Use of an active polypeptide in maintaining the stemness of umbilical cord mesenchymal stem cells

Abstract

The present application relates to the technical field of biology, and particularly relates to application of an active polypeptide in maintaining stemness of umbilical cord mesenchymal stem cells. The active polypeptide is PNC-27, and the amino acid sequence of the PNC-27 is shown as SEQ ID NO:1. The present application finds through experiments that the PCN-27 polypeptide can effectively promote proliferation of the umbilical cord mesenchymal stem cells by regulating expression of cyclin Cyclin-D1 and CDK1, and can effectively maintain stemness of the umbilical cord mesenchymal stem cells by promoting expression of the stemness gene Oct-4 protein in the umbilical cord mesenchymal stem cells. The present application fully proves through experiments that the PCN-27 polypeptide can effectively maintain stemness of the umbilical cord mesenchymal stem cells, and can stimulate potential application value of the umbilical cord mesenchymal stem cells in the fields of cell culture, biomedicine and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to the application of an active polypeptide in maintaining the stemness of umbilical cord mesenchymal stem cells. Background Technology

[0002] Stem cells are a special cell population with self-renewal, high proliferation, and multi-lineage differentiation potential. They can continuously generate daughter cells identical to themselves in specific microenvironments and can differentiate into almost all types of functional cells in the body under signal induction, thus providing a continuous source of cells for tissue development, homeostasis maintenance, and damage repair. However, embryonic stem cells are subject to ethical controversies, iPSCs pose tumorigenic risks, and difficulties in obtaining them and immune rejection limit their widespread application. Among them, umbilical cord mesenchymal stem cells (UC-MSCs) are a type of pluripotent stem cells. They are abundant, easy to collect and isolate, avoid ethical controversies, and possess strong proliferative capacity and excellent multi-lineage differentiation potential. Under suitable in vitro culture conditions, they can differentiate into various cell types such as bone, cartilage, fat, nerve, and cardiomyocytes, and have become highly promising seed cells in the fields of regenerative medicine, tissue engineering, and cell therapy. Furthermore, UC-MSCs possess significant immunomodulatory capabilities. They can effectively regulate the activity of immune cells and influence the immune system's response by secreting various soluble factors, such as IDO, PGE2, and IL-10, or through cell-cell interactions. This demonstrates unique advantages in treating autoimmune diseases and mitigating transplant rejection. Therefore, UC-MSCs have immeasurable value in both basic stem cell research and clinical translational applications.

[0003] Stem cell stemness refers to the ability of stem cells to maintain their undifferentiated state and differentiate into various cell types. Stem cell stemness primarily refers to the potential for high proliferation, multi-lineage differentiation, and self-renewal. It provides the body with cell renewal and repair capabilities, enabling the regeneration of tissues with specific morphology and function. Maintaining stem cell stemness not only ensures a sufficient quantity, functional homogeneity, and high quality of stem cells for tissue engineering applications, guaranteeing the activity and function of the constructed tissues, but also significantly enhances the therapeutic efficacy of stem cells in cell therapy while reducing costs. Therefore, maintaining the stemness of umbilical cord mesenchymal stem cells is particularly important.

[0004] However, existing technologies and methods still have certain limitations in maintaining the stemness of umbilical cord mesenchymal stem cells. During large-scale in vitro expansion, UC-MSCs inevitably face a slowdown in proliferation rate and a decrease in cell activity, leading to the loss of stemness of UC-MSCs.

[0005] Therefore, the need to effectively maintain the stemness of umbilical cord mesenchymal stem cells is urgent and of great significance. This can not only fully guarantee the efficacy of umbilical cord mesenchymal stem cells in cell therapy and clinical applications, but also promote their development in the medical field. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the present invention aims to provide an application of an active polypeptide in maintaining the stemness of umbilical cord mesenchymal stem cells. The present invention demonstrates that, within a certain range, the concentration of PCN-27 polypeptide is positively correlated with the proliferative activity of umbilical cord mesenchymal stem cells, and that it can effectively regulate the expression of cyclin-D1 and CDK1, and upregulate the protein expression of the stemness gene Oct-4.

[0007] To achieve the above applications, the objective of this invention is realized through the following technical solution:

[0008] The application of an active polypeptide in maintaining the stemness of umbilical cord mesenchymal stem cells, wherein the active polypeptide is PNC-27, and the amino acid sequence of PNC-27 is shown in SEQ ID NO: 1:

[0009] Pro-Pro-Leu-Ser-Gln-Glu-Thr-Phe-Ser-Asp-Leu-Trp-Lys-Leu-Leu-Lys-Lys-Trp-Lys-Met-Arg-Arg-Asn-Gln-Phe-Trp-Val-Lys-Val-Gln-Arg-Gly.

[0010] Furthermore, the application specifically involves culturing umbilical cord mesenchymal stem cells in a culture medium containing PCN-27.

[0011] Furthermore, the culture medium is DMEM / F12 medium.

[0012] Furthermore, the concentration of PNC-27 in the culture medium is 20-90 μg / mL.

[0013] Furthermore, the concentration of PNC-27 in the culture medium is 60 μg / mL.

[0014] Furthermore, the umbilical cord mesenchymal stem cells are P2-P6 generation cells.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] This invention provides the application of the PCN-27 peptide in maintaining the expression of stem cell genes in umbilical cord mesenchymal stem cells. Relevant experimental data demonstrate that the PCN-27 peptide can enhance the proliferative activity of umbilical cord mesenchymal stem cells by regulating cyclin expression, providing favorable conditions for their large-scale culture and application. Furthermore, this invention further demonstrates through Western blotting experiments that the PCN-27 peptide can upregulate the protein expression of the cell stem cell marker gene Oct-4 to maintain the stemness of umbilical cord mesenchymal stem cells, fully ensuring its application in cell therapy and clinical settings. Attached Figure Description

[0017] Figure 1 This is a morphological characteristic diagram of the P3 generation umbilical cord mesenchymal stem cells obtained in Example 1.

[0018] Figure 2 This image shows the effect of PCN-27 peptide on the proliferation of umbilical cord mesenchymal stem cells.

[0019] Figure 3 The figure shows the effect of PCN-27 peptide on cyclin expression in umbilical cord mesenchymal stem cells.

[0020] Figure 4 The figure shows the effect of PCN-27 peptide on the expression of stem gene proteins in umbilical cord mesenchymal stem cells. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. Various examples are provided by way of explanation and not by way of limitation. In fact, those skilled in the art will understand that modifications and variations can be made to the present invention without departing from its scope or spirit. Therefore, it is desirable that the present invention encompass such modifications and variations falling within the scope of the appended claims and their equivalents. Unless otherwise specified, the experimental methods used in the embodiments and comparative examples of the present invention are conventional methods, and the materials, reagents, etc., used are commercially available unless otherwise specified.

[0022] Example 1

[0023] Detecting the regulation of umbilical cord mesenchymal stem cell proliferation by PNC-27 peptide.

[0024] (1) Extraction and culture of umbilical cord mesenchymal stem cells: The umbilical cord tissue was rinsed with a mixture containing 1% penicillin and streptomycin, and the umbilical cord was cut into 1.5 mm pieces with sterilized surgical scissors. 3Tissue blocks of varying sizes were resuspended in 10% fetal bovine serum + DMEM / F12 medium and plated atop 10cm culture dishes. The cells were cultured at 37℃ under 5% CO2 conditions until 80% confluence. The original culture medium was discarded, and the cells were washed twice with PBS buffer. After digestion with trypsin containing 0.25% EDTA, primary umbilical cord mesenchymal stem cells were collected. The primary umbilical cord mesenchymal stem cells were resuspended in complete culture medium at a ratio of 1×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 10% fetal bovine serum and DMEM / F12 medium into T25 culture flasks. When the cell confluence reached 90% under a microscope, the cells were digested with trypsin containing 0.25% EDTA and passaged at a ratio of 1:2. P3 generation umbilical cord mesenchymal stem cells were taken and observed under a microscope to examine their morphological characteristics.

[0025] Figure 1 The image shows the morphology of P3 generation umbilical cord mesenchymal stem cells. It can be seen that P3 generation umbilical cord mesenchymal stem cells exhibit good growth morphology, with spindle-shaped cells and a high degree of consistency in overall morphology, which is consistent with the typical morphological characteristics of umbilical cord mesenchymal stem cells.

[0026] (2) CCK-8 assay for cell proliferation: The P3 generation umbilical cord mesenchymal stem cells obtained from step (1) were digested with 0.25% trypsin to adjust the density of umbilical cord mesenchymal stem cells to 1×10⁻⁶. 4 The concentration of PCN-27 peptide was seeded at 0 μg / mL into each well of a 96-well plate. DMEM / F12 medium containing PCN-27 peptide was added to each well for incubation. The final concentrations of PCN-27 peptide in DMEM / F12 medium were 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 90 μg / mL, respectively. Each well was incubated in groups with a final volume of 150 μL. Each group had three replicates. The plates were incubated at 37°C with 5% CO2 for 3 days. After incubation, 10 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for another 3.5 h. The absorbance at 450 nm was measured using a multi-mode microplate reader. The results are shown below. Figure 2 As shown.

[0027] Figure 2The graph shows the absorbance results of different concentrations of PCN-27 peptide on the proliferation of umbilical cord mesenchymal stem cells. As can be seen from the graph, the absorbance value reaches its maximum when the concentration of PCN-27 peptide in the culture medium is 60 μg / mL. With increasing concentration, the absorbance value does not significantly increase. The absorbance values ​​at concentrations of 20 μg / mL and 40 μg / mL are also higher than those at 0 μg / mL. This indicates that the absorbance value increases with increasing PCN-27 peptide concentration, and overall, the proliferation activity of umbilical cord mesenchymal stem cells shows a dose-dependent relationship with the PCN-27 peptide concentration. Therefore, the above experimental results show that PCN-27 peptide can effectively improve the proliferation capacity of umbilical cord mesenchymal stem cells, and that a concentration of 60 μg / mL in the culture medium represents the minimum suitable concentration for promoting umbilical cord mesenchymal stem cell proliferation.

[0028] Example 2

[0029] Detection of PNC-27 peptide on the expression of cell cycle-related proteins in umbilical cord mesenchymal stem cells

[0030] The P3 generation umbilical cord mesenchymal stem cells obtained in step (1) of Example 1 were seeded into a 6-well plate. When the cell confluence reached 85%, the umbilical cord mesenchymal stem cells were cultured in DMEM / F12 medium containing 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 90 μg / mL PCN-27 peptide, respectively. Five replicates were set for each group, and then the cells were placed in a 37°C biochemical incubator for continuous culture for 3 days.

[0031] Protein sample preparation: After culture, discard the original culture medium and wash the cells with PBS buffer to remove residual culture medium. Then, add 200 μL of protein lysis buffer to the cells and incubate at 4°C for 10 min to fully lyse the cells and extract total cell protein. Then, scrape the cells off with a cell scraper and collect the cell lysis buffer into a centrifuge tube to obtain total cell lysis buffer. Add protein loading buffer in a 5:1 ratio (the volume ratio of extracted protein to protein loading buffer is 5:1), and boil for 10 min to fully denature the protein sample. After cooling to room temperature, centrifuge at 12000g for 3 min and store at 4°C.

[0032] Sample loading: Then prepare a 10% SDS-PAGE gel, and place the denatured protein samples of the experimental group and the blank control group containing different concentrations of PCN-27 peptide into the SDS-PAGE gel for electrophoretic separation. The electrophoresis is stopped when the bromophenol blue reaches the bottom of the electrophoresis tank.

[0033] Transfer: Cut the SDS-PAGE gel to the appropriate size as needed, and then transfer the protein from the gel to the PVDF membrane using a wet transfer technique.

[0034] Blocking: The transferred PVDF membrane was washed three times with TBST (5 min each time), and then placed in 2% bovine serum albumin blocking solution and incubated at room temperature in the dark for 2 h. The membrane was then washed three times with TBST (5 min each time).

[0035] Antibody incubation and development: The membrane was placed in a solution containing primary antibodies against Cyclin-D1, CDK1, and β-actin and incubated overnight at 4°C. The primary antibodies were then removed, and the membrane was washed three times (5 min each) with TBST. The next day, the corresponding secondary antibodies were added, and the membrane was incubated at room temperature for 2 h with low-speed shaking. The membrane was then washed three times (5 min each) with TBST. Finally, the chemiluminescent reaction solution was coated onto the PVDF membrane, developed, and the experimental results were photographed and recorded.

[0036] Figure 3 The Western blot data clearly demonstrate that, compared to the blank control group without PCN-27 peptide treatment, umbilical cord mesenchymal stem cells treated with DMEM / F12 medium containing 60 μg / mL PCN-27 peptide exhibited significantly increased expression levels of cyclins (Cyclin-D1 and CDK1). This result not only confirms that PCN-27 peptide can promote the expression of Cyclin-D1 and CDK1 cyclins, but also further verifies the effectiveness of PCN-27 peptide in promoting the proliferation of umbilical cord mesenchymal stem cells, corroborating the experimental results obtained in Example 1.

[0037] Example 3

[0038] Detection of PNC-27 peptide-mediated expression of stem cell-related genes in umbilical cord mesenchymal stem cells

[0039] The P3 generation umbilical cord mesenchymal stem cells obtained in step (1) of Example 1 were seeded into a 6-well plate. When the cell confluence reached 85%, the experimental group of umbilical cord mesenchymal stem cells were cultured in DMEM / F12 medium containing 0 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 90 μg / mL PCN-27 peptide. Five replicates were set for each group, and then the cells were placed in a 37°C biochemical incubator for continuous culture for 3 days.

[0040] Protein sample preparation: After culture, discard the original culture medium and wash the cells with PBS buffer to remove residual culture medium. Then, add 200 μL of protein lysis buffer to the cells and incubate them at 4°C for 10 min to lyse the cells and extract the total cell protein. Then, scrape the cells off with a cell scraper and collect the cell lysis buffer into a centrifuge tube to obtain the total cell lysis buffer. Add protein loading buffer in a ratio of 5:1 (extracted protein: protein loading buffer). Then boil for 10 min to obtain the denatured protein sample. After cooling to room temperature, centrifuge at 12000g for 3 min and store at 4°C.

[0041] Sample loading: Then prepare a 10% SDS-PAGE gel, and place the protein samples treated in the experimental group and the blank control group into the SDS-PAGE gel for electrophoretic separation. The electrophoresis is stopped when the bromophenol blue reaches the bottom of the electrophoresis tank.

[0042] Transfer: Cut the SDS-PAGE gel to the appropriate size as needed, and then transfer the protein from the gel to the PVDF membrane using a wet transfer technique. Then wash the transferred PVDF membrane three times with TBST (5 min each time).

[0043] Blocking: Then place it in 2% bovine serum albumin blocking solution and incubate at room temperature in the dark for 2 hours. Wash the membrane 3 times with TBST (5 min each time).

[0044] Antibody incubation and development: The membrane was placed in a solution containing primary antibodies against Oct-4 protein and β-actin and incubated overnight at 4°C. The primary antibody was then removed, and the membrane was washed three times (5 min each) with TBST. The next day, the corresponding secondary antibody was added, and the membrane was incubated at room temperature for 2 h with low-speed shaking. The membrane was then washed three times (5 min each) with TBST. Finally, the chemiluminescent reaction solution was coated onto the PVDF membrane, developed, and the experimental results were photographed and recorded.

[0045] Western blot analysis can be used to detect the expression of stem cell marker genes and proteins in umbilical cord mesenchymal stem cells. Figure 4 The experimental results clearly show that the PCN-27 peptide provided by this invention has a significant effect on promoting the expression of Oct-4 protein. When the concentration of PCN-27 peptide is 60 μg / mL, the expression level of Oct-4 protein is significantly higher than that of the blank control group. That is to say, PCN-27 can effectively promote the protein expression of the stem marker gene Oct-4 at a concentration of 60 μg / mL.

[0046] In summary, this invention demonstrated through CCK-8 assays that PCN-27 peptide can promote the proliferation of umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells), and within a certain range (PCN-27 peptide concentration in culture medium: 20-90 μg / mL), the concentration of PCN-27 peptide and the proliferative activity of umbilical cord mesenchymal stem cells were positively correlated. Western blot experiments further explored its effect on cell cycle proteins, demonstrating that PCN-27 peptide can regulate the expression of cell cycle-related proteins Cyclin-D1 and CDK1. Furthermore, the results of western blot experiments showed that PCN-27 peptide can effectively regulate the expression of the stemness gene Oct-4, and that PCN-27 at a concentration of 60 μg / mL effectively promoted the expression of the stemness marker gene Oct-4. These results fully demonstrate that PCN-27 peptide can promote the proliferation of umbilical cord mesenchymal stem cells and effectively maintain their stemness, stimulating its potential application value in cell culture, biomedicine, and other fields.

[0047] The foregoing has described and evaluated some embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the present invention, still fall within the protection scope of the present invention.

Claims

1. The application of an active polypeptide in the preparation of a culture medium for maintaining the stemness of umbilical cord mesenchymal stem cells, characterized in that, The active polypeptide is PNC-27, and the amino acid sequence of PNC-27 is shown in SEQ ID NO: 1: Pro-Pro-Leu-Ser-Gln-Glu-Thr-Phe-Ser-Asp-Leu-Trp-Lys-Leu-Leu-Lys-Lys-Trp-Lys-Met-Arg-Arg-Asn-Gln-Phe-Trp-Val-Lys-Val-Gln-Arg-Gly; The concentration of PNC-27 in the culture medium is 20-90 μg / mL.

2. The application of the active polypeptide according to claim 1 in the preparation of a culture medium for maintaining the stemness of umbilical cord mesenchymal stem cells, characterized in that, The culture medium is DMEM / F12 medium.

3. The application of the active polypeptide according to claim 1 in the preparation of a culture medium for maintaining the stemness of umbilical cord mesenchymal stem cells, characterized in that, The concentration of PNC-27 in the culture medium was 60 μg / mL.

4. The application of the active polypeptide according to claim 1 in the preparation of a culture medium for maintaining the stemness of umbilical cord mesenchymal stem cells, characterized in that, The umbilical cord mesenchymal stem cells are P2-P6 generation cells.

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