Recombinant polypeptide for improving 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 recombinant polypeptide for improving the activity of bone marrow mesenchymal stem cells and application thereof. The amino acid sequence of the recombinant polypeptide is shown as SEQ ID NO. 3. The recombinant polypeptide of the application is obtained by integrating the extracellular matrix amino acid sequence and the heparin amino acid sequence. The recombinant polypeptide of the application can promote the expression of the Ki67 gene, improve the proliferation activity of the bone marrow mesenchymal stem cells, and has the effect of inhibiting the replicative senescence of the 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 recombinant polypeptide that enhances the activity of bone marrow mesenchymal stem cells and its application. Background Technology

[0002] Stem cells are a type of cell with unlimited or even immortal self-renewal capabilities, holding immense potential in regenerative cell therapy. Bone marrow mesenchymal stem cells (BMSCs) have become a key focus of 21st-century life science research, possessing functions such as self-proliferation, multi-lineage differentiation, damage repair, and immune regulation, showing broad application prospects and holding promise for use in cell therapy. Furthermore, BMSCs can also serve as delivery vehicles for targeted delivery of biological agents to treat certain diseases. Currently, BMSCs have already shown promise in treating heart disease, liver injury, and spinal cord injury.

[0003] Peptides are bioactive substances involved in various cellular functions within organisms. They are formed by the dehydration of amino acids and contain carboxyl and amino groups. They possess specificity, high stability, low production cost, and ease of customization and immobilization. They also enhance stem cell adhesion, proliferation, and directed differentiation. Currently, hundreds of peptides have been discovered in organisms, playing an essential role in various complex physiological activities. Peptides are involved in hormones, nerves, cell growth, and reproduction, primarily regulating various systems, organs, and cells within the body. With the development of genetic engineering technology, genes expressing bioactive peptides can be cloned into certain microorganisms or animals, allowing for direct expression of desired peptides and significantly increasing yield and purity. Recombinant peptides have broad potential value and applications in many fields. They can be used not only to improve plant varieties and produce enzymes, antibodies, and hormones, but also to effectively enhance stem cell adhesion, promote mesenchymal stem cell proliferation and directed differentiation, providing strong evidence for the development and application of novel bioactive peptides. Therefore, further research into peptide functions can advance their clinical application in improving stem cell quality and tissue regeneration.

[0004] RGD integrin-binding peptide and YIGSR integrin-binding peptide are short peptide sequences derived from the extracellular matrix and possess the ability to specifically bind to integrin receptors. Integrins, as important receptors on the cell membrane, regulate various cellular functions by activating signal transduction pathways. The binding of RGD peptide and YIGSR peptide to integrins can trigger a series of signal transduction events, affecting processes such as cell proliferation, apoptosis, and migration. KRSR heparin-binding peptide is a peptide molecule that can bind to heparin, widely distributed in the extracellular matrix, and plays an important role in various biological processes. The binding of heparin-binding peptide to heparin may trigger a series of signal transduction processes, activating or inhibiting specific signaling pathways to regulate processes such as cell proliferation, migration, or apoptosis.

[0005] Bone marrow mesenchymal stem cells (BMSCs) have broad application prospects in both basic research and clinical applications, but the limited number of directly isolated BMSCs has become a major problem restricting their large-scale clinical application. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a recombinant polypeptide that enhances the activity of bone marrow mesenchymal stem cells, which can promote the proliferation activity of bone marrow mesenchymal stem cells.

[0007] The second objective of this invention is to provide an application of a recombinant polypeptide that enhances the activity of bone marrow mesenchymal stem cells.

[0008] One of the objectives of this invention is achieved through the following technical solution:

[0009] A recombinant polypeptide for enhancing the activity of bone marrow mesenchymal stem cells, the amino acid sequence of which is shown in SEQ ID NO.3.

[0010] The second objective of this invention is achieved by the following technical solution:

[0011] Application of a recombinant polypeptide that enhances the activity of bone marrow mesenchymal stem cells, wherein the recombinant polypeptide is used in a culture medium for bone marrow mesenchymal stem cells.

[0012] Furthermore, the bone marrow mesenchymal stem cells are P2-P5 generation bone marrow mesenchymal stem cells.

[0013] Furthermore, the concentration of the recombinant polypeptide in the culture medium is 25-200 μg / mL.

[0014] Furthermore, the optimal concentration of the recombinant polypeptide in the culture medium is 100 μg / mL.

[0015] Furthermore, the bone marrow mesenchymal stem cells were cultured at 37°C and 5% CO2.

[0016] Furthermore, the seeding density of the bone marrow mesenchymal stem cells is 1-4 × 10⁻⁴. 4 per mL.

[0017] Compared with existing technologies, the main advantages of this invention are as follows: This invention obtains the recombinant polypeptide by integrating the extracellular matrix amino acid sequence with the heparin amino acid sequence. The recombinant polypeptide of this invention can promote Ki67 gene expression, enhance the proliferative activity of bone marrow mesenchymal stem cells, and also inhibit the replicative senescence of bone marrow mesenchymal stem cells, thus possessing broad application prospects. Attached Figure Description

[0018] Figure 1 Morphological diagram of P3 generation bone marrow mesenchymal stem cells;

[0019] Figure 2 The figure shows the effect of recombinant peptides on Ki67 gene expression in bone marrow mesenchymal stem cells.

[0020] Figure 3 The figure shows the effect of recombinant peptides on the percentage of β-gal positive cells in bone marrow mesenchymal stem cells. Detailed Implementation

[0021] 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.

[0022] Example 1

[0023] Isolation and culture of bone marrow mesenchymal stem cells (BMSCs):

[0024] Wash the bone marrow tissue with PBS buffer and cut it into 1mm pieces using surgical scissors. 3 Bone marrow tissue of varying sizes was repeatedly pipetted into a suspension using a 24-gauge syringe. The suspension was then filtered through a 200-mesh cell sieve and centrifuged at 6000g for 5 minutes. The supernatant was carefully aspirated, retaining the cell pellet. The cell pellet was resuspended in DMEM / F12 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. The pellet was then transferred to a culture dish and cultured at 37°C and 5% CO2. The culture medium was changed every 48 hours. Once the cell confluence reached 80%, the cells were passaged. The tissue fragments and culture medium were discarded, and the cells were digested with 0.25% trypsin. After centrifugation to remove the digestive fluid, the cells were resuspended in DMEM / F12 medium containing 10% 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). The morphology of the obtained BMSCs was observed under a microscope, and the results are as follows: Figure 1 As shown, the cells are uniform in size, consistent with the typical characteristics of bone marrow mesenchymal stem cells. Therefore, this invention has yielded bone marrow mesenchymal stem cells.

[0025] Example 2

[0026] Synthesis of recombinant peptides:

[0027] The recombinant polypeptide of this invention is obtained by integrating the amino acid sequence of the extracellular matrix with the amino acid sequence of heparin. The synthesis process of the recombinant polypeptide is as follows:

[0028] (1) Synthesis of peptide resin: Weigh Wang's resin and place it in the reactor of SYMPHONY 12-channel peptide synthesizer.

[0029] (2) Bonding of carboxyl groups to resin: Weigh 1 mmol each of protected diaminocarboxylic acids (including Fmoc-L-Lys(Fmoc)-OH, Fmoc-L-Orn(Fmoc)-OH, Fmoc-2,4-DAB(Fmoc)-OH, or DI-Fmoc-2,6-Diaminoheptanedioic acid) and bottle them separately. This allows the carboxyl groups of the diaminocarboxylic acids to covalently bond with Wang's resin, laying the foundation for peptide chain synthesis.

[0030] (3) Synthesizing recombinant polypeptide: Weigh 1 mmol of amino acid monomers with protecting groups and bottle them. According to the predetermined amino acid sequence (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3), from the C-terminus to the N-terminus, the synthesis instrument is automatically controlled by computer program to remove Fmoc protection, activate and link the amino acid monomers. Then, the next cycle is carried out until all amino acid monomers are linked to obtain recombinant polypeptides with side chain protecting groups.

[0031] (4) Purification of recombinant peptides: The recombinant peptides with side-chain protecting groups obtained in step (3) were separated and purified by high performance liquid chromatography, and then freeze-dried to obtain three recombinant peptides named KYR-1, KYR-2, and KYR-3, respectively. The amino acid sequences of KYR-1, KYR-2, and KYR-3 are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0032] Table 1 Sequence List

[0033]

[0034]

[0035] Experimental Example 1

[0036] An investigation into the effects of recombinant peptides on the proliferation of bone marrow mesenchymal stem cells:

[0037] An experimental group was set up, consisting of a blank control group and experimental groups with concentrations of KYR-1, KYR-2, and KYR-3 of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, with three replicates for each concentration. Bone marrow mesenchymal stem cells in the logarithmic growth phase prepared in Example 1 were digested with trypsin, and the cell suspension concentration was adjusted to 1 × 10⁻⁶. 4 Cells / mL were seeded at 100 μL into 96-well culture plates and cultured in DMEM / F12 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin in a cell culture incubator at 37°C and 5% CO2. After cell adhesion, the above-mentioned peptides were added, and after 4 days of culture, the culture medium was discarded, and the total number of BMSCs was determined. Cell counting: After discarding the culture medium, the cells were washed twice with PBS, fixed at room temperature for 20 min, stained with 0.1% crystal violet solution for 10 min, and observed and photographed under a high-power microscope.

[0038] Table 2. Effects of recombinant peptides on the proliferation of bone marrow mesenchymal stem cells.

[0039] Group Bone marrow mesenchymal stem cell proliferation rate Blank control group 100％ KYR-1 25 μg / mL (104.51±3.9)％ 50 μg / mL KYR-1 (111.46±2.7)％ 100 μg / mL KYR-1 (126.82±2.4)％ 200 μg / mL KYR-1 (121.26±1.8)％ KYR-2 25 μg / mL (109.26±4.1)％ 50 μg / mL KYR-2 (116.58±3.2)％ 100 μg / mL KYR-2 (136.74±3.5)％ 200 μg / mL KYR-2 (131.37±3.9)％ KYR-3 25 μg / mL (134.62±4.8)％ 50 μg / mL KYR-3 (149.57±6.7)％ 100 μg / mL KYR-3 (171.3±5.3)％ 200 μg / mL KYR-3 (162.6±5.8)％

[0040] The results are shown in Figure 2. Compared with the blank control group, the proliferation rates of bone marrow mesenchymal stem cells (BMSCs) in the experimental groups (25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL) were significantly increased. Among them, 100 μg / mL KYR-3 showed the most significant effect in promoting BMSC proliferation, with a BMSC proliferation rate of (171.3±5.3)%. This indicates that the recombinant polypeptide KRSRGGSGGSRGDGSYIGSR of the present invention has a significant effect on promoting the proliferation of bone marrow mesenchymal stem cells. In conclusion, the culture medium supplemented with KYR-3 can better promote the proliferation activity of bone marrow mesenchymal stem cells.

[0041] Experimental Example 2

[0042] Effects of recombinant peptides on Ki67 gene expression in bone marrow mesenchymal stem cells:

[0043] A blank control group and experimental groups were set up with KYR-1, KYR-2, and KYR-3 at concentrations of 100 μg / mL and 200 μg / mL, respectively, with three replicates for each concentration. Bone marrow mesenchymal stem cells in the logarithmic growth phase prepared in Example 1 were digested with trypsin, and the cell suspension concentration was adjusted to 1 × 10⁻⁶. 4Cells were seeded at a density of 100 μL / mL into 96-well culture plates and cultured in DMEM / F12 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37°C, 5% CO2 cell culture incubator. After cell adhesion, the aforementioned peptides were added. When the cell density reached 60%, bone marrow mesenchymal stem cells were collected, and total mRNA was extracted using the RNeasy Mini kit. RNA samples were reverse transcribed using a reverse transcriptase kit. The expression level of the Ki67 gene in BMSCs in the blank control group and the recombinant peptide experimental group was detected by real-time quantitative PCR.

[0044] (1) Primer design for Ki67 gene and internal reference gene β-actin

[0045] Primers were designed based on the Ki67 gene. The upstream primer sequence of the Ki67 gene primer pair is shown in SEQ ID NO.4, and the downstream primer sequence is shown in SEQ ID NO.5. Using β-actin as an internal reference gene, the target gene Ki67 was normalized. The upstream primer sequence of the primer pair for amplifying the internal reference gene β-actin is shown in SEQ ID NO.6, and the downstream primer sequence is shown in SEQ ID NO.7.

[0046] Table 3 Sequence List

[0047] SEQ ID NO.4 Ki67-F 5'-CACCGGACAGTGGACACAA-3' SEQ ID NO.5 Ki67-R 5'-GTGGGATACCTCGTTGGGTAGTC-3' SEQ ID NO.6 β-actin-F 5'-AGAGATGGCCACGGCTGCTT-3' SEQ ID NO.7 β-actin-R 5'-ATTTCGCCTGGACGTGGAG-3'

[0048] (2) Real-time PCR reaction procedure

[0049] Using the SYB Green method and primers for the Ki67 gene and β-actin internal reference gene from step (1), a qPCR reaction was performed on a real-time PCR instrument to amplify the cDNA sequence corresponding to the Ki67 gene. The real-time PCR reaction system is shown in Table 4. Using cDNA as a template, upstream and downstream primers for the Ki67 gene, SYB Green dye, and Water Nuclease-Free were added and mixed well to prepare the reaction system. The PCR reaction program for the real-time PCR instrument was set to amplify the cDNA sequence corresponding to the Ki67 gene. The real-time PCR reaction program is shown in Table 5. Each sample was divided into three replicates. The relative expression level of Ki67 was calculated using the Ct method, with the formula Ct = Ct. 目的基因 -Ct β-actin Statistical analysis of the quantitative fluorescence data was performed using SPSS statistical analysis software. The relative expression levels of the Ki67 gene were plotted using Origin graphing software to create bar charts.

[0050] Table 4. Real-time PCR reaction system

[0051]

[0052]

[0053] Table 5. Quantitative PCR reaction procedure

[0054]

[0055] The results are as follows Figure 2 As shown, compared with the blank control group, the expression level of Ki67 gene in bone marrow mesenchymal stem cells (BMSCs) at concentrations of 100 μg / mL, 200 μg / mL, and KYR-3 in the experimental group was significantly increased. Among them, the highest relative expression level of Ki67 gene in BMSCs was achieved at a KYR-3 concentration of 100 μg / mL (2.34 ± 0.24). This indicates that the recombinant polypeptide KRSRGGSGGSRGDGSYIGSR of the present invention has a significant effect on promoting Ki67 gene expression in BMSCs. In conclusion, the culture medium supplemented with KYR-3 can better promote Ki67 gene expression in bone marrow mesenchymal stem cells.

[0056] Experimental Example 3

[0057] The effect of recombinant peptides on the aging of bone marrow mesenchymal stem cells:

[0058] A blank control group and experimental groups of KYR-1, KYR-2, and KYR-3 at concentrations of 100 μg / mL and 200 μg / mL were set up, with three replicates for each concentration. Passage 20 BMSCs obtained from conventional culture were digested with trypsin at a concentration of 2 × 10⁻⁶. 4 Cells were seeded in 96-well plates and cultured in DMEM / F12 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin, incubated at 37°C in a 5% CO2 cell culture incubator. After cell attachment, the aforementioned peptides were added to the culture plates. After 7 days of culture, the senescence status of cells was observed using a cell senescence β-galactosidase staining kit. Different fields of view were randomly selected, with 300 cells per well, and the proportion of senescent cells was calculated. The β-galactosidase positive cell rate was further calculated. The cell senescence β-galactosidase staining kit is based on the upregulation of β-galactosidase activity levels during senescence, and is used to stain and detect senescent cells or tissues. Cell or tissue senescence can be observed under a regular optical microscope; positive cells appear blue-green.

[0059] The results are as follows Figure 3As shown, compared with the blank control group, the experimental group showed a significant decrease in the β-gal positive cell rate in bone marrow mesenchymal stem cells (BMSCs) at concentrations of 100 μg / mL, KYR-2, and KYR-3, and concentrations of 200 μg / mL. The inhibitory effect on BMSC senescence was most pronounced at a KYR-3 concentration of 100 μg / mL, with a β-gal positive cell rate of (32.14 ± 0.24)%. This indicates that the recombinant polypeptide KRSRGGSGGSRGDGSYIGSR of this invention has a significant inhibitory effect on the replicative senescence of BMSCs. In conclusion, culture medium supplemented with KYR-3 can better inhibit the replicative senescence of BMSCs, and the optimal concentration of KYR-3 in the culture medium is 100 μg / mL.

[0060] 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 recombinant polypeptide for enhancing the activity of bone marrow mesenchymal stem cells, characterized in that, The amino acid sequence of the recombinant polypeptide is shown in SEQ ID NO.

3.

2. The application of the recombinant polypeptide for enhancing the activity of bone marrow mesenchymal stem cells as described in claim 1, characterized in that, The recombinant polypeptide is used to prepare a culture medium for bone marrow mesenchymal stem cells.

3. The application according to claim 2, characterized in that, The bone marrow mesenchymal stem cells mentioned are P2-P5 generation bone marrow mesenchymal stem cells.

4. The application according to claim 2, characterized in that, The concentration of the recombinant polypeptide in the culture medium is 25-200 μg / mL.

5. The application according to claim 4, characterized in that, The concentration of the recombinant polypeptide in the culture medium was 100 μg / mL.

6. The application according to claim 2, characterized in that, The bone marrow mesenchymal stem cells were cultured at 37°C and 5% CO2.

7. The application according to claim 2, characterized in that, The seeding density of the bone marrow mesenchymal stem cells is 1-4 × 10⁻⁴. 4 per mL.

Images