Application of protopanoxadiol in aspect of improving osteogenic differentiation capacity of bone marrow mesenchymal stem cells

Abstract

The invention discloses a culture method for improving osteogenic differentiation capacity of bone marrow mesenchymal stem cells. The method comprises the following steps: S1, adding a certain amount of protopanoxadiol into a culture medium; s2, the culture medium containing protopanoxadiol is used for flushing and suspending the bone marrow mesenchymal stem cells, and cell suspension is obtained; and S3, adjusting the cell density in the cell suspension, inoculating the cell suspension into a cell culture device, and placing the cell culture device in an incubator for multiplication culture. According to the culture method, the culture medium containing the protopanoxadiol with the concentration of the protopanoxadiol being 5-50 [mu] M is used for culturing the mesenchymal stem cells, so that the cell osteogenic differentiation related gene expression of the mesenchymal stem cells is effectively increased, and the osteogenic differentiation capability of the mesenchymal stem cells is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of bone marrow mesenchymal stem cell culture, and particularly relates to a culture method for improving the osteogenic differentiation ability of bone marrow mesenchymal stem cells. Background Art

[0002] Osteoporosis is a metabolic bone disease caused by abnormal bone remodeling. Osteoporosis can be divided into two major categories, primary and secondary, according to its causes. The former includes postmenopausal osteoporosis (5 - 10 years after menopause in women), senile osteoporosis (over 70 years old), and idiopathic osteoporosis (adolescents, with unknown causes); the latter refers to osteoporosis caused by any disease / drug affecting bone metabolism and other unknown causes [see the prior art Nature 2003, 423, 349 - 355]. Postmenopausal osteoporosis, which has been studied more in the existing research, is mainly driven by a sudden drop in estrogen, resulting in uncontrolled osteoclast activity and hyperactive bone resorption, and an imbalance in osteoblast - osteoclast coupling; while the occurrence mechanism of senile osteoporosis is centered around multi - factor systemic aging, involving a decline in osteogenic differentiation ability caused by the aging of bone marrow mesenchymal stem cells (BMSCs), abnormally active adipogenic differentiation (bone marrow fat accumulation), accompanied by cumulative oxidative stress, mitochondrial dysfunction, and a chronic inflammatory state [see the prior art Ageing Res.Rev.2024, 99, 102235]. Therefore, the research on how to improve the osteogenic differentiation ability of BMSCs is also a hot topic in the study of senile osteoporosis, and natural product molecules have safety advantages in promoting the osteogenic differentiation of BMSCs.

[0003] Ginsenosides are the main pharmacological active components in ginseng, belonging to triterpenoid glycoside compounds. According to their different structural characteristics, they can be divided into protopanaxadiol - type saponins, protopanaxatriol - type saponins, and oleanane - type. Different types of ginsenosides may regulate osteogenic or osteoclastic differentiation through multiple signaling pathways, thereby improving osteoporosis symptoms. It has been found that an extract of rice seeds rich in protopanaxadiol (PPD) can inhibit RANKL - induced osteoclast differentiation [see the prior art Life (Basel) 2022, 12, 1886]. Our research found that protopanaxadiol has significant activity in promoting the osteogenic differentiation of BMSCs, and this osteogenic promotion effect has not been reported in the research.

[0004] The chemical structural formula of protopanaxadiol is as follows:

[0005]

[0006] Molecular formula: C 30 H 52 O3

[0007] Molecular weight: 460.73 Summary of the Invention

[0008] In view of this, the object of the present invention is to provide a culture method for enhancing the osteogenic differentiation ability of bone marrow mesenchymal stem cells, so as to provide potential therapeutic drugs for solving the insufficient osteogenic differentiation ability of BMSCs in senile osteoporosis patients.

[0009] To solve the above problems, the technical solution adopted by the present invention is: a culture method for enhancing the osteogenic differentiation ability of bone marrow mesenchymal stem cells by adding protopanaxadiol, adding protopanaxadiol to the culture medium; specifically including the following steps:

[0010] S1. Add fetal bovine serum and protopanaxadiol to a medium without animal component source (for example ) to prepare a medium containing protopanaxadiol;

[0011] S2. Suspend the bone marrow mesenchymal stem cells of passages P2 to P6 with the medium containing protopanaxadiol prepared in S1 to obtain a cell suspension;

[0012] S3. Adjust the cell density in the cell suspension obtained in S2, and inoculate the cell suspension into a cell culture device and place it in an incubator for amplification culture.

[0013] Preferably, in S1, the concentration of protopanaxadiol in the medium containing protopanaxadiol is 5 μM to 50 μM, for example, 5 μM, 20 μM or 50 μM (where μM refers to μMol / L).

[0014] Preferably, in S1, the volume ratio of fetal bovine serum in the medium containing protopanaxadiol is 10%.

[0015] Preferably, in S3, the cell density in the cell suspension is 1×10 6 ~4×10 6 cells / mL.

[0016] Preferably, in S3, the cell culture device is a cell culture plate or a cell culture flask, and the cell density inoculated in the cell culture plate or cell culture flask is 0.5 to 1×10 4 cells / cm 2 .

[0017] Preferably, in S3, the incubator is a CO2 incubator, and the temperature during the culture is 35°C to 38°C.

[0018] Preferably, the volume percentage concentration of CO2 in the CO2 incubator is 4.5 to 5.5%.

[0019] Compared with the prior art, the culture method of the present invention for culturing bone marrow mesenchymal stem cells of passages P2 to P6 by using a MesenGro medium containing protopanaxadiol with a concentration of 1 μM to 50 μM of protopanaxadiol can effectively increase the expression of genes related to osteogenic differentiation of bone marrow mesenchymal stem cells, thereby improving the osteogenic differentiation ability of bone marrow mesenchymal stem cells. Description of the Drawings

[0020] Figure 1 After culturing BMSCs with different concentrations of protopanaxadiol (0 μM, 1 μM, 5 μM, 20 μM, 50 μM, 100 μM) for 3 days, the mRNA expression level of the osteogenic differentiation gene RUNX2 was detected by qPCR;

[0021] Figure 2 After culturing BMSCs with different concentrations of protopanaxadiol (0 μM, 1 μM, 5 μM, 20 μM, 50 μM, 100 μM) for 3 days, the mRNA expression level of the osteogenic differentiation gene ALPL was detected by qPCR;

[0022] Figure 3 After culturing BMSCs with different concentrations of protopanaxadiol (0 μM, 1 μM, 5 μM, 20 μM, 50 μM, 100 μM) for 3 days, the mRNA expression level of the osteogenic differentiation gene COL1A1 was detected by qPCR;

[0023] Figure 4 After culturing BMSCs with different concentrations of protopanaxadiol (0 μM, 1 μM, 5 μM, 20 μM, 50 μM, 100 μM) for 3 days, the mRNA expression level of the osteogenic differentiation gene OPN was detected by qPCR. Detailed Embodiments

[0024] In the following examples, the MesenGro medium was purchased from StemRD, with the product number MGro-500; the fetal bovine serum was purchased from Gibco, with the product number 10099141C; the protopanaxadiol was purchased from MCE, with the product number HY-N0797.

[0025] A culture method for improving the antioxidant capacity of bone marrow mesenchymal stem cells provided in this example includes the following steps:

[0026] S1. Add a certain amount of protopanaxadiol to the MesenGro medium containing 10% (volume ratio) fetal bovine serum to prepare a medium containing protopanaxadiol with a concentration of 1 μM to 50 μM of protopanaxadiol; the concentration of protopanaxadiol in the medium containing protopanaxadiol is preferably 5 μM, 20 μM or 50 μM;

[0027] S2. Suspend the bone marrow mesenchymal stem cells of passages P2 to P6 with the culture medium containing protopanaxadiol prepared in S1 to obtain a cell suspension;

[0028] S3. Adjust the cell density in the cell suspension obtained in S2 to 1×10 6 ~4×10 6 cells / mL, and inoculate the cell suspension into a culture device with a cell density of 0.5~1×10 4 cells / cm 2 , and then place it in a CO2 incubator at 35℃~38℃ with a volume percentage concentration of CO2 of 4.5~5.5% for amplification culture.

[0029] The following are specific examples:

[0030] Example 1

[0031] A culture method for improving the antioxidant capacity of bone marrow mesenchymal stem cells by adding protopanaxadiol provided in this example includes the following steps:

[0032] S1. Add 10% (volume ratio) fetal bovine serum and a certain amount of protopanaxadiol to the MesenGro culture medium to prepare a culture medium containing protopanaxadiol with a concentration of 5 μM of protopanaxadiol;

[0033] S2. Suspend the bone marrow mesenchymal stem cells of passages P2 to P6 with the culture medium containing protopanaxadiol prepared in S1 to obtain a cell suspension;

[0034] S3. Adjust the cell density in the cell suspension obtained in S2 to 1×10 6 ~4×10 6 cells / mL, and inoculate the cell suspension into a culture device with a cell density of 0.5~1×10 4 cells / cm 2 , and then place it in a CO2 incubator at 37℃ with a volume percentage concentration of CO2 of 5% for amplification culture for 3 days.

[0035] Example 2

[0036] A culture method for improving the antioxidant capacity of bone marrow mesenchymal stem cells by adding protopanaxadiol provided in this example includes the following steps:

[0037] S1. Add 10% (volume ratio) fetal bovine serum and a certain amount of protopanaxadiol to the MesenGro culture medium to prepare a culture medium containing protopanaxadiol with a concentration of 20 μM of protopanaxadiol;

[0038] S2. Suspend the bone marrow mesenchymal stem cells of passages P2 to P6 with the culture medium containing protopanaxadiol prepared in S1 to obtain a cell suspension;

[0039] S3. Adjust the cell density in the cell suspension obtained in S2 to 1×10 6 ~4×10 6 cells / mL, and inoculate the cell suspension into a culture device with a cell density of 0.5~1.0×10 4 cells / cm 2 , and then place it in a CO₂ incubator at 37°C with a volume percentage concentration of CO₂ of 5% for amplification culture for 3 days.

[0040] Example 3

[0041] A culture method for improving the antioxidant capacity of bone marrow mesenchymal stem cells by adding protopanaxadiol provided in this example includes the following steps:

[0042] S1. Add 10% (volume ratio) fetal bovine serum and a certain amount of protopanaxadiol to the MesenGro culture medium to prepare a culture medium containing protopanaxadiol with a concentration of 50 μM;

[0043] S2. Suspend the bone marrow mesenchymal stem cells of passages P2 to P6 with the culture medium containing protopanaxadiol prepared in S1 to obtain a cell suspension;

[0044] S3. Adjust the cell density in the cell suspension obtained in S2 to 1×10 6 ~4×10 6 cells / mL, and inoculate the cell suspension into a culture device with a cell density of 0.5~1×10 4 cells / cm 2 , and then place it in a CO₂ incubator at 37°C with a volume percentage concentration of CO₂ of 5% for amplification culture for 3 days.

[0045] Comparative Example 1 (Control Example)

[0046] S1. Suspend the bone marrow mesenchymal stem cells of passages P2 to P6 with the MesenGro culture medium added with 10% (volume ratio) fetal bovine serum to obtain a cell suspension;

[0047] S2. Adjust the cell density in the cell suspension obtained in S2 to 1×10 6 ~4×10 6 cells / mL, and inoculate the cell suspension into a culture device with a cell density of 0.5~1×10 4 cells / cm 2In a culture device, and then placed in a CO₂ incubator at 37°C with a volume percentage concentration of CO₂ of 5% for 3 days of amplification culture.

[0048] Comparative Example 2

[0049] A culture method for improving the antioxidant capacity of bone marrow mesenchymal stem cells by adding protopanaxadiol provided in this comparative example, the method includes the following steps:

[0050] S1. Add 10% (volume ratio) fetal bovine serum and a certain amount of protopanaxadiol to MesenGro medium to prepare a medium containing protopanaxadiol with a concentration of 1 μM of protopanaxadiol;

[0051] S2. Suspend the bone marrow mesenchymal stem cells of passages P2 to P6 with the medium containing protopanaxadiol prepared in S1 to obtain a cell suspension;

[0052] S3. Adjust the cell density in the cell suspension obtained in S2 to 1×10 6 ~4×10 6 cells / mL, and inoculate the cell suspension into a culture device with a cell density of 0.5~1×10 4 cells / cm 2 In a culture device, and then placed in a CO₂ incubator at 37°C with a volume percentage concentration of CO₂ of 5% for 3 days of amplification culture.

[0053] Comparative Example 3

[0054] A culture method for improving the antioxidant capacity of bone marrow mesenchymal stem cells by adding protopanaxadiol provided in this comparative example, the method includes the following steps:

[0055] S1. Add 10% (volume ratio) fetal bovine serum and a certain amount of protopanaxadiol to MesenGro medium to prepare a medium containing protopanaxadiol with a concentration of 100 μM of protopanaxadiol;

[0056] S2. Suspend the bone marrow mesenchymal stem cells of passages P2 to P6 with the medium containing protopanaxadiol prepared in S1 to obtain a cell suspension;

[0057] S3. Adjust the cell density in the cell suspension obtained in S2 to 1×10 6 ~4×10 6 cells / mL, and inoculate the cell suspension into a culture device with a cell density of 0.5~1×10 4 cells / cm 2 In a culture device, and then placed in a CO₂ incubator at 37°C with a volume percentage concentration of CO₂ of 5% for 3 days of amplification culture.

[0058] It can be seen from Examples 1-3 and Comparative Examples 1-3 that the difference between them is the concentration of protopanaxadiol in the selected culture medium. Specifically, in Example 1, a culture medium containing protopanaxadiol with a concentration of 5 μM of protopanaxadiol was used; in Example 2, a culture medium containing protopanaxadiol with a concentration of 20 μM of protopanaxadiol was used; in Example 3, a culture medium containing protopanaxadiol with a concentration of 50 μM of protopanaxadiol was used; the culture medium in Comparative Example 1 was a culture medium without protopanaxadiol; in Comparative Example 2, a culture medium containing protopanaxadiol with a concentration of 1 μM of protopanaxadiol was used; in Comparative Example 3, a culture medium containing protopanaxadiol with a concentration of 100 μM of protopanaxadiol was used.

[0059] To verify the osteogenic differentiation ability of bone marrow mesenchymal stem cells cultured after only adding protopanaxadiol to the MesenGro culture medium containing 10% (volume ratio) fetal bovine serum in the present invention, qPCR detection was performed on the mRNA expressions of osteogenic differentiation marker genes RUNX2, ALPL, COL1A1, and OPN of bone marrow mesenchymal stem cells cultured in Examples 1-3 and Comparative Examples 1-3. The detection results are as Figure 1 shown:

[0060] In addition, in the above detection method, the intracellular mRNA sample was to lyse cells with Trizol reagent and extract total RNA. Equal amounts of RNA were taken from each group for reverse transcription and then qPCR was performed using the Real-time PCR detection reagent (CAT: RR420A) of Takara Company. The mRNA expression level of the ACTIN gene was used as an internal reference, and after normalization, the expression of each gene was compared.

[0061] Trizol reagent was purchased from Invitrogen Company, the reverse transcription reagent was purchased from Thermo Scientific Company, and the primers for each gene were purchased from Shanghai Sangon Company.

[0062] From Figures 1-4 the results in, it can be seen that adding 5 μM, 20 μM, and 50 μM of protopanaxadiol to the culture medium is beneficial to the increase in the mRNA expressions of osteogenic differentiation genes RUNX2, ALPL, COL1A1, and OPN of BMSCs; and 20 μM of protopanaxadiol is the concentration with the strongest promotion of the expression of osteogenic differentiation genes of BMSCs among the three concentrations; compared with Comparative Example 1 (control example), the culture medium added with 1 μM or 100 μM of protopanaxadiol has less obvious effect on improving the osteogenic differentiation ability of BMSCs than the culture medium added with 5 μM - 50 μM of protopanaxadiol.

[0063] In summary, the present invention provides a culture method for culturing bone marrow mesenchymal stem cells of passages P2 to P6 by using a MesenGro medium containing protopanaxadiol with a concentration of 5 μM to 50 μM and 10% (volume ratio) fetal bovine serum. This method can effectively increase the expression of genes related to osteogenic differentiation of bone marrow mesenchymal stem cells, thereby improving the osteogenic differentiation ability of bone marrow mesenchymal stem cells.

[0064] The above embodiments are the preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A culture method for improving the osteogenic differentiation ability of bone marrow mesenchymal stem cells, which adds protopanaxadiol to the culture medium.

2. The method according to claim 1, specifically including the following steps: S1 Add fetal bovine serum and protopanaxadiol to the culture medium to prepare a culture medium containing protopanaxadiol. S2 Use the culture medium containing protopanaxadiol prepared in S1 to resuspend bone marrow mesenchymal stem cells of passages P2 to P6 to obtain a cell suspension. S3 Adjust the cell density in the cell suspension obtained in S2, and inoculate the cell suspension into a cell culture device and place it in an incubator for amplification culture.

3. The method according to claim 2, in S1, the concentration of protopanaxadiol in the culture medium containing protopanaxadiol is 5 μM to 50 μM.

4. The method according to claim 2, in S1, the volume ratio of fetal bovine serum in the culture medium containing protopanaxadiol is 10%.

5. According to the method described in claim 2, in S3, the cell density in the cell suspension is 1×10 6 ~4×10 6 cells / mL.

6. According to the method described in claim 2, in S3, the cell culture device is a cell culture plate or a cell culture flask, and the cell density inoculated in the cell culture plate or the cell culture flask is 0.5 to 1×10 4 cells / cm 2 .

7. The method according to claim 2, in S3, the incubator is a CO2 incubator, the temperature during culture is 35°C to 38°C, and the volume percentage concentration of CO2 in the CO2 incubator is 4.5 to 5.5%.

8. The method according to claim 2 includes the following steps S1 Add 10% (volume ratio) fetal bovine serum and a certain amount of protopanaxadiol to MesenGro medium to prepare a culture medium containing protopanaxadiol with a protopanaxadiol concentration of 5 μM, 20 μM or 50 μM. S2 Use the culture medium containing protopanaxadiol prepared in S1 to resuspend bone marrow mesenchymal stem cells of passages P2 to P6 to obtain a cell suspension. Adjust the cell density in the cell suspension obtained in S2 to 1×10 6 ~4×10 6 cells / mL, and inoculate the cell suspension into a culture device with a cell density of 0.5~1×10 4 cells / cm 2 , and then place it in a CO2 incubator at 37°C with a volume percentage concentration of CO2 of 5% for amplification culture for 3 days.

9. The method according to claim 1 can effectively increase the expression of genes related to osteogenic differentiation of bone marrow mesenchymal stem cells.

10. The method according to claim 9, the osteogenic differentiation genes include at least one of RUNX2, ALPL, COL1A1 and OPN.

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