Recombinant polypeptide for improving activity of bone marrow mesenchymal stem cells and application of recombinant polypeptide

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a recombinant polypeptide for improving the activity of bone marrow mesenchymal stem cells and application of the recombinant polypeptide. The amino acid sequence of the recombinant polypeptide is as shown in SEQ ID NO.3. The recombinant polypeptide is obtained by integrating an extracellular matrix amino acid sequence and a heparin amino acid sequence. The recombinant polypeptide provided by the invention can promote the expression of the Ki67 gene and improve the proliferative activity of the bone marrow mesenchymal stem cells, also has the effect of inhibiting the replicative aging of the bone marrow mesenchymal stem cells, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a recombinant polypeptide for improving the activity of bone marrow mesenchymal stem cells and its application. Background Art

[0002] Stem cells are a class of cells with unlimited or immortal self-renewal ability and have great potential in regenerative cell therapy. Bone marrow mesenchymal stem cells have currently become the focus of life science research in the 21st century. They have functions such as self-proliferation, multi-directional differentiation, repair of injuries, and immune regulation, and have broad application prospects and are expected to be used in cell therapy. In addition, bone marrow mesenchymal stem cells can also be used as a carrier to deliver biological agents to treat some diseases. Currently, bone marrow mesenchymal stem cells have made certain breakthroughs in heart diseases, liver injuries, spinal cord injuries, etc.

[0003] Peptide is a bioactive substance involved in various cellular functions in organisms. It is formed by dehydration of amino acids, contains carboxyl and amino groups, and has the characteristics of specificity, high stability, low production cost, and easy customization and immobilization. It also has the ability to enhance the adhesion, proliferation, and directed differentiation of stem cells. Currently, hundreds of peptides have been discovered in organisms and are essential participants in the body's various complex physiological activities. Peptides are involved in various fields such as hormones, nerves, cell growth, and reproduction, mainly for regulating various systems, organs, and cells in the body. With the development of genetic engineering technology, cloning the gene expressing the active peptide into some microorganisms or animals and directly expressing the required peptides through the organism can greatly increase their yield and purity. Recombinant polypeptides have extensive potential value and applications in many fields. They can not only be used to improve plant varieties, produce enzymes, antibodies, and hormones, but also effectively enhance the adhesion of stem cells, promote the proliferation and directed differentiation of mesenchymal stem cells, providing a strong basis for the development and application of new active polypeptides. Therefore, we can promote the clinical application of peptides in improving the quality of stem cells and tissue regeneration by further studying the functions of peptides.

[0004] RGD integrin-binding peptide and YIGSR integrin-binding peptide are short peptide sequences derived from the extracellular matrix and have the ability to specifically bind to integrin receptors. Integrin, as an important receptor on the cell membrane, regulates various functions of cells by activating signal transduction pathways. The binding of RGD peptide and YIGSR peptide to integrin 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 exists 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 signal pathways to regulate processes such as cell proliferation, migration, or apoptosis.

[0005] Due to the broad application prospects of bone marrow mesenchymal stem cells (BMSCs) in basic research and clinical applications, the number of directly isolated bone marrow mesenchymal stem cells is limited, which has become the main problem restricting their large-scale clinical applications. Summary of the Invention

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

[0007] Another objective of the present invention is to provide an application of a recombinant polypeptide that can improve the activity of bone marrow mesenchymal stem cells.

[0008] One of the objectives of the present invention is achieved by the following technical solution:

[0009] A recombinant polypeptide that can improve the activity of bone marrow mesenchymal stem cells, and the amino acid sequence of the recombinant polypeptide is as shown in SEQ ID NO.3.

[0010] Another objective of the present invention is achieved by the following technical solution:

[0011] An application of a recombinant polypeptide that can improve the activity of bone marrow mesenchymal stem cells, and the recombinant polypeptide is used in the culture medium of bone marrow mesenchymal stem cells.

[0012] Further, the bone marrow mesenchymal stem cells are passage 2-5 bone marrow mesenchymal stem cells.

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

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

[0015] Further, the culture of the bone marrow mesenchymal stem cells is carried out in an environment of 37°C and 5% CO2.

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

[0017] Compared with the prior art, the beneficial effects of the present invention mainly lie in: the present invention integrates the extracellular matrix amino acid sequence and the heparin amino acid sequence to obtain the recombinant polypeptide of the present invention. The recombinant polypeptide of the present invention can promote the expression of the Ki67 gene, improve the proliferation activity of bone marrow mesenchymal stem cells, and at the same time also has the effect of inhibiting the replicative senescence of bone marrow mesenchymal stem cells, and has broad application prospects. Brief Description of the Drawings

[0018] Figure 1 It is the morphological diagram of passage 3 bone marrow mesenchymal stem cells;

[0019] Figure 2 It is the result diagram of the influence of the recombinant polypeptide on the expression of Ki67 gene in bone marrow mesenchymal stem cells;

[0020] Figure 3 It is the result diagram of the influence of the recombinant polypeptide on the positive cell rate of β-gal in bone marrow mesenchymal stem cells. Specific implementation manners

[0021] The technical solution of the present invention will be further described below in combination with specific implementation manners. However, those skilled in the art should understand that the following examples are only used to illustrate the present invention and should not be regarded as a limitation to the present invention. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are all conventional products obtained through commercial channels.

[0022] Example 1

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

[0024] The bone marrow tissue was washed with PBS buffer and cut into pieces of 1 mm 3 size with surgical scissors. The bone marrow tissue was repeatedly pipetted into a bone marrow tissue suspension through a 24-gauge syringe. The obtained bone marrow tissue suspension was filtered through a 200-mesh cell sieve, centrifuged at 6000 g for 5 min, the supernatant was carefully aspirated, and the cell pellet was retained. After resuspending the cell pellet with DMEM / F12 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin, it was transferred to a culture dish and cultured under the conditions of 37 °C and 5% CO2. The culture medium was changed every 48 hours. After the cell confluence reached 80%, passage culture was carried out. The tissue mass and the culture medium were discarded, digested with 0.25% trypsin, the digestion solution was removed by centrifugation, and then resuspended with DMEM / F12 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin for passage culture to obtain passage 3 bone marrow mesenchymal stem cells (BMSCs). The morphology of the obtained bone marrow mesenchymal stem cells was observed under a microscope, and the results were as Figure 1 shown. The cell sizes were uniform, conforming to the typical characteristics of bone marrow mesenchymal stem cells. That is, bone marrow mesenchymal stem cells were obtained in the present invention.

[0025] Example 2

[0026] Synthesis of recombinant polypeptide:

[0027] Integrate the extracellular matrix amino acid sequence with the heparin amino acid sequence to obtain the recombinant polypeptide of the present invention. The synthesis process of the recombinant polypeptide is as follows:

[0028] (1) Polypeptide resin synthesis: Weigh Wang resin and place it in the reactor of a SYMPHONY type 12-channel polypeptide synthesizer.

[0029] (2) Binding of carboxyl group to resin: Weigh 1 mmol of each protected diamino carboxylic acid (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. Covalently bind the carboxyl group of the diamino carboxylic acid to Wang resin to lay the foundation for peptide chain synthesis.

[0030] (3) Synthesis of recombinant polypeptide: Weigh another 1 mmol of protected amino acid monomers and bottle them. According to the predetermined amino acid sequences (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3), automatically carry out Fmoc deprotection, activation, and connection from the C-terminus to the N-terminus in the synthesizer by a computer program, and then perform the next cycle until all amino acid monomers are completely connected to obtain a recombinant polypeptide with side chain protecting groups.

[0031] (4) Purification of recombinant polypeptide: Separate and purify the recombinant polypeptide with side chain protecting groups obtained in step (3) by high performance liquid chromatography, and then freeze-dry it to obtain three recombinant polypeptides named KYR-1, KYR-2, and KYR-3 respectively. The amino acid sequences of KYR-1, KYR-2, and KYR-3 are as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0032] Table 1 Sequence Listing

[0033]

[0034]

[0035] Experimental Example 1

[0036] Investigation on the proliferation effect of recombinant polypeptide on bone marrow mesenchymal stem cells:

[0037] Set up a blank control group and experimental groups of KYR-1, KYR-2, and KYR-3 at concentrations of 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, with 3 replicate wells for each concentration. Take the bone marrow mesenchymal stem cells in the logarithmic growth phase prepared in Example 1, digest them with trypsin, and adjust the cell suspension concentration to 1×10 4 cells / mL. Pipette 100 μL and inoculate it into a 96-well culture plate. Culture it 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 the cells adhered, add the above-mentioned polypeptides respectively. After culturing for 4 days, discard the culture medium and measure the total number of BMSCs cells. Cell counting: After discarding the culture medium, wash twice with PBS, fix at room temperature for 20 min, stain with 0.1% crystal violet solution for 10 minutes, and observe and take pictures under a high-power microscope.

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

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

[0040] The results are shown in Table 2. Compared with the blank control group, the proliferation rates of bone marrow mesenchymal stem cells of KYR-1, KYR-2, and KYR-3 at 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL in the experimental groups were significantly increased. Among them, KYR-3 at 100 μg / mL had the most obvious effect on promoting the proliferation of BMSCs, and the BMSCs proliferation rate was (171.3 ± 5.3)%. It shows that the recombinant polypeptide KRSRGGSGGSRGDGSYIGSR of the present invention has a significant effect on promoting the proliferation of bone marrow mesenchymal stem cells. In summary, the culture medium supplemented with KYR-3 can better promote the proliferation activity of bone marrow mesenchymal stem cells.

[0041] Test Example 2

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

[0043] Set up 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, with 3 replicate wells for each concentration. Take the bone marrow mesenchymal stem cells in the logarithmic growth phase prepared in Example 1, digest them with trypsin, and adjust the cell suspension concentration to 1×10 4Cells were inoculated at a density of 1×10⁵ cells / mL. 100 μL was aspirated and inoculated into a 96-well culture plate, 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 the cells adhered, the above-mentioned polypeptides were added respectively. When the cell density reached 60%, the bone marrow mesenchymal stem cells were collected. The total cellular mRNA was extracted using the RNeasy Mini kit, and the RNA samples were reverse transcribed using a reverse transcriptase kit. The expression levels of the Ki67 gene in the blank control group and the recombinant polypeptide experimental group of BMSCs were detected by real-time fluorescence quantitative PCR technology.

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

[0045] Primers were designed according to the Ki67 gene. The upstream primer sequence of the primer pair for the Ki67 gene is shown as SEQ ID NO.4, and the downstream primer sequence is shown as SEQ ID NO.5. Using β-actin as the internal reference gene to normalize the target gene Ki67, the upstream primer sequence of the primer pair for amplifying the internal reference gene β-actin is shown as SEQ ID NO.6, and the downstream primer sequence is shown as 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) Fluorescence quantitative PCR reaction procedure

[0049] Using the SYB Green method and the primers for the Ki67 gene and β-actin internal reference gene in step (1), qPCR reaction was carried out on a fluorescence quantitative PCR instrument to amplify the cDNA sequence corresponding to the Ki67 gene. The fluorescence quantitative PCR reaction system is shown in Table 4. Using cDNA as a template, the upstream and downstream primers of the Ki67 gene, SYBR Green dye, and Water Nuclease-Free were added respectively, and the reaction system was mixed and configured. The PCR reaction procedure was set in the fluorescence quantitative PCR instrument to amplify the cDNA sequence corresponding to the Ki67 gene. The fluorescence quantitative PCR reaction procedure is shown in Table 5. Each sample had 3 replicates, and the relative expression level of Ki67 was calculated using the Ct method. The formula is Ct = Ct 目的基因 -Ct β-actin . Statistical analysis of the fluorescence quantitative data was performed using SPSS statistical analysis software. The relative expression level of the Ki67 gene was achieved by plotting a bar chart using Origin plotting software.

[0050] Table 4 Fluorescence quantitative PCR reaction system

[0051]

[0052]

[0053] Table 5 Fluorescent quantitative PCR reaction program

[0054]

[0055] The results are as Figure 2 shown. Compared with the blank control group, the expression levels of Ki67 gene in bone marrow mesenchymal stem cells of KYR-1, KYR-2, and KYR-3 at the concentrations of 100 μg / mL and 200 μg / mL in the experimental group were significantly increased. Among them, when the concentration of KYR-3 was 100 μg / mL, the relative expression level of Ki67 gene in bone marrow mesenchymal stem cells was the highest, which was (2.34 ± 0.24). It shows that the recombinant polypeptide KRSRGGSGGSRGDGSYIGSR of the present invention has a significant effect on promoting the expression of Ki67 gene in BMSCs. In summary, the medium supplemented with KYR-3 can better promote the expression level of Ki67 gene in bone marrow mesenchymal stem cells.

[0056] Test Example 3

[0057] Effect of recombinant polypeptide on the senescence of bone marrow mesenchymal stem cells:

[0058] A blank control group and experimental groups of KYR-1, KYR-2, and KYR-3 at the concentrations of 100 μg / mL and 200 μg / mL were set up, with 3 replicate wells for each concentration. The 20th passage of BMSCs obtained by conventional culture was digested with trypsin and inoculated into a 96-well culture plate at 2×10 4 / well, 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 the cells adhered, the above-mentioned polypeptides were added to the culture plate respectively. After culturing for 7 days, a cell senescence β-galactosidase staining kit was used to observe the senescence of the cells. Different fields of view were randomly selected, 300 cells were counted for each well, and the proportion of senescent cells was calculated, and further the β-gal positive cell rate was calculated. The cell senescence β-galactosidase staining kit is a kit for staining and detecting senescent cells or tissues based on the up-regulation of the activity level of related β-galactosidase during senescence, and the senescence of cells or tissues can be observed under an ordinary optical microscope, and the positive cells are blue-green.

[0059] The results are as Figure 3As shown, compared with the blank control group, the percentage of β-gal positive cells in bone marrow mesenchymal stem cells of KYR-1, KYR-2, and KYR-3 at concentrations of 100 μg / mL and 200 μg / mL in the experimental group was significantly reduced. When the concentration of KYR-3 was 100 μg / mL, the effect of inhibiting the senescence of bone marrow mesenchymal stem cells was the most obvious, and the percentage of β-gal positive cells was (32.14±0.24)%. It shows that the recombinant polypeptide KRSRGGSGGSRGDGSYIGSR of the present invention has a significant effect of inhibiting the replicative senescence of bone marrow mesenchymal stem cells. In summary, the culture medium supplemented with KYR-3 can better inhibit the replicative senescence of bone marrow mesenchymal stem cells, and the optimal choice of the present invention is when the 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, rather than to limit them. The basic principles and main features of the present invention have been described in specific implementation manners above. On the basis of the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the present invention claimed.

Claims

1. A recombinant polypeptide for improving 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. An application of a recombinant polypeptide for improving the activity of bone marrow mesenchymal stem cells, characterized in that: The recombinant polypeptide is used in the culture medium of bone marrow mesenchymal stem cells.

3. The use of a recombinant polypeptide for improving the activity of bone marrow mesenchymal stem cells according to claim 2, characterized in that: The bone marrow mesenchymal stem cells are P2-P5 generation bone marrow mesenchymal stem cells.

4. The use of a recombinant polypeptide for improving the activity of bone marrow mesenchymal stem cells according to claim 2, characterized in that: The concentration of the recombinant polypeptide in the culture medium is 25-200 μg / mL.

5. The use of a recombinant polypeptide for improving the activity of bone marrow mesenchymal stem cells according to claim 4, characterized in that: The optimal concentration of the recombinant polypeptide in the culture medium is 100 μg / mL.

6. The use of a recombinant polypeptide for improving the activity of bone marrow mesenchymal stem cells according to claim 2, characterized in that: The bone marrow mesenchymal stem cells were cultured at 37° C. and 5% CO 2 .

7. The use of a recombinant polypeptide for improving the activity of bone marrow mesenchymal stem cells according to claim 2, characterized in that: The seeding density of the bone marrow mesenchymal stem cells is 1-4×10 4 Pieces / mL.

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