce a synergistic effect in promoting osteogenic differentiation.

[0134] Meanwhile, it can be seen that there is a certain concentration dependence between different concentrations of deer antler polypeptide and rhodioloside. Increasing the polypeptide concentration significantly increases the q-value, while increasing the rhodioloside concentration does not significantly increase the q-value. This may be because, in combined application, 20 μg / ml of rhodioloside already exhibits the optimal effect, and further increasing the concentration does not improve the combined effect. However, at 20 μg / ml, the deer antler polypeptide has not yet reached its optimal concentration; therefore, increasing the concentration leads to a more significant combined effect. However, when the concentration of deer antler polypeptide is increased to 80 μg / ml, there is no significant change in the effect, whether used alone or in combination.

Claims

1. A method for preparing bone marrow mesenchymal stem cells with enhanced osteogenic differentiation capacity, characterized in that, The method includes the following steps: (1) Prepare a basic high-efficiency osteogenic induction medium, which includes ordinary osteogenic induction medium, deer antler polypeptide and rhodioloside; (2) The basic high-efficiency osteogenic induction culture medium was filtered and sterilized using a filter membrane; (3) Supplement with fetal bovine serum and antibiotics to obtain a complete and efficient osteogenic induction culture medium; (4) Seed bone marrow mesenchymal stem cells into a culture plate and replace it with a complete and efficient osteogenic induction medium after the cells reach a suitable degree of fusion. (5) Place the culture medium in a cell culture incubator and incubate for 7-21 days; In the aforementioned basic high-efficiency osteogenic induction culture medium, the concentration of the deer antler polypeptide is 20 μg / ml-80 μg / ml, and the concentration of the rhodioloside is 20 μg / ml-40 μg / ml. The amino acid sequence of the deer antler polypeptide is VLSATDKTNVLAAWGKVGGNAPAFGAEALERM; The CAS number for the rhodioloside is 10338-51-9.

2. The method for preparing bone marrow mesenchymal stem cells with enhanced osteogenic differentiation capacity according to claim 1, characterized in that, The conventional osteogenic induction medium consists of DMEM / F12 medium, 10 mM sodium β-glycerophosphate, 50 μg / ml ascorbic acid, and 100 nM dexamethasone.

3. A culture medium for enhancing the osteogenic differentiation capacity of bone marrow mesenchymal stem cells, characterized in that, The culture medium consists of ordinary osteogenic induction medium, deer antler polypeptide, rhodioloside, fetal bovine serum and antibiotics; The concentration of the deer antler polypeptide is 20 μg / ml-80 μg / ml, and the concentration of the rhodioloside is 20 μg / ml-40 μg / ml; The amino acid sequence of the deer antler polypeptide is VLSATDKTNVLAAWGKVGGNAPAFGAEALERM; The CAS number for the rhodioloside is 10338-51-9.

4. The culture medium for enhancing the osteogenic differentiation capacity of bone marrow mesenchymal stem cells according to claim 3, characterized in that, The conventional osteogenic induction medium consisted of DMEM / F12 medium, 10 mM sodium β-glycerophosphate, 50 μg / ml ascorbic acid, and 100 nM dexamethasone. The concentration of the fetal bovine serum is 10%, the concentration of the antibiotic is 1%, and the antibiotic is penicillin-streptomycin.

5. The use of a composition of deer antler polypeptide and rhodioloside in the preparation of a culture medium that promotes the mineralization of bone marrow mesenchymal stem cells, characterized in that, The composition contains 40 μg / ml deer antler polypeptide and 40 μg / ml rhodioloside; The amino acid sequence of the deer antler polypeptide is VLSATDKTNVLAAWGKVGGNAPAFGAEALERM; The CAS number of the rhodioloside is 10338-51-9; The culture medium also includes: DMEM / F12 medium, 10 mM β-glycerophosphate sodium, 50 μg / ml ascorbic acid, 100 nM dexamethasone, 10% fetal bovine serum and 1% penicillin-streptomycin solution.

Images