Polypeptide for promoting proliferation of mesenchymal stem cells and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to polypeptide for promoting proliferation of mesenchymal stem cells and application of the polypeptide. The amino acid sequence of the polypeptide for promoting proliferation of the mesenchymal stem cells is shown as SEQ ID NO: 1. The active polypeptide for promoting proliferation of the umbilical cord mesenchymal stem cells is obtained by adopting a solid-phase synthesis method. Umbilical cord mesenchymal stem cells obtained by culturing the polypeptide have good adherence and multiplication capacities. In addition, the polypeptide can promote Nrf2 gene expression, so that proliferation of the umbilical cord mesenchymal stem cells is effectively promoted.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a polypeptide for promoting the proliferation of mesenchymal stem cells and an application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) have both strong multidirectional differentiation capabilities and strong immunosuppressive functions, and can secrete cytokines and immunomodulatory substances to regulate immune responses. In addition, mesenchymal stem cells have low immunogenicity and do not require strict allogeneic matching. Therefore, they have attracted much attention in the medical field. In particular, umbilical cord mesenchymal stem cells (UC-MSCs), derived from the umbilical cord tissue of newborns, are considered to be highly promising seed cells due to their easy access, rich yield and low immunogenicity. They show broad application prospects in tissue repair, treatment of immune diseases and regenerative medicine.

[0003] However, despite the many advantages of UC-MSCs, their low natural abundance has become a major bottleneck limiting their large-scale clinical application. To meet the needs of clinical trials, researchers generally use in vitro expansion culture techniques to obtain sufficient quantities of UC-MSCs. However, currently widely used in vitro expansion culture media still have many deficiencies in promoting the efficient proliferation of UC-MSCs, such as poor cell adhesion and low proliferation efficiency. These problems seriously affect the quality and quantity of cell growth, thereby limiting their further application in clinical and scientific research.

[0004] In response to the above challenges, scholars at home and abroad have actively explored various strategies to improve the in vitro expansion efficiency of UC-MSCs. Among them, adding active peptides to the culture medium as an emerging method has shown good application prospects. For example, Chinese patent CN106367384A discloses a velvet antler peptide isolated from deer antler, which has the effect of promoting the proliferation of adipose mesenchymal stem cells. Chinese patent CN114702548A discloses a polypeptide composition that promotes the growth of mesenchymal stem cells, which has the ability to maintain a high proliferation capacity of amniotic mesenchymal stem cells after multiple passages. These studies not only confirm the effectiveness of active peptides in promoting the proliferation of mesenchymal stem cells, but also provide a strong basis for the development and application of new active peptides.

[0005] KRSR heparin-binding peptide (HBP) is a peptide molecule that binds to heparin. It is widely present in the extracellular matrix and plays an important role in various biological processes. Binding of KRSR HBP to heparin may trigger a series of signaling processes, affecting cellular biological behavior. For example, it may regulate cell proliferation, migration, or apoptosis by activating or inhibiting specific signaling pathways.

[0006] RGD integrin-binding peptides are short peptide sequences derived from the extracellular matrix that 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 peptides to integrins can trigger a series of signaling events, influencing processes such as cell proliferation, apoptosis, and migration.

[0007] Based on the above background, the present invention provides a novel active polypeptide to enhance the proliferation ability of umbilical cord mesenchymal stem cells. Summary of the Invention

[0008] The first object of the present invention is to provide a polypeptide that promotes the proliferation of mesenchymal stem cells, wherein the polypeptide can promote the proliferation of umbilical cord mesenchymal stem cells.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] A polypeptide for promoting the proliferation of mesenchymal stem cells, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

[0011] The second purpose of the present invention is to provide an application of a polypeptide for promoting the proliferation of mesenchymal stem cells, which has broad application prospects.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] The invention discloses an application of a polypeptide for promoting the proliferation of mesenchymal stem cells, which is used for preparing a culture medium or a medicine for promoting the proliferation of mesenchymal stem cells.

[0014] Furthermore, based on the final concentration of the culture medium, the concentration of the polypeptide in the culture medium is 80-120 μg / mL.

[0015] Furthermore, the culture medium also includes DMEM basal culture medium and 1-5% FBS.

[0016] Furthermore, the drug is composed of the polypeptide and a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is a carrier, a diluent, or an excipient.

[0017] Furthermore, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0018] The third object of the present invention is to provide a culture method for promoting the proliferation of mesenchymal stem cells. The method is simple, easy to operate, and suitable for the culture of umbilical cord mesenchymal stem cells.

[0019] In order to achieve the above object, the technical solution adopted by the present invention is:

[0020] A culture method for promoting the proliferation of mesenchymal stem cells comprises the following steps:

[0021] Umbilical cord mesenchymal stem cells in the logarithmic growth phase are seeded in a culture medium and cultured for proliferation to obtain;

[0022] The culture medium includes DMEM basal medium, 1-5% FBS and the above-mentioned polypeptide. The concentration of the polypeptide is 80-120 μg / mL based on the final concentration of the culture medium.

[0023] Furthermore, the seeding density of the umbilical cord mesenchymal stem cells in the culture medium is 1-5×10 4 pieces / mL.

[0024] Furthermore, the umbilical cord mesenchymal stem cells are P2-P5 generation cells.

[0025] Furthermore, the culture is carried out at 37° C. and 5% CO 2 ; the culture time is 4-6 days.

[0026] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0027] The present invention successfully prepared an active polypeptide (named polypeptide 1, with the sequence KRSRGGSFFRGD) using a solid-phase synthesis method. This polypeptide can significantly promote the proliferation of umbilical cord mesenchymal stem cells. Experimental results showed that when the concentration of polypeptide 1 in the culture medium was 100 μg / mL, the adhesion rate of umbilical cord mesenchymal stem cells reached 94.16%, and the relative proliferation rate was as high as 133.41%. In other words, the polypeptide provided by the present invention can not only promote the adhesion of umbilical cord mesenchymal stem cells but also significantly increase their proliferation rate.

[0028] Furthermore, when the concentration of polypeptide 1 in the culture medium is 100 μg / mL, the polypeptide can also effectively promote the secretion of vascular endothelial growth factor, epidermal growth factor, and fibroblast growth factor by umbilical cord mesenchymal stem cells. These growth factors play a key role in cell repair, tissue regeneration and the treatment of various diseases, which is of far-reaching significance for the clinical application of umbilical cord mesenchymal stem cells.

[0029] In addition, in-depth studies have shown that polypeptide 1 effectively promotes the proliferation of umbilical cord mesenchymal stem cells by upregulating the expression of the Nrf2 gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a morphological diagram of umbilical cord mesenchymal stem cells according to Example 2 of the present invention;

[0031] Figure 2 This is an analysis of the effects of different culture conditions on Nrf2 protein expression;

[0032] Figure 3 This is an analysis chart of the expression level of Nrf2 mRNA under different culture conditions. DETAILED DESCRIPTION

[0033] The technical solution of the present invention will be further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only intended to illustrate the present invention and should not be construed as limiting the present invention. Specific conditions not specified in the examples are to be followed according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments used, unless otherwise specified, are conventional products obtained from commercial channels.

[0034] 1. Example

[0035] Example 1

[0036] Example 1 provides a polypeptide that promotes Nrf2 gene expression. The present invention prepares the polypeptide by solid-phase synthesis, and the polypeptide synthesis process is illustrated using a 0.25 mmol scale as an example. The specific process is as follows:

[0037] (1) Weigh 0.25 g of Wang resin and place it in a reactor on a SYMPHONY 12-channel peptide synthesizer. Add DMF solvent to the reactor so that the Wang resin is completely immersed in the DMF solvent and swell for 30 minutes;

[0038] (2) Weigh 1 mmol of diaminocarboxylic acid with a protected group (Fmoc-L-Lys(Fmoc)-OH) and place it in a reactor to allow the carboxyl group of the diaminocarboxylic acid with a protected group to bind to the amino group of the Wang resin. Then weigh 1 mmol of other amino acid monomers with protected groups and bottle them. According to the amino acid sequences in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, at 25°C, in the order from the C-terminus to the N-terminus of the target polypeptide amino acid sequence, the Fmoc protection is automatically removed, activated, and connected in a synthesizer controlled by a computer program. Then, the next cycle is carried out until all the amino acid monomers are connected to obtain a polypeptide resin with side chain protecting groups.

[0039] (3) Removal of protecting groups and cleavage of resin: The obtained polypeptide resin with side chain protecting groups is placed in a stoppered conical flask, and a cleavage reagent (consisting of 0.25 ml of water, 0.25 ml of ether, i.e., 1,2-diethanol, 1 mL of TIS, i.e., triisopropylsilane, and 9.45 mL of trihydroxymethylphenol) is added. The mixture is stirred at 30°C for 2 h, and the reaction liquid is filtered and the filtrate is collected. The resin is then washed with trifluoroacetic acid, and the collected liquid and the washing liquid are combined and then ether is added to produce a precipitate. The precipitate is filtered, washed with ether, and dried to obtain a crude product.

[0040] (4) The crude product was separated and purified by preparative high performance liquid chromatography (HPLC), and then freeze-dried to obtain the corresponding polypeptide. The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1-3, and the specific amino acid sequence is as follows:

[0041] KRSRGGSFFRGD (SEQ ID NO: 1, denoted as polypeptide 1);

[0042] KRSRGSGSFFRGD (SEQ ID NO: 2, denoted as polypeptide 2);

[0043] KRSRGGSGGSFFRGD (SEQ ID NO: 3, denoted as polypeptide 3).

[0044] Example 2

[0045] Example 2 provides an umbilical cord mesenchymal stem cell, and the specific process is as follows:

[0046] The umbilical cord tissue was washed with PBS buffer and cut into 2 mm pieces using surgical scissors. 3 Tissues of different sizes were digested with 0.1% collagenase II in a constant temperature shaker, and physiological saline was added. After mixing evenly, the cells were centrifuged at 2000 rpm for 15 min, and the supernatant was discarded to obtain the cell pellet. 4 The cell pellet was inoculated with DMEM medium (containing 5% FBS) at a seeding density of 100 μg / mL and cultured in a cell culture incubator at 37°C and 5% CO2 until the cell confluence reached 80%. When the cell confluence reached 80%, the cells were digested with 0.25% EDTA trypsin solution and passaged in a cell culture incubator at 37°C and 5% CO2 at a passage ratio of 1:2 to obtain P3 umbilical cord mesenchymal stem cells.

[0047] 2. Experimental Examples

[0048] Experimental Example 1

[0049] The morphology of the umbilical cord mesenchymal stem cells obtained in Example 2 was observed using a microscope. The results were as follows: Figure 1 shown.

[0050] Figure 1 This is a morphological diagram of umbilical cord mesenchymal stem cells according to Example 2 of the present invention. Figure 2 It can be seen that the cells are long spindle-shaped and uniform in size, which is consistent with the typical characteristics of umbilical cord mesenchymal stem cells. In other words, the present invention has obtained umbilical cord mesenchymal stem cells.

[0051] Experimental Example 2

[0052] The three polypeptides obtained in Example 1 were added to DMEM basal culture medium containing 5% FBS, respectively, to obtain final polypeptide concentrations of 50, 80, 100, 120, and 150 μg / mL culture medium, respectively.

[0053] The P3 umbilical cord mesenchymal stem cells obtained in Example 2 were resuspended in the above-mentioned culture medium and inoculated into T75 cell culture flasks at a cell density of 1×10 4 Cells were cultured at 400 nmol / mL in a 37°C, 5% CO2 incubator. After 9 hours of adherence, the cells were digested with TrypLE enzyme and counted. Adhesion efficiency = (number of adherent cells / total number of seeded cells) × 100%. The results are shown in Figure 1.

[0054] Table 1

[0055]

[0056]

[0057] Table 1 shows the results of the analysis of the adhesion rate of umbilical cord mesenchymal stem cells after culture in different culture media. As can be seen from Table 1, compared with the culture media supplemented with peptides 2-3, the culture media supplemented with peptide 1 can better promote the adhesion of umbilical cord mesenchymal stem cells.

[0058] Experimental Example 3

[0059] The three polypeptides obtained in Example 1 were added to DMEM basal culture medium containing 5% FBS, respectively, to obtain final polypeptide concentrations of 50, 80, 100, 120, and 150 μg / mL culture medium, respectively.

[0060] The P3 umbilical cord mesenchymal stem cells of Example 2 were grown in DMEM medium (containing 5% FBS) to the logarithmic phase, digested and diluted to form a cell suspension. 4 Cells were inoculated into the above culture medium at a concentration of 100 cells / mL and cultured in a cell culture incubator at 37°C and 5% CO2 for 5 days, with the culture medium replaced every 2 days. A control group was also set up, with the culture medium consisting of DMEM basal medium + 5% FBS.

[0061] (1) After the culture, the proliferation capacity of each group of cells was analyzed using the MTT assay. The relative proliferation rate was calculated as: experimental group / control group × 100%. The results are shown in Table 2.

[0062] (2) After the culture was completed, the supernatant of each group after centrifugation was collected after culture in a medium with a final polypeptide concentration of 100 μg / mL. The supernatant was dialyzed through a 50KD ultrafiltration membrane, and the dialyzate was taken and passed through a 100D ultrafiltration membrane. The retentate was collected and the content of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) secreted by the cells of each group was detected using an enzyme-linked immunosorbent assay (ELISA kit). The results are shown in Table 3.

[0063] Table 2

[0064]

[0065] Table 3

[0066]

[0067] Table 2 shows the relative proliferation rates of umbilical cord mesenchymal stem cells after culture in different culture media. As can be seen from Table 2, polypeptides 1-3 provided by the present invention are all capable of promoting the proliferation of umbilical cord mesenchymal stem cells. However, compared to culture media supplemented with polypeptides 2-3, culture media supplemented with polypeptide 1 is more capable of promoting the proliferation of umbilical cord mesenchymal stem cells.

[0068] Further observations revealed that the relative proliferation rate of umbilical cord mesenchymal stem cells gradually increased with increasing polypeptide dosage in the culture medium. When the concentration of polypeptide 1 in the culture medium was 100 μg / mL, the effect on promoting the proliferation of umbilical cord mesenchymal stem cells was optimal. When the concentration of polypeptide 1 in the culture medium exceeded 100 μg / mL, the proliferation of umbilical cord mesenchymal stem cells tended to decrease. Therefore, a concentration of 100 μg / mL of polypeptide 1 in the culture medium was the preferred choice in the present invention.

[0069] Table 3 shows the analysis of cytokine secretion by umbilical cord mesenchymal stem cells after culture in different culture media. As shown in Table 3, culture media supplemented with peptide 1 significantly enhanced the secretion of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) by umbilical cord mesenchymal stem cells compared to culture media supplemented with peptides 2-3. This has far-reaching implications for the clinical application of umbilical cord mesenchymal stem cells.

[0070] The following experimental examples illustrate the technical effects that can be achieved by the polypeptide of the present invention by using a concentration of 100 μg / mL of polypeptide 1 in the culture medium.

[0071] Experimental Example 4

[0072] The P3 umbilical cord mesenchymal stem cells of Example 2 were grown in DMEM medium (containing 5% FBS) to the logarithmic phase, digested and diluted to form a cell suspension.4 The concentration of each cell was inoculated into the culture medium, which consisted of DMEM culture medium, 100 μg / mL polypeptide 1, and 5% FBS added to the culture medium. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 4 hours, and the total cell mRNA was extracted using the RNeasy Mini kit. After treatment with DNAse I, the RNA sample was reverse transcribed using a reverse transcriptase kit. SYBRgreenPCR MasterMix (ABI) was used for qRT-PCR to detect the expression of Nrf2 mRNA. In addition, a control group was set up, and the culture medium consisted of DMEM basal culture medium + 5% FBS. The present invention uses the verified housekeeping gene GAPDH as the internal reference gene. And 2 -△△CT Methods The relative expression of genes was calculated. The primer sequences are shown in Table 4, and the protein expression differences are shown in Figure 2 The relative expression of Nrf2 mRNA is shown in Figure 3 shown.

[0073] Table 4

[0074]

[0075] Figure 2 This is the analysis of the effects of different culture conditions on Nrf2 protein expression. Figure 2 It can be seen that compared with the control group, the culture medium containing the polypeptide 1 of the present invention can promote the expression of Nrf2 protein.

[0076] Figure 3 This is the result analysis of the expression of Nrf2 mRNA under different culture conditions. Figure 3 It can be seen that compared with the control group, polypeptide 1 can promote the expression of Nrf2 mRNA.

[0077] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the same. The basic principles and main features of the present invention have been described above using specific implementation schemes. Modifications or substitutions may be made based on the present invention, but such modifications or substitutions do not detract from the essence of the corresponding technical solutions from the scope of protection claimed by the present invention.

Claims

1. A polypeptide for promoting the proliferation of mesenchymal stem cells, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The use of the polypeptide for promoting mesenchymal stem cell proliferation according to claim 1, characterized in that: Used for preparing culture medium or medicine for promoting proliferation of mesenchymal stem cells.

3. The use of the polypeptide for promoting mesenchymal stem cell proliferation according to claim 2, characterized in that: Based on the final concentration in the culture medium, the concentration of the polypeptide in the culture medium is 80-120 μg / mL.

4. The use of the polypeptide for promoting mesenchymal stem cell proliferation according to claim 2, characterized in that: The culture medium also includes DMEM basal medium and 1-5% FBS.

5. The use of the polypeptide for promoting mesenchymal stem cell proliferation according to claim 2, characterized in that: The drug is composed of the polypeptide and a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is a carrier, a diluent, and an excipient.

6. The use of the polypeptide for promoting mesenchymal stem cell proliferation according to claim 2, characterized in that: The mesenchymal stem cells are umbilical cord mesenchymal stem cells.

7. A method for promoting the proliferation of mesenchymal stem cells, characterized in that: The following steps are involved: Umbilical cord mesenchymal stem cells in the logarithmic growth phase are seeded in a culture medium and cultured for proliferation to obtain; The culture medium comprises DMEM basal medium, 1-5% FBS and the polypeptide according to claim 1, and the concentration of the polypeptide is 80-120 μg / mL based on the final concentration of the culture medium.

8. The method for promoting the proliferation of mesenchymal stem cells according to claim 7, wherein: The seeding density of the umbilical cord mesenchymal stem cells in the culture medium is 1-5×10 4 pieces / mL.

9. The method for promoting the proliferation of mesenchymal stem cells according to claim 7, wherein: The umbilical cord mesenchymal stem cells are P2-P5 generation cells.

10. The method for promoting the proliferation of mesenchymal stem cells according to claim 7, wherein: The culture is carried out at 37° C. and 5% CO 2 ; the culture time is 4-6 days.

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