Polypeptide for improving osteogenic differentiation activity of bone marrow mesenchymal stem cells and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a polypeptide for improving osteogenic differentiation activity of bone marrow mesenchymal stem cells and application thereof, wherein an amino acid sequence of the polypeptide is shown as SEQ ID NO. 1; the polypeptide is fused from a chicken gall protein amino acid sequence and a oyster protein amino acid sequence. The polypeptide is added in DMEM / F12 basic culture medium, can provide necessary nutrient support for the expansion process of bone marrow mesenchymal stem cells, improve the proliferation activity of bone marrow mesenchymal stem cells, and can also improve the osteogenic differentiation activity of bone marrow mesenchymal stem cells, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide that enhances the osteogenic differentiation activity of bone marrow mesenchymal stem cells and its applications. Background Technology

[0002] In clinical practice, the treatment and prevention of bone defects and osteoporosis have become urgent problems in orthopedics. Traditional bone grafting surgery is invasive and has limited bone resources, thus necessitating a less invasive, safe, and effective alternative. Bone marrow mesenchymal stem cells (BMSCs) are pluripotent stem cells with self-renewal and multi-lineage differentiation potential. Due to their wide availability, ease of in vitro culture and expansion, and low immunogenicity, they are considered one of the most ideal seed cells for bone tissue engineering. After osteogenic induction, BMSCs can form new bone tissue to repair various bone defects. Currently, tissue engineering techniques often face the problem of insufficient osteoblasts; therefore, screening and developing bioactive substances that can efficiently and specifically promote osteogenic differentiation of BMSCs is a current research focus.

[0003] Currently, the main inducing factors for promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) include chemical drugs (such as dexamethasone and estrogen), cytokines, physical stimulation (such as electromagnetic fields and ultrasound), and traditional Chinese medicine extracts, which have become research hotspots. However, these methods still have certain limitations: chemical drugs may cause systemic side effects; cytokines are expensive; physical stimulation equipment is complex and parameters are difficult to control, limiting clinical application; and traditional Chinese medicine extracts have complex components, unclear mechanisms of action, and are difficult to standardize. In recent years, peptides have been widely used in the biomedical field due to their high bioactivity, high specificity, and few side effects. Studies have shown that specific peptide sequences can significantly promote osteogenic differentiation of BMSCs. Therefore, developing peptides that can efficiently enhance the osteogenic differentiation activity of BMSCs has significant clinical application value.

[0004] To achieve the above objectives, this invention provides a polypeptide that enhances the osteogenic differentiation activity of bone marrow mesenchymal stem cells, its application, preparation method, and application. Summary of the Invention

[0005] The primary objective of this invention is to provide a polypeptide that enhances the osteogenic differentiation activity of bone marrow mesenchymal stem cells.

[0006] The second objective of this invention is to provide an application of a polypeptide that enhances the osteogenic differentiation activity of bone marrow mesenchymal stem cells.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A polypeptide that enhances the osteogenic differentiation activity of bone marrow mesenchymal stem cells, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Furthermore, the polypeptide is formed by fusing the amino acid sequences of chicken gluconein and oyster protein.

[0009] The above-described polypeptide for enhancing the osteogenic differentiation activity of bone marrow mesenchymal stem cells is used to prepare a culture medium for osteogenic differentiation of bone marrow mesenchymal stem cells.

[0010] Furthermore, the culture medium is a DMEM / F12 basal medium containing 8-12 v / v% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0011] Furthermore, the concentration of the polypeptide in the culture medium is 50-150 μg / mL.

[0012] Furthermore, the bone marrow mesenchymal stem cells are P2-P4 generation bone marrow mesenchymal stem cells.

[0013] Furthermore, the seeding density of the bone marrow mesenchymal stem cells in the culture medium is 4-6 × 10⁻⁶. 4 per mL.

[0014] Furthermore, the culture conditions for the bone marrow mesenchymal stem cells were: 37°C, 5v / v% CO2.

[0015] Compared with the prior art, the main advantages of the present invention are as follows: This invention integrates the amino acid sequences of chicken gluconeogenesis protein and oyster protein to obtain a fusion polypeptide with the amino acid sequence Lys-Ala-Pro-Asp-Phe-Val-Leu-Leu-Glu-Tyr-Ser-Ile. Adding this fusion polypeptide to DMEM / F12 basal medium provides essential nutritional support for the expansion of bone marrow mesenchymal stem cells (BMSCs), enhances their proliferative activity, and also improves their osteogenic differentiation activity, demonstrating broad application prospects. Attached Figure Description

[0016] Figure 1 Morphological diagram of P3 generation bone marrow mesenchymal stem cells; Figure 2 The effect of the fusion peptide prepared in Example 1 (at a concentration of 100 μg / mL) on osteogenic differentiation of bone marrow mesenchymal stem cells is shown in the figure. Figure 3 The effect of the fusion peptide prepared in Comparative Example 1 (100 μg / mL) on osteogenic differentiation of bone marrow mesenchymal stem cells is shown in the figure. Figure 4 The effect of the fusion peptides prepared in two comparative groups (100 μg / mL) on osteogenic differentiation of bone marrow mesenchymal stem cells is shown in the figure. Figure 5 The figure shows the effect of different fusion peptides on the expression level of the RUNX2 gene in bone marrow mesenchymal stem cells. Detailed Implementation

[0017] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of 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, all reagents or instruments used are conventional products obtained through commercial channels.

[0018] Example 1 Synthesis of fusion peptides: (1) Add Fmoc-Ile-Wang resin into the polypeptide synthesizer.

[0019] (2) Remove the Fmoc protecting group on the Fmoc-Ile-Wang resin with 20 v / v% piperidine (DMF: hexahydropyridine = 4:1), react for 20 minutes, wash the resin with DMF, and the blue color of ninhydrin confirms that the Fmoc group is completely deprotected.

[0020] (3) DIC (N,N'-diisopropylcarbodiimide), HOBT (1-hydroxybenzotriazole) and FMOC-Ser(tBu)-OH (N-fluorenylmethoxycarbonyl-O-tert-butyl-L-serine) were added to the synthesizer and the condensation reaction was carried out for 50 minutes. The resin was washed with DMF (dimethylformamide) and tested with ninhydrin. The resin was colorless, which confirmed that the amino acid was successfully attached, and the next amino acid could be attached.

[0021] (4) Repeat steps (2) and (3) to sequentially combine the following amino acids from the C-terminus to the N-terminus in the synthesizer: Lys-Ala-Pro-Asp-Phe-Val-Leu-Leu-Glu-Tyr-Ser-Ile. After the last amino acid is deprotected and indene is detected, the residual liquid is dried, and the resin is washed with DCM (dichloromethane) and then dried until it is loose and free of lumps.

[0022] (5) Add the cutting agent trifluoroacetic acid to the resin to cut the polypeptide off the resin, then extract with ether and freeze dry to obtain crude polypeptide.

[0023] (6) The obtained crude fusion peptide was separated and purified by a semi-preparative high-performance liquid chromatography (HPLC) instrument to remove unreacted raw materials and byproducts. The purified fusion peptide was frozen and stored at -80°C, and then freeze-dried using a freeze dryer to obtain the peptide of the present invention that enhances the osteogenic differentiation activity of bone marrow mesenchymal stem cells. The amino acid sequence of the peptide obtained in this invention is shown in SEQ ID NO.1: Lys-Ala-Pro-Asp-Phe-Val-Leu-Leu-Glu-Tyr-Ser-Ile.

[0024] Example 2 Isolation and culture of bone marrow mesenchymal stem cells (BMSCs): Bone marrow tissue was harvested, washed with PBS buffer, and then minced to approximately 1 mm using surgical scissors. 3 Bone marrow tissue was repeatedly pipetted into a suspension using a syringe. The suspension was then filtered through a 200-mesh sieve to remove impurities. The cells were centrifuged at 6000g for 5 minutes, the supernatant was discarded, and the cell pellet was retained. The cell pellet was resuspended in DMEM / F12 medium containing 10 v / v% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. The pellet was then transferred to a culture dish and incubated at 37°C in a 5 v / v% CO2 incubator. The culture medium was changed every 48 hours. Once the cells reached 80% confluence, they were passaged. The original culture medium and any remaining tissue fragments were discarded. The cells were digested with 0.25% trypsin, centrifuged to remove the digestive fluid, and then resuspended in DMEM / F12 medium containing 10 v / v% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were then passaged to obtain P3 generation bone marrow mesenchymal stem cells (BMSCs). Microscopic observation revealed that the obtained cells were of uniform size and conformed to the general morphological characteristics of myeloid mesenchymal stem cells. Figure 1 The results show that the present invention successfully isolated and cultured bone marrow mesenchymal stem cells.

[0025] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the amino acid sequence of the Lys-Ala-Pro-Asp-Phe-Val-Leu-Leu-Glu-Tyr-Ser-Ile polypeptide in step (4) of Example 1 is replaced with the chicken gizzard membrane polypeptide: Lys-Ala-Pro-Asp-Phe-Val (SEQ ID NO.2), and all other steps are the same as in Example 1.

[0026] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the amino acid sequence of the Lys-Ala-Pro-Asp-Phe-Val-Leu-Leu-Glu-Tyr-Ser-Ile polypeptide in step (4) of Example 1 is replaced with the oyster polypeptide: Leu-Leu-Glu-Tyr-Ser-Ile (SEQ ID NO.3), while all other steps are the same as in Example 1.

[0027] Experimental Example 1 Effects of recombinant peptides on the proliferation of bone marrow mesenchymal stem cells: (1) The three polypeptides obtained in Example 1, Comparative Example 1 and Comparative Example 2 were added to DMEM / F12 basal medium containing 10 v / v% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL) respectively, and cell culture media containing 50, 75, 100 and 125 μg / mL of the corresponding group polypeptides were obtained as experimental groups; at the same time, DMEM / F12 basal medium containing 10% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL) without the addition of polypeptides was prepared as negative control group.

[0028] (2) Take P3 generation bone marrow mesenchymal stem cells in the logarithmic growth phase prepared in Example 2, digest them with trypsin, and terminate the digestion with complete culture medium containing 10 v / v% FBS, according to 5 × 10 4 Cells were seeded at a density of cells / mL into the experimental groups (50 μg / mL, 75 μg / mL, 100 μg / mL, 125 μg / mL) and the negative control group prepared in step (1), respectively. Additionally, 100 μL of DMEM / F12 medium containing 10% FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL) was added to the blank wells of a 96-well culture plate without adding cells, serving as the OD blank group. At least three replicate wells were set up for each concentration group.

[0029] (3) After the addition was completed, the cells were placed in a 37℃, 5% CO2 cell culture incubator for 3 days. After the culture was completed, 10 μL of CCK-8 reagent was added to each well and incubated for 4 hours. The absorbance of each group of cells at 450 nm was detected by an ELISA reader, and the cell proliferation rate was calculated. The calculation formula is as follows: Cell proliferation rate (%) = [(OD experimental group - OD blank group) / (OD negative control group - OD blank group)] × 100%. The results are shown in Table 1.

[0030] Table 1. Effects of recombinant peptides on the proliferation of bone marrow mesenchymal stem cells. The results are shown in Table 1, illustrating the effect of different concentrations of the fusion peptide on the proliferation of bone marrow mesenchymal stem cells (BMSCs). As shown in Table 1, compared to the blank control group, the BMSC proliferation rates of the culture media containing 50 μg / mL, 75 μg / mL, 100 μg / mL, and 125 μg / mL peptides in Example 1, Comparative Example 1, and Comparative Example 2 were significantly increased. Among them, the peptide containing 100 μg / mL in Example 1 showed the most significant effect in promoting BMSC proliferation, with a BMSC proliferation rate of (150.69 ± 2.7)%. This indicates that the fusion peptide Lys-Ala-Pro-Asp-Phe-Val-Leu-Leu-Glu-Tyr-Ser-Ile of the present invention has a significant effect on promoting the proliferation of BMSCs.

[0031] Experimental Example 2 Effects of recombinant peptides on osteogenic differentiation of bone marrow mesenchymal stem cells: (1) Take the P3 generation bone marrow mesenchymal stem cells prepared in Example 1, digest them with trypsin, and terminate the digestion with complete culture medium containing 10 v / v% FBS. 4 Cells were inoculated at a density of 100 μg / mL into the cell culture medium containing the corresponding peptides of Example 1, Comparative Example 1, and Comparative Example 2 prepared in step (1) of Experimental Example 1, with 100 μg / mL of peptides and 100 μg / mL of peptides. Each group was divided into 3 replicates. When the cell confluence reached 70%, the old culture medium in each well was discarded, and osteogenic induction differentiation medium (DMEM / F12 medium containing dexamethasone (0.15 μmol / L), sodium β-glycerophosphate (10 mmol / L), sodium ascorbate (50 mg / L), 10% (v / v) FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL) was added. The osteogenic induction differentiation medium was changed every 2 days. On day 10 of osteogenic induction culture, alkaline phosphatase (ALP) staining was performed according to the alkaline phosphatase kit instructions. After staining, the working solution was discarded, and the cells were washed three times with PBS. Images were taken under a microscope, and the osteogenic differentiation capacity of bone marrow mesenchymal stem cells in each group was assessed based on the staining results. The results are as follows: Figure 2 , Figure 3 , Figure 4 As shown.

[0032] The results are as follows Figure 2 , Figure 3 , Figure 4 The figure shown illustrates the effect of the fusion peptides prepared in three groups (100 μg / mL, Example 1, Comparative Example 1, and Comparative Example 2) on osteogenic differentiation of bone marrow mesenchymal stem cells. Figure 2-4It can be seen that, compared with Comparative Examples 1 and 2, the osteogenic differentiation capacity of bone marrow mesenchymal stem cells obtained by culturing the polypeptide prepared in Example 1 of this invention is enhanced. This indicates that by adding the fusion polypeptide Lys-Ala-Pro-Asp-Phe-Val-Leu-Leu-Glu-Tyr-Ser-Ile of this invention to the basal culture, the necessary nutritional support for the expansion process of bone marrow mesenchymal stem cells can be provided, significantly promoting the osteogenic differentiation potential of bone marrow mesenchymal stem cells.

[0033] (2) To further detect the expression of osteogenic differentiation markers, bone marrow mesenchymal stem cells were collected on day 14 of osteogenic induction culture. Total mRNA was extracted from the cells using the RNeasy Mini kit, and cDNA was synthesized using a reverse transcriptase kit. The expression level of the RUNX2 gene in Example 1, Comparative Example 1, and Comparative Example 2 was detected using real-time quantitative PCR (qPCR). The specific experimental steps are as follows: GAPDH was used as an internal reference gene. The upstream primer sequence for amplifying RUNX2 is shown in SEQ ID NO.4, and the downstream primer sequence is shown in SEQ ID NO.5; the upstream primer sequence for amplifying GAPDH is shown in SEQ ID NO.6, and the downstream primer sequence is shown in SEQ ID NO.7. The relative expression level of the RUNX2 gene was calculated using the 2^(-ΔΔCt) method. The relative expression level of the RUNX2 gene was obtained by plotting a bar chart using Origin plotting software. The results are shown in the figure below. Figure 5 As shown.

[0034] Table 2 Sequence List The results are as follows Figure 5 The figure shows the effect of different fusion peptides on the expression level of the RUNX2 gene in bone marrow mesenchymal stem cells. RUNX2 is a core master-controlling transcription factor for osteogenic differentiation. Figure 5 It was found that, compared with Comparative Examples 1 and 2, the 100 μg / mL polypeptide prepared in Example 1 could effectively promote the expression of the osteogenic differentiation gene RUNX2 in bone marrow mesenchymal stem cells. This indicates that adding the fusion polypeptide Lys-Ala-Pro-Asp-Phe-Val-Leu-Leu-Glu-Tyr-Ser-Ile of the present invention to the basal culture medium can provide the necessary nutritional support for the expansion process of bone marrow mesenchymal stem cells and significantly promote the osteogenic differentiation potential of bone marrow mesenchymal stem cells.

[0035] 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 polypeptide that enhances the osteogenic differentiation activity of bone marrow mesenchymal stem cells, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The polypeptide for enhancing osteogenic differentiation activity of bone marrow mesenchymal stem cells according to claim 1, characterized in that, The polypeptide is formed by the fusion of chicken gizzard protein and oyster protein; the amino acid sequence of the chicken gizzard protein is SEQ ID NO.2; the amino acid sequence of the oyster protein is SEQ ID NO.

3.

3. The application of the polypeptide according to claim 1 that enhances the osteogenic differentiation activity of bone marrow mesenchymal stem cells, characterized in that, Culture medium used to prepare osteogenic differentiation of bone marrow mesenchymal stem cells.

4. The application of the polypeptide according to claim 3 that enhances the osteogenic differentiation activity of bone marrow mesenchymal stem cells, characterized in that, The concentration of the polypeptide in the culture medium is 50-150 μg / mL.

5. The application of the polypeptide according to claim 3 that enhances the osteogenic differentiation activity of bone marrow mesenchymal stem cells, characterized in that, The culture medium is DMEM / F12 medium containing 8-12 v / v% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin and 50-150 μg / mL of the polypeptide.

6. The application of the polypeptide according to claim 3 that enhances the osteogenic differentiation activity of bone marrow mesenchymal stem cells, characterized in that, The bone marrow mesenchymal stem cells mentioned are P2-P4 generation bone marrow mesenchymal stem cells.

Images