A fusion polypeptide for improving the activity of umbilical cord mesenchymal stem cells and its application

Abstract

This invention belongs to the field of biotechnology and discloses a fusion polypeptide for enhancing the activity of umbilical cord mesenchymal stem cells and its applications. The amino acid sequence of the fusion polypeptide provided by this invention is any one of the sequences shown in SEQ ID NO. 1-3. This invention obtains the fusion polypeptide by integrating the extracellular matrix amino acid sequence with the heparin amino acid sequence. Umbilical cord mesenchymal stem cells cultured using this fusion polypeptide exhibit good proliferative capacity, can promote Ki67 gene expression, and simultaneously inhibit aging of umbilical cord mesenchymal stem cells, showing broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a fusion polypeptide that enhances the activity of umbilical cord mesenchymal stem cells and its applications. Background Technology

[0002] Mesenchymal stem cells (MSCs) include those derived from bone marrow, umbilical cord, and adipose tissue. They possess a certain degree of self-renewal capacity, allowing for large-scale expansion as well as senescence and apoptosis. Human umbilical cord MSCs, isolated from human umbilical cord tissue, have strong differentiation potential and, under specific induction conditions, can differentiate into various tissue cells such as nerve, muscle, liver, and cardiomyocytes. Due to their low immunogenicity, human umbilical cord MSCs can reduce immune rejection responses, thus holding broad application prospects in both basic research and clinical applications.

[0003] To meet the needs of clinical trials, researchers commonly employ in vitro expansion culture techniques to obtain sufficient quantities of umbilical cord mesenchymal stem cells (UC-MSCs). However, existing expansion culture methods have several drawbacks. For example, traditional culture medium components may not adequately meet the growth requirements of human UC-MSCs, leading to slow cell proliferation and reduced cell viability. Furthermore, with increasing cell passage numbers, the aging and apoptosis of human UC-MSCs gradually intensify, further limiting their effectiveness in clinical applications.

[0004] To address these issues, researchers have promoted the proliferation and differentiation of human umbilical cord mesenchymal stem cells by adding bioactive peptides to the culture medium. Peptides are bioactive substances involved in various cellular functions within an organism; they are formed by the dehydration of amino acids and contain carboxyl and amino groups. Recombinant peptides have broad potential value and applications in many fields, not only for the production of enzymes, antibodies, and hormones, but also for effectively promoting the proliferation and directed differentiation of mesenchymal stem cells. Therefore, further research into the functions of peptides can advance their clinical applications in improving stem cell quality and tissue regeneration.

[0005] RGD integrin-binding peptides are short peptide sequences derived from the extracellular matrix that have the ability to specifically bind to integrin receptors and regulate various cellular functions. KRSR heparin-binding peptides are heparin-binding peptides that are widely distributed in the extracellular matrix and play important roles in various biological processes. The binding of heparin-binding peptides 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.

[0006] Based on the above background, the present invention provides a novel fusion peptide to enhance the activity of umbilical cord mesenchymal stem cells. Summary of the Invention

[0007] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a fusion polypeptide that enhances the activity of umbilical cord mesenchymal stem cells, which can promote the proliferation activity of umbilical cord mesenchymal stem cells.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A fusion polypeptide for enhancing the activity of umbilical cord mesenchymal stem cells, wherein the amino acid sequence of the fusion polypeptide is any one of the sequences shown in SEQ ID NO. 1-3.

[0010] The second objective of this invention is to provide an application of a fusion polypeptide that enhances the activity of umbilical cord mesenchymal stem cells.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] Application of a fusion peptide that enhances the activity of umbilical cord mesenchymal stem cells, used in the preparation of culture media for umbilical cord mesenchymal stem cells.

[0013] Preferably, the concentration of the fusion polypeptide in the culture medium is 50-400 μg / mL.

[0014] Preferably, the concentration of the fusion peptide in the culture medium is 200 μg / mL.

[0015] Preferably, the culture medium further includes DMEM basal medium and 5-10% FBS.

[0016] Preferably, the umbilical cord mesenchymal stem cells are P1-P5 generation umbilical cord mesenchymal stem cells.

[0017] Preferably, the seeding density of the umbilical cord mesenchymal stem cells in the culture medium is 1-6 × 10⁻⁶. 4 per mL.

[0018] Preferably, the culture conditions for the umbilical cord mesenchymal stem cells are: 37°C and 5% CO2.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention prepares a fusion polypeptide by integrating the amino acid sequence of the extracellular matrix with the amino acid sequence of heparin using a solid-phase synthesis method. The fusion polypeptide of this invention can promote Ki67 gene expression, enhance the proliferative activity of umbilical cord mesenchymal stem cells, and also inhibit the senescence of umbilical cord mesenchymal stem cells, showing broad application prospects. Attached Figure Description

[0021] Figure 1 Morphological diagram of P3 generation umbilical cord mesenchymal stem cells;

[0022] Figure 2 A schematic diagram showing the results of β-lactosidase positivity rate in umbilical cord mesenchymal stem cells under different culture conditions;

[0023] Figure 3 A schematic diagram showing the expression of Ki67 protein in umbilical cord mesenchymal stem cells under different culture conditions;

[0024] Figure 4 This is a schematic diagram showing the relative expression levels of the Ki67 gene in umbilical cord mesenchymal stem cells under different culture conditions. Detailed Implementation

[0025] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. 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.

[0026] Example 1

[0027] Preparation of umbilical cord mesenchymal stem cells:

[0028] (1) Wash the umbilical cord tissue with PBS, peel the Wharton jelly from the umbilical cord tissue, cut the Wharton jelly into tissue pieces, wash three times with physiological saline, and place the tissue pieces in DMEM medium supplemented with 10% FBS, penicillin (100U / mL) and streptomycin (100μg / mL) in a 37℃, 5% CO2 cell culture incubator and change half of the medium every 2 days;

[0029] (2) Observe that umbilical cord mesenchymal stem cells appear around the tissue block, change the medium, and record it as P0 generation;

[0030] (3) When the cell fusion rate reaches 80%, discard the umbilical cord tissue block and culture medium, add 0.25% trypsin solution for digestion, centrifuge to remove digestion solution, add DMEM culture medium for resuspending, and perform passage culture to obtain P3 generation umbilical cord mesenchymal stem cells.

[0031] The morphology of the obtained P3 generation umbilical cord mesenchymal stem cells was observed under a microscope, and the results are as follows: Figure 1 As shown, from Figure 1 As can be seen, the cells are elongated spindle-shaped and uniform in size, consistent with the typical characteristics of umbilical cord mesenchymal stem cells. Therefore, this invention has successfully obtained umbilical cord mesenchymal stem cells.

[0032] Example 2

[0033] Synthesis of fusion peptides:

[0034] This invention integrates the amino acid sequence of the extracellular matrix with the amino acid sequence of heparin to obtain fusion polypeptides A, B, and C. The specific steps of the synthesis process of the fusion polypeptides of this invention are as follows:

[0035] (1) Accurately weigh 200 mg of Wang's resin and place it in the solid phase synthesis tube of the SYMPHONY 12-channel peptide synthesizer. Add 5 mL of DMF solvent and allow it to swell for 20 min. Weigh 1 mmol of the first amino acid with a protecting group (Fmoc-L-Lys(Fmoc)-OH) and place it in the solid phase reaction tube so that the carboxyl group of the first amino acid covalently binds to the amino group of Wang's resin, laying the foundation for peptide chain synthesis.

[0036] (2) Weigh 1 mmol of the second and subsequent amino acid monomers with protecting groups and bottle them. At 25°C, according to the predetermined amino acid sequences in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, perform the de-Fmoc protection, activation, and ligation reactions from the C-terminus to the N-terminus in a synthesizer. Then, perform the next cycle until all amino acid monomers are ligated. After the ligation of the entire amino acid sequence is completed, cleave the peptide from the resin to obtain the crude fusion peptide with side-chain protecting groups.

[0037] (3) The obtained crude fusion peptide was separated and purified by a semi-preparative high-performance liquid chromatography (HPLC) system to remove unreacted raw materials and byproducts. The purified fusion peptide was then frozen at -80°C and freeze-dried to obtain a dried, high-purity fusion peptide product. The specific amino acid sequences of the fusion peptides obtained in this invention are shown in SEQ ID NO. 1-3:

[0038] KRSRGSGSIKLLLGSRGD (SEQ ID NO.1, denoted as polypeptide A);

[0039] KRSRGSGSIKLLL (SEQ ID NO.2, denoted as polypeptide B);

[0040] KRSRGSGSRGD (SEQ ID NO.3, denoted as polypeptide C).

[0041] Test Example 1

[0042] Effects of fusion peptides on the proliferation of umbilical cord mesenchymal stem cells:

[0043] The three polypeptides obtained in Example 2 were added to DMEM medium containing 10% FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL), respectively, to obtain cell culture media containing 50, 100, 200, and 400 μg / mL of fusion polypeptide A, fusion polypeptide B, and fusion polypeptide C, respectively, which served as the experimental groups; the blank control was DMEM medium containing 10% FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL) without the addition of polypeptides.

[0044] P3 generation umbilical cord mesenchymal stem cells in the logarithmic growth phase prepared in Example 1 were digested with trypsin, and the cell suspension concentration was adjusted to 5 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL / mL into 96-well culture plates and cultured in DMEM medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. After overnight culture, the above-mentioned medium was added. The cells were then cultured at 37°C in a 5% CO2 incubator for 3 days. After the incubation period, 10 μL of LCK-8 reagent was added to each well, and the cells were incubated for 4 hours. The absorbance at 450 nm was then measured using a microplate reader. The results are shown in Table 1.

[0045] Table 1. Effects of fusion peptides A, B, and C on the proliferation of umbilical cord mesenchymal stem cells.

[0046] Group 450nm absorbance Blank control group 0.343±0.025 50 μg / mL fusion peptide A 0.406±0.019 100 μg / mL fusion peptide A 0.548±0.027 200 μg / mL fusion peptide A 0.607±0.031 400 μg / mL fusion peptide A 0.562±0.035 50 μg / mL fusion peptide B 0.364±0.021 100 μg / mL fusion peptide B 0.452±0.032 200 μg / mL fusion peptide B 0.506±0.037 400 μg / mL fusion peptide B 0.472±0.024 50 μg / mL fusion peptide C 0.354±0.017 100 μg / mL fusion peptide C 0.438±0.019 200 μg / mL fusion peptide C 0.497±0.023 400 μg / mL fusion peptide C 0.462±0.028

[0047] Table 1 shows the proliferation results of umbilical cord mesenchymal stem cells after culture in different culture media. Compared with the blank control group, the culture media containing 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL of fusion peptide A, fusion peptide B, and fusion peptide C significantly affected the proliferation of umbilical cord mesenchymal stem cells. Among them, the culture medium supplemented with 200 μg / mL of fusion peptide A showed the most significant effect on promoting the proliferation of umbilical cord mesenchymal stem cells, with an OD value of (0.607±0.031).

[0048] Test Example 2

[0049] The effect of fusion peptides on the aging of umbilical cord mesenchymal stem cells:

[0050] (1) The three polypeptides obtained in Example 2 were added to DMEM medium containing 10% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL) respectively to obtain cell culture media containing 100, 200 and 400 μg / mL fusion polypeptide A, fusion polypeptide B and fusion polypeptide C respectively, which were used as experimental groups; the blank control was DMEM medium containing 10% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL) without the addition of polypeptides.

[0051] (2) Take the 9th generation umbilical cord mesenchymal stem cells obtained by conventional culture, digest them with trypsin, and adjust the cell suspension concentration to 6×10⁻⁶. 4 Cells / mL were seeded at a concentration of 100 μL into 96-well culture plates and cultured in DMEM medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. When the cell density reached 80-90%, the cell culture medium from step (1) was added to treat the cells. Each group was set up with 3 replicates. After the addition was completed, the cells were placed in a 37°C, 5% CO2 cell culture incubator.

[0052] (3) After culturing umbilical cord mesenchymal stem cells for 2 days, wash them with PBS, add 1 mL of cell fixation solution to each well, and fix them at room temperature for 20 min.

[0053] (4) Remove the fixative, gently wash the cells with PBS for 2 min each time, add 1 mL of β-galactosidase staining solution to each well, seal the wells with plastic wrap, and incubate at 37°C for 10 h.

[0054] (5) After incubation, the staining solution was removed, and the stained cells were counted under an optical microscope. Different fields of view were randomly selected, with 200 cells counted per well, and the β-lactosidase positivity rate was calculated. The results are as follows: Figure 2 As shown.

[0055] Figure 2 The study investigated the β-galactosidase positivity rate of umbilical cord mesenchymal stem cells (UCMSCs) after culture in different culture media. Compared with the blank control group, the β-galactosidase positivity rate of UCMSCs was significantly reduced after culture in media supplemented with 100 μg / mL, 200 μg / mL, and 400 μg / mL of fusion peptide A, fusion peptide B, and fusion peptide C, respectively. Among these, the culture medium supplemented with 200 μg / mL of fusion peptide A significantly reduced the β-galactosidase positivity rate of UCMSCs.

[0056] Experimental Example 3

[0057] Effect of fusion peptides on Ki67 gene expression in umbilical cord mesenchymal stem cells:

[0058] (1) The three polypeptides obtained in Example 2 were added to DMEM medium containing 10% FBS, penicillin (100U / mL) and streptomycin (100μg / mL) respectively to obtain cell culture medium containing 200μg / mL fusion polypeptide A, fusion polypeptide B and fusion polypeptide C respectively, which were used as experimental groups; a blank control group was set up, which was DMEM medium containing 10% FBS, penicillin (100U / mL) and streptomycin (100μg / mL) without polypeptides.

[0059] (2) P3 generation umbilical cord mesenchymal stem cells in the logarithmic growth phase prepared in Example 1 were seeded into 96-well plates, digested with trypsin, and the cell suspension concentration was adjusted to 2 × 10⁻⁶. 4 Cells / mL, 100 μL was seeded into a 96-well culture plate, and the culture medium of each group in step (1) was added. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 4 h.

[0060] (3) Collect the umbilical cord mesenchymal stem cells obtained after culture, extract total mRNA from the cells using the RNeasy Mini kit, and reverse transcribe the RNA samples using a reverse transcriptase kit to obtain cDNA.

[0061] (4) The expression level of the Ki67 gene in umbilical cord mesenchymal stem cells obtained from the blank control group and each experimental group was detected by real-time quantitative PCR. The validated housekeeping gene β-actin was used as an internal reference gene, and 2... -△△CT The relative expression levels of genes were calculated. Primer sequences are shown in Table 2, and differences in protein expression levels are shown in Table 3. Figure 3 As shown, the relative expression levels of the Ki67 gene are as follows: Figure 4 As shown.

[0062] Table 2

[0063]

[0064] Figure 3 This is a graph showing the results of the analysis of the effects of different culture conditions on Ki67 protein expression. Compared with the blank control group, the culture medium supplemented with 200 μg / mL of fusion peptide A, fusion peptide B, and fusion peptide C promoted Ki67 protein expression in umbilical cord mesenchymal stem cells. Among them, the culture medium supplemented with 200 μg / mL of fusion peptide A showed a more significant effect in promoting Ki67 protein expression in umbilical cord mesenchymal stem cells.

[0065] Figure 4This is a graph showing the analysis of the relative expression levels of the Ki67 gene under different culture conditions. Compared with the blank control group, the culture medium supplemented with 200 μg / mL of fusion peptide A, fusion peptide B, and fusion peptide C promoted the expression level of the Ki67 gene in umbilical cord mesenchymal stem cells. Among them, the culture medium supplemented with 200 μg / mL of fusion peptide A promoted the highest relative expression level of the Ki67 gene in umbilical cord mesenchymal stem cells, which was 19.02 ± 0.39. This indicates that the fusion peptide AC synthesized in this invention has a significant effect on promoting the expression of the Ki67 gene in umbilical cord mesenchymal stem cells.

[0066] In summary, the culture medium supplemented with the fusion polypeptide AC synthesized in this invention can better promote the proliferation of umbilical cord mesenchymal stem cells, inhibit the senescence of umbilical cord mesenchymal stem cells, and promote the expression level of Ki67 gene in umbilical cord mesenchymal stem cells. Among these, the optimal choice is when the concentration of fusion polypeptide A in the culture medium is 200 μg / mL.

[0067] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A fusion polypeptide for enhancing the activity of umbilical cord mesenchymal stem cells, characterized in that, The amino acid sequence of the fusion polypeptide is any one of the sequences shown in SEQ ID NO.1-3.

2. The application of the fusion polypeptide for enhancing the activity of umbilical cord mesenchymal stem cells as described in claim 1, characterized in that, Culture medium used to prepare umbilical cord mesenchymal stem cells.

3. The application of the fusion polypeptide for enhancing the activity of umbilical cord mesenchymal stem cells according to claim 2, characterized in that, The concentration of the fusion peptide in the culture medium is 50-400 μg / mL.

4. The application of the fusion polypeptide for enhancing the activity of umbilical cord mesenchymal stem cells according to claim 3, characterized in that, The concentration of the fusion peptide in the culture medium was 200 μg / mL.

5. The application of the fusion polypeptide for enhancing the activity of umbilical cord mesenchymal stem cells according to claim 2, characterized in that, The culture medium also includes DMEM basal medium and 5-10% FBS.

6. The application of the fusion polypeptide for enhancing the activity of umbilical cord mesenchymal stem cells according to claim 2, characterized in that, The umbilical cord mesenchymal stem cells are P1-P5 generation umbilical cord mesenchymal stem cells.

7. The application of the fusion polypeptide for enhancing the activity of umbilical cord mesenchymal stem cells according to claim 2, characterized in that, The seeding density of the umbilical cord mesenchymal stem cells in the culture medium was 1-6 × 10⁻⁶. 4 per mL.

8. The application of the fusion polypeptide for enhancing the activity of umbilical cord mesenchymal stem cells according to claim 2, characterized in that, The culture conditions for the umbilical cord mesenchymal stem cells were: 37°C and 5% CO2.

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