A composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells and application thereof

Abstract

The present application belongs to the technical field of stem cells, and particularly relates to a composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells and application thereof. The composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells provided by the present application comprises the following raw materials: DMEM / F12 serum-free medium, topiroxostat, calanolide D, recombinant human epidermal growth factor, insulin, vitamin C, sodium selenite and glutathione. The topiroxostat in the composition can promote the expression of OPN, a bone marrow mesenchymal stem cell osteoblast-related gene, and the calanolide D can further enhance the promoting effect of the topiroxostat, thereby achieving the purpose of promoting osteogenic differentiation of bone marrow mesenchymal stem cells, which is of great significance for the clinical application of bone marrow mesenchymal stem cells.

Description

Technical Field

[0001] This invention belongs to the field of stem cell technology, specifically relating to a composition that promotes osteogenic differentiation of bone marrow mesenchymal stem cells and its application. Background Technology

[0002] Stem cells are a type of cell with unlimited or immortal self-renewal capacity, capable of producing highly differentiated daughter cells. They are increasingly favored by researchers due to their advantages such as multi-lineage differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation, and self-replication. Based on developmental stage, stem cells are divided into two categories: embryonic stem cells and adult stem cells. Bone marrow mesenchymal stem cells (BMSCs) are a type of adult stem cell derived from the mesoderm, possessing advantages such as wide availability, ease of culture, high differentiation potential, and the ability to be autologously transplanted. Further clinical trials have revealed that BMSCs are closely related to bone tissue formation, regeneration, and remodeling, and that a dynamic balance exists between osteogenic and adipogenic differentiation, mutually restricting and transforming each other, which is crucial for maintaining bone homeostasis.

[0003] The differentiation direction of bone marrow mesenchymal stem cells (BMSCs) is closely related to their microenvironment, which includes the bone marrow microenvironment and the local mesenchymal tissue microenvironment. These microenvironments are highly susceptible to the influence of the culture medium, thus affecting the differentiation direction of BMSCs. Traditional culture media often contain fetal bovine serum, reducing safety; furthermore, existing induction methods suffer from low differentiation efficiency and long induction times, all of which pose significant challenges to the clinical use of BMSCs. Therefore, a new, safe, and efficient BMSC induction composition is urgently needed, which is of great significance for the clinical application of BMSCs. Summary of the Invention

[0004] The first objective of this invention is to provide a composition that promotes osteogenic differentiation of bone marrow mesenchymal stem cells.

[0005] The second objective of this invention is to provide an application of a composition that promotes osteogenic differentiation of bone marrow mesenchymal stem cells, which is of great significance for the clinical application of bone marrow mesenchymal stem cells.

[0006] To achieve one of the above objectives, the technical solution adopted by the present invention is: A composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, comprising the following ingredients: DMEM / F12 serum-free culture medium, topipusta, crotonin D, recombinant human epidermal growth factor, insulin, vitamin C, sodium selenite, and glutathione. The contents of topipirostat, crocin D, recombinant human epidermal growth factor, insulin, vitamin C, sodium selenite, and glutathione in DMEM / F12 serum-free medium are as follows: 7~12 μg / mL topipirostat, 1~5 μg / mL crocin D, 16.5~39 ng / mL recombinant human epidermal growth factor, 15~23 μg / mL insulin, 4~9 μg / mL vitamin C, 30~44.5 ng / mL sodium selenite, and 55~70 μg / mL glutathione.

[0007] Furthermore, the contents of topipirostat, crocin D, recombinant human epidermal growth factor, insulin, vitamin C, sodium selenite, and glutathione in DMEM / F12 serum-free culture medium are as follows: 9 μg / mL topipirostat, 2.5 μg / mL crocin D, 27 ng / mL recombinant human epidermal growth factor, 18 μg / mL insulin, 6 μg / mL vitamin C, 34 ng / mL sodium selenite, and 65 μg / mL glutathione.

[0008] To achieve the second objective mentioned above, the technical solution adopted by the present invention is as follows: The application of a composition that promotes osteogenic differentiation of bone marrow mesenchymal stem cells includes the following steps: seeding bone marrow mesenchymal stem cells obtained from passage culture into the above-described composition for culture.

[0009] Furthermore, the bone marrow mesenchymal stem cells are P3-P5 generation bone marrow mesenchymal stem cells.

[0010] Furthermore, the density of the bone marrow mesenchymal stem cells seeded into the composition is (2~6.5)×10⁻⁶. 5 per mL.

[0011] Furthermore, the culture conditions are 37°C and CO2 with a volume concentration of 5%.

[0012] Furthermore, the culture period is 13-14 days.

[0013] Compared with the prior art, the main advantages of the present invention are as follows: The expression level of osteopontin (OPN), an osteoblast-related gene, in bone marrow mesenchymal stem cells induced by the composition of the present invention was detected. It was found that topipustine in the composition can promote the expression of OPN in bone marrow mesenchymal stem cells, and crotonin D can further enhance the promoting effect of topipustine, thereby achieving the purpose of promoting osteogenic differentiation of bone marrow mesenchymal stem cells. This is of great significance for the clinical application of bone marrow mesenchymal stem cells. Attached Figure Description

[0014] Figure 1These are morphological characteristic diagrams of P3-P5 generation bone marrow mesenchymal stem cells. Figure 2 This is a graph showing the results of detecting the expression level of osteoblast-related gene OPN in bone marrow mesenchymal stem cells in Examples 1-3 and Comparative Examples 1-2; Figure 3 These are ALP staining results of bone marrow mesenchymal stem cells in Examples 1-3 and Comparative Examples 1-2. Detailed Implementation

[0015] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0016] The preparation process of P3~P5 generation bone marrow mesenchymal stem cells is as follows: Human bone marrow fluid and PBS were mixed at a volume ratio of 1:1 and centrifuged at 1800 rpm for 5 min. An equal volume of Ficoll separation medium was added to the diluted bone marrow fluid, and the mixture was centrifuged at 2000 rpm for 40 min. Mononuclear cells with a white membrane at the interface were aspirated, and the cells were gently washed with PBS. The cells were then resuspended in LG-DMEM medium (with 10% fetal bovine serum added) at a cell density of 2 × 10⁶ cells / mL. 5 Cells / mL, inoculated into a substrate with a bottom area of ​​25 cm² 2 The cells were cultured in culture flasks at 37°C in a cell culture incubator with 5% CO2. The medium was changed every 2 days. When the cell confluence reached 80%, the cells were digested with trypsin-EDTA solution and passaged at a ratio of 1:2 to obtain passaged P3-P5 generation bone marrow mesenchymal stem cells for later use.

[0017] Example Example 1 A composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, comprising DMEM / F12 serum-free medium, topiprostine, crotonin D, recombinant human epidermal growth factor, insulin, vitamin C, sodium selenite, and glutathione. The concentrations of each component in the DMEM / F12 serum-free medium are as follows: 9 μg / mL topiprostine, 2.5 μg / mL crotonin D, 27 ng / mL recombinant human epidermal growth factor, 18 μg / mL insulin, 6 μg / mL vitamin C, 34 ng / mL sodium selenite, and 65 μg / mL glutathione.

[0018] The application of a composition that promotes osteogenic differentiation of bone marrow mesenchymal stem cells, the specific operation process of which is as follows: Press 2×10 5 P3 generation bone marrow mesenchymal stem cells were seeded into 24-well plates at a density of cells / mL. The 24-well plates were supplemented with the above-mentioned osteogenic differentiation composition and placed in a cell culture incubator at 37°C and 5% CO2 for induced differentiation culture for 14 days, with the medium changed every 2 days.

[0019] Example 2 A composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, comprising DMEM / F12 serum-free medium, topiprostine, crotonin D, recombinant human epidermal growth factor, insulin, vitamin C, sodium selenite, and glutathione. The concentrations of each component in the DMEM / F12 serum-free medium are as follows: 7 μg / mL topiprostine, 1 μg / mL crotonin D, 16.5 ng / mL recombinant human epidermal growth factor, 15 μg / mL insulin, 4 μg / mL vitamin C, 30 ng / mL sodium selenite, and 55 μg / mL glutathione.

[0020] The application of a composition that promotes osteogenic differentiation of bone marrow mesenchymal stem cells, the specific operation process of which is as follows: 4×10 5 P4 generation bone marrow mesenchymal stem cells were seeded into 24-well plates at a density of cells / mL. The 24-well plates were supplemented with the above-mentioned osteogenic differentiation composition and placed in a cell culture incubator at 37°C and 5% CO2 for induced differentiation culture for 13 days, with the medium changed every 2 days.

[0021] Example 3 A composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, comprising DMEM / F12 serum-free medium, topiprestat, crocin D, recombinant human epidermal growth factor, insulin, vitamin C, sodium selenite, and glutathione. The concentrations of each component in the DMEM / F12 serum-free medium are as follows: 12 μg / mL topiprestat, 5 μg / mL crocin D, 39 ng / mL recombinant human epidermal growth factor, 23 μg / mL insulin, 9 μg / mL vitamin C, 44.5 ng / mL sodium selenite, and 70 μg / mL glutathione.

[0022] The application of a composition that promotes osteogenic differentiation of bone marrow mesenchymal stem cells, the specific operation process of which is as follows: According to 6.5×10 5 P5 generation bone marrow mesenchymal stem cells were seeded into 24-well plates at a density of cells / mL. The 24-well plates were supplemented with the above-mentioned osteogenic differentiation composition and placed in a cell culture incubator at 37°C and 5% CO2 for induced differentiation culture for 14 days, with the medium changed every 2 days.

[0023] Comparative Example Comparative Example 1 The difference between this comparative example and Example 1 is that the composition consists of DMEM / F12 serum-free culture medium, crotonin D, recombinant human epidermal growth factor, insulin, vitamin C, sodium selenite, and glutathione. The contents of each component in DMEM / F12 serum-free culture medium are as follows: 2.5 μg / mL crotonin D, 27 ng / mL recombinant human epidermal growth factor, 18 μg / mL insulin, 6 μg / mL vitamin C, 34 ng / mL sodium selenite, and 65 μg / mL glutathione.

[0024] Comparative Example 2 The difference between this comparative example and Example 1 is that the composition consists of DMEM / F12 serum-free culture medium, topirostat, recombinant human epidermal growth factor, insulin, vitamin C, sodium selenite, and glutathione. The concentrations of each component in the DMEM / F12 serum-free culture medium are as follows: 9 μg / mL topirostat, 27 ng / mL recombinant human epidermal growth factor, 18 μg / mL insulin, 6 μg / mL vitamin C, 34 ng / mL sodium selenite, and 65 μg / mL glutathione.

[0025] Test case Experimental Example 1 The obtained P3-P5 generation bone marrow mesenchymal stem cells were observed under an inverted microscope (magnification 20×) and photographed. The results are as follows: Figure 1 As shown.

[0026] Figure 1 This is a morphological diagram of P3-P5 generation bone marrow mesenchymal stem cells. Figure 1 Images (A)-(C) show the morphological characteristics of bone marrow mesenchymal stem cells from generations P3 to P5, respectively. (Observation) Figure 1 It can be seen that the cells have a uniform morphology and exhibit a long spindle-shaped structure, which is consistent with the typical characteristics of bone marrow mesenchymal stem cells.

[0027] Experimental Example 2 Real-time RT-PCR was used to detect the expression level of osteopontin (OPN), an osteoblast-related gene. The specific procedure is as follows: (1) Collect bone marrow mesenchymal stem cells that have undergone induced differentiation culture in Examples 1-3 and Comparative Examples 1-2, discard the composition, and wash the cells three times with PBS; (2) Total RNA was extracted from each group of cells for PCR detection. The primer sequences are shown in Table 1. Table 1 (3) 2 -ΔΔCt Quantitative analysis was performed using this method, and the results are as follows: Figure 2 .

[0028] Figure 2 This is a graph showing the results of detecting the expression level of the osteoblast-related gene OPN in bone marrow mesenchymal stem cells in Examples 1-3 and Comparative Examples 1-2. (Observation) Figure 2 It can be seen that the composition provided by the present invention can increase the expression level of the osteoblast-related gene OPN in bone marrow mesenchymal stem cells. This result also shows that the composition provided by the present invention can better promote osteogenic differentiation of bone marrow mesenchymal stem cells.

[0029] Compared with Examples 1-3, the expression levels of the osteoblast-related gene OPN in bone marrow mesenchymal stem cells of Comparative Examples 1-2 were all reduced, with a particularly significant decrease in Comparative Example 1. Furthermore, a comparison of the compositions of Comparative Examples 1-2 with those of Examples 1-3 revealed that the composition of Comparative Example 1 lacked topipistat, and the composition of Comparative Example 2 lacked crocin D. These results indicate that topipistat can increase the expression of the osteoblast-related gene OPN in bone marrow mesenchymal stem cells, and crocin D can further enhance the promoting effect of topipistat.

[0030] Experimental Example 3 The ALP staining assay was used to detect the osteogenic differentiation of bone marrow mesenchymal stem cells in Examples 1-3 and Comparative Examples 1-2. The specific procedure is as follows: Bone marrow mesenchymal stem cells (BMSCs) induced by osteogenic differentiation in Examples 1-3 and Comparative Examples 1-2 were collected. Cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, and then washed again with PBS to remove the fixative. Cells were stained with alkaline phosphatase staining reagent according to the manufacturer's instructions for 30 min. After staining, the cells were observed under a microscope and photographed. The results are as follows: Figure 3 .

[0031] Figure 3 These are ALP staining results of bone marrow mesenchymal stem cells from Examples 1-3 and Comparative Examples 1-2. Figure 3 Images (A) through (C) show the ALP staining results of bone marrow mesenchymal stem cells from Examples 1 through 3, respectively. Figure 3 Images (D) through (E) show the ALP staining results of bone marrow mesenchymal stem cells in comparisons 1 and 2, respectively. (Observation) Figure 3 It can be seen that, compared with Examples 1-3, the positive staining rate of bone marrow mesenchymal stem cells in Comparative Examples 1-2 was significantly reduced. These results also demonstrate that the composition provided by the present invention can better promote osteogenic differentiation of bone marrow mesenchymal stem cells.

[0032] In summary, topipustine in the composition of this invention can promote the expression of the osteoblast-related gene OPN in bone marrow mesenchymal stem cells, and crotonin D can further enhance the promoting effect of topipustine, thereby achieving the goal of promoting osteogenic differentiation of bone marrow mesenchymal stem cells. This is of great significance for the clinical application of bone marrow mesenchymal stem cells.

[0033] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells, characterized in that, The ingredients include: DMEM / F12 serum-free culture medium, topirostat, crocin D, recombinant human epidermal growth factor, insulin, vitamin C, sodium selenite, and glutathione. The contents of topipirostat, crocin D, recombinant human epidermal growth factor, insulin, vitamin C, sodium selenite, and glutathione in DMEM / F12 serum-free medium are as follows: 7~12 μg / mL topipirostat, 1~5 μg / mL crocin D, 16.5~39 ng / mL recombinant human epidermal growth factor, 15~23 μg / mL insulin, 4~9 μg / mL vitamin C, 30~44.5 ng / mL sodium selenite, and 55~70 μg / mL glutathione.

2. The composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells according to claim 1, characterized in that, The concentrations of topipirostat, crocin D, recombinant human epidermal growth factor, insulin, vitamin C, sodium selenite, and glutathione in DMEM / F12 serum-free medium were as follows: 9 μg / mL topipirostat, 2.5 μg / mL crocin D, 27 ng / mL recombinant human epidermal growth factor, 18 μg / mL insulin, 6 μg / mL vitamin C, 34 ng / mL sodium selenite, and 65 μg / mL glutathione.

3. The application of a composition that promotes osteogenic differentiation of bone marrow mesenchymal stem cells, characterized in that, Includes the following steps: Bone marrow mesenchymal stem cells obtained from passage culture are seeded into the composition of claim 1 or 2 and cultured.

4. The application of the composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells according to claim 3, characterized in that, The bone marrow mesenchymal stem cells mentioned are P3-P5 generation bone marrow mesenchymal stem cells.

5. The application of the composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells according to claim 3, characterized in that, The density of the bone marrow mesenchymal stem cells seeded into the composition is (2~6.5)×10⁻⁶. 5 per mL.

6. The application of the composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells according to claim 3, characterized in that, The culture conditions were 37°C and 5% CO2 (by volume).

7. The application of the composition for promoting osteogenic differentiation of bone marrow mesenchymal stem cells according to claim 3, characterized in that, The incubation period is 13-14 days.

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