Fusion type polypeptide for improving activity of umbilical cord mesenchymal stem cells and application of fusion type polypeptide

Abstract

The invention belongs to the technical field of biology, and discloses a fusion type polypeptide for improving the activity of umbilical cord mesenchymal stem cells and application of the fusion type polypeptide. The amino acid sequence of the fusion type polypeptide provided by the invention is any one sequence as shown in SEQ ID NO.1-3. The fusion type polypeptide is obtained by integrating an extracellular matrix amino acid sequence and a heparin amino acid sequence. The umbilical cord mesenchymal stem cells obtained by culturing the fusion type polypeptide have good multiplication capacity, can promote expression of the Ki67 gene and inhibit aging of the umbilical cord mesenchymal stem cells, and have wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a fusion polypeptide for enhancing the activity of umbilical cord mesenchymal stem cells and its application. Background Art

[0002] Mesenchymal stem cells include bone marrow, umbilical cord, and adipose-derived mesenchymal stem cells, which have a certain self-renewal ability, can be amplified in large numbers, and can also senesce and apoptose. Human umbilical cord mesenchymal stem cells are a type of stem cells isolated from human umbilical cord tissue, which have strong differentiation potential and can differentiate into various tissue cells such as nerve, muscle, liver, and myocardium under specific induction conditions. Since human umbilical cord mesenchymal stem cells have low immunogenicity and can reduce immune rejection reactions, they have broad application prospects in basic research and clinical applications.

[0003] In order to meet the needs of clinical experiments, scientific researchers generally use in vitro amplification and culture techniques to obtain sufficient umbilical cord mesenchymal stem cells. However, there are some problems with existing amplification and culture methods. For example, the components of traditional culture media may not fully meet the growth requirements of human umbilical cord mesenchymal stem cells, resulting in slow cell proliferation rate and reduced cell activity. In addition, with the increase in the number of cell passages, the senescence and apoptosis of human umbilical cord mesenchymal stem cells gradually intensify, which further limits their effects in clinical applications.

[0004] To improve these problems, researchers add bioactive polypeptides to the culture medium to promote the proliferation and differentiation of human umbilical cord mesenchymal stem cells. Peptide is a bioactive substance involved in various cell functions in vivo, formed by dehydration of amino acids, and contains carboxyl and amino groups. Recombinant polypeptides have extensive potential value and applications in many fields. They can not only be used for the production of enzymes, antibodies, and hormones, but also effectively promote the proliferation and directed differentiation of mesenchymal stem cells. Therefore, we can advance the application of peptides in improving the quality of stem cells and tissue regeneration in clinical practice by further studying the functions of polypeptides.

[0005] RGD integrin-binding peptide is a short peptide sequence derived from the extracellular matrix, which has the ability to specifically bind to integrin receptors and can regulate various functions of cells. 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 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 polypeptide to enhance the activity of umbilical cord mesenchymal stem cells. Summary of the Invention

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

[0008] In order to achieve the above objective, the technical solution adopted by the present invention is:

[0009] A fusion polypeptide for improving 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] Another objective of the present invention is to provide an application of a fusion polypeptide for improving the activity of umbilical cord mesenchymal stem cells.

[0011] In order to achieve the above objective, the technical solution adopted by the present invention is:

[0012] An application of a fusion polypeptide for improving the activity of umbilical cord mesenchymal stem cells, which is used for preparing a culture medium 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 polypeptide in the culture medium is 200 μg / mL.

[0015] Preferably, the culture medium further comprises 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 4 cells / mL.

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

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

[0020] In the present invention, the extracellular matrix amino acid sequence and the heparin amino acid sequence are integrated, and the fusion polypeptide of the present invention is prepared by a solid-phase synthesis method. The fusion polypeptide of the present invention can promote the expression of the Ki67 gene, improve the proliferation activity of umbilical cord mesenchymal stem cells, and at the same time has the effect of inhibiting the senescence of umbilical cord mesenchymal stem cells, and has broad application prospects. Description of the Drawings

[0021] Figure 1 It is a morphological diagram of P3 generation umbilical cord mesenchymal stem cells;

[0022] Figure 2 Schematic diagram of the positive rate of β-galactosidase in umbilical cord mesenchymal stem cells under different culture conditions;

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

[0024] Figure 4 Schematic diagram of the relative expression level of Ki67 gene in umbilical cord mesenchymal stem cells under different culture conditions. Detailed implementation manners

[0025] Next, in combination with the accompanying drawings and specific implementation manners, the present invention will be further described. It should be noted that, on the premise of no conflict, the following-described embodiments or technical features can be arbitrarily combined to form new embodiments. The specific conditions not specified in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, 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, strip the Wharton's jelly from the umbilical cord tissue, cut the Wharton's jelly into tissue blocks, wash three times with physiological saline, take the tissue blocks and place them in DMEM medium supplemented with 10% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL) in a 37 °C, 5% CO2 cell culture incubator for culture, and change the medium by half every 2 days;

[0029] (2) Observe that umbilical cord mesenchymal stem cells appear around the tissue blocks, and perform medium change treatment, which is recorded as the P0 generation;

[0030] (3) When the cell confluence reaches 80%, discard the umbilical cord tissue blocks and culture medium, add 0.25% trypsin solution for digestion, centrifuge to remove the digestion solution, and then resuspend with DMEM medium for subculture to obtain P3 generation umbilical cord mesenchymal stem cells.

[0031] Use a microscope to observe the morphology of the obtained P3 generation umbilical cord mesenchymal stem cells. The results are as Figure 1 shown. It can be seen from Figure 1 that the cells are spindle-shaped and the cell sizes are uniform, which conforms to the typical characteristics of umbilical cord mesenchymal stem cells. That is, the present invention successfully obtains umbilical cord mesenchymal stem cells.

[0032] Example 2

[0033] Synthesis of the fusion polypeptide:

[0034] By integrating the extracellular matrix amino acid sequence and the heparin amino acid sequence, the fusion polypeptides A, B, and C of the present invention are obtained. The specific steps of the synthesis process of the fusion polypeptides of the present invention are as follows:

[0035] (1) Accurately weigh 200 mg of Wang resin, place it in the solid-phase synthesis tube of a SYMPHONY type 12-channel polypeptide synthesizer, add 5 mL of solvent DMF, and 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 resin, laying the foundation for peptide chain synthesis.

[0036] (2) Weigh another 1 mmol of the second and subsequent amino acid monomers with protecting groups and put them in a bottle. At 25 °C, according to the amino acid sequences in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, from the C-terminus to the N-terminus, the Fmoc deprotection, activation, and coupling reactions are completed in the synthesizer, and then the next cycle is carried out until all the amino acid monomers are completely coupled. After completing the coupling of the entire amino acid sequence, the polypeptide is cleaved from the resin to obtain a crude fusion polypeptide with side-chain protecting groups.

[0037] (3) The obtained crude fusion polypeptide is separated and purified by a semi-preparative high-performance liquid chromatograph to remove unreacted raw materials and by-products. The purified fusion polypeptide is stored frozen at -80 °C, and then freeze-dried by a freeze dryer to obtain a dry high-purity fusion polypeptide product. The specific amino acid sequences of the fusion polypeptides obtained in the present 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] Experimental Example 1

[0042] Effect of the fusion polypeptide on the proliferation of umbilical cord mesenchymal stem cells:

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

[0044] Take the P3 generation of umbilical cord mesenchymal stem cells in the logarithmic growth phase prepared in Example 1, digest them with trypsin, and adjust the cell suspension concentration to 5×10 4 cells / mL. Pipette 100 μL and inoculate it into a 96-well culture plate, and culture it in DMEM medium containing 10% FBS, 100 U / mL of penicillin, and 100 μg / mL of streptomycin. After culturing overnight, add the above-mentioned culture media for each group. After the addition is completed, place the cells in a cell culture incubator at 37°C and 5% CO2 for 3 days. After the culture is completed, add 10 μL of CCK-8 reagent to each well. After incubating for 4 h, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance of each group of cells at 450 nm. The results are shown in Table 1.

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

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

[0047] Table 1 shows the results of the proliferation of umbilical cord mesenchymal stem cells after culturing with 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 polypeptide A, fusion polypeptide B, and fusion polypeptide C had a significant effect on the proliferation of umbilical cord mesenchymal stem cells. Among them, the culture medium added with 200 μg / mL of fusion polypeptide A promoted the proliferation of umbilical cord mesenchymal stem cells most significantly, and the OD value was (0.607 ± 0.031).

[0048] Test Example 2

[0049] Effect of fusion polypeptide on the senescence of umbilical cord mesenchymal stem cells:

[0050] (1) Add the three polypeptides obtained in Example 2 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 of fusion polypeptide A, fusion polypeptide B, and fusion polypeptide C respectively as experimental groups; the blank control is DMEM medium containing 10% FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL) without adding polypeptides.

[0051] (2) Take the 9th generation of umbilical cord mesenchymal stem cells obtained by conventional culture, digest them with trypsin, and adjust the cell suspension concentration to 6×10 4 cells / mL. Pipette 100 μL and inoculate it into a 96-well culture plate, and culture it in DMEM medium containing 10% FBS, 100 U / mL of penicillin, and 100 μg / mL of streptomycin. When the cell density reaches 80 - 90%, add the cell culture media of each group in step (1) to treat the cells. Each group has 3 replicates. After adding, place the cells in a 37°C, 5% CO2 cell culture incubator for culture.

[0052] (3) After culturing the umbilical cord mesenchymal stem cells for 2 days, wash them with PBS, add 1 mL of cell fixative 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 them at 37°C for 10 h.

[0054] (5) After the incubation is completed, remove the staining solution, count the stained cells under an optical microscope, randomly select different fields of view, count 200 cells per well, and calculate the positive rate of β-lactosidase. The results are as Figure 2 shown.

[0055] Figure 2 The positive rate of β-galactosidase in umbilical cord mesenchymal stem cells after culturing with different media. Compared with the blank control group, after culturing with media containing 100 μg / mL, 200 μg / mL, and 400 μg / mL of fusion polypeptide A, fusion polypeptide B, and fusion polypeptide C respectively, the positive rate of β-galactosidase in umbilical cord mesenchymal stem cells decreased significantly. Among them, the medium containing 200 μg / mL of fusion polypeptide A can significantly reduce the positive rate of β-galactosidase in umbilical cord mesenchymal stem cells.

[0056] Experimental Example 3

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

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

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

[0060] (3) The umbilical cord mesenchymal stem cells obtained after culture in each group were collected, and total cellular mRNA was extracted using the RNeasy Mini kit, and the RNA samples were reverse transcribed into cDNA using a reverse transcriptase kit.

[0061] (4) Real-time fluorescence quantitative PCR technology was used to detect the expression levels of the Ki67 gene in umbilical cord mesenchymal stem cells obtained in the blank control group and each experimental group. The validated housekeeping gene β-actin was used as an internal reference gene, and the 2 -△△CT method was used to calculate the relative expression levels of the genes. The primer sequences are shown in Table 2, and the results of the analysis of the influence of the expression differences at the protein level are as Figure 3 shown, and the relative expression levels of the Ki67 gene are as Figure 4 shown.

[0062] Table 2

[0063]

[0064] Figure 3 is a result analysis chart of the influence of Ki67 protein expression under different culture conditions. Compared with the blank control group, the media supplemented with 200 μg / mL of fusion polypeptide A, fusion polypeptide B, and fusion polypeptide C could promote the expression of Ki67 protein in umbilical cord mesenchymal stem cells. Among them, the medium supplemented with 200 μg / mL of fusion polypeptide A had a more obvious effect on promoting the expression of Ki67 protein in umbilical cord mesenchymal stem cells.

[0065] Figure 4It is a result analysis chart of the relative expression levels of the Ki67 gene under different culture conditions. Compared with the blank control group, the culture media supplemented with 200 μg / mL of fusion polypeptide A, fusion polypeptide B, and fusion polypeptide C can promote 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 polypeptide A has the highest relative expression level of the Ki67 gene in umbilical cord mesenchymal stem cells, which is 19.02 ± 0.39. This indicates that the synthesized fusion polypeptides A-C of the present invention have 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 synthesized fusion polypeptides A-C of the present invention can better promote the proliferation ability of umbilical cord mesenchymal stem cells, inhibit the senescence of umbilical cord mesenchymal stem cells, and promote the expression level of the Ki67 gene in umbilical cord mesenchymal stem cells. The optimal choice is when the concentration of fusion polypeptide A in the culture medium is 200 μg / mL.

[0067] The above embodiments are only the preferred embodiments of the present invention and cannot be used to limit 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 fall within the scope of protection required 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. Use of the fusion polypeptide for enhancing the activity of umbilical cord mesenchymal stem cells according to claim 1, characterized in that, A culture medium for preparing umbilical cord mesenchymal stem cells.

3. Use 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 polypeptide in the culture medium is 50-400 μg / mL.

4. Use 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 polypeptide in the culture medium is 200 μg / mL.

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

6. Use 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 passage P1-P5 umbilical cord mesenchymal stem cells.

7. Use 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 is 1 - 6×10 4 cells / mL.

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

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