Polypeptide for promoting mesenchymal stem cell proliferation and application thereof

Abstract

The application belongs to the technical field of biology and particularly relates to a polypeptide for promoting mesenchymal stem cell proliferation and application thereof. The amino acid sequence of the polypeptide for promoting mesenchymal stem cell proliferation is shown as SEQ ID NO:1. The polypeptide for promoting umbilical cord mesenchymal stem cell proliferation is obtained by using a solid-phase synthesis method. The umbilical cord mesenchymal stem cell obtained by using the polypeptide for culture has good adhesion and proliferation capacity. In addition, the polypeptide can also promote Nrf2 gene expression, thereby effectively promoting the proliferation of umbilical cord mesenchymal stem cells.

Description

TECHNICAL FIELD

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

[0002] Mesenchymal stem cells (MSCs) have strong multi-directional differentiation ability and strong immunosuppressive function, and can also secrete cytokines and immunoregulatory substances to regulate immune response. In addition, mesenchymal stem cells have low immunogenicity and do not require strict matching of allogeneic cells. Therefore, they are highly concerned in the medical field. In particular, umbilical cord mesenchymal stem cells (UC-MSCs) are derived from the umbilical cord tissue of newborns and are considered as a potential seed cell due to their easy acquisition, high yield and low immunogenicity. They have shown a broad application prospect in tissue repair, treatment of immune diseases and regenerative medicine.

[0003] However, although UC-MSCs have many advantages, their low natural content is the main bottleneck limiting their large-scale clinical application. In order to meet the needs of clinical experiments, researchers generally use in vitro expansion culture technology to obtain sufficient UC-MSCs. However, the current widely used in vitro expansion culture medium still has many shortcomings in promoting the efficient proliferation of UC-MSCs, such as poor cell adhesion ability, low proliferation efficiency and other problems, which seriously affect the growth quality and quantity of cells, and further limit their further application in clinical and scientific research.

[0004] In view of the above challenges, scholars at home and abroad actively explore various strategies to improve the in vitro expansion efficiency of UC-MSCs. Among them, adding active polypeptides to the culture medium as a new method has shown good application prospects. For example, Chinese patent CN106367384A discloses a deer 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 for promoting the growth of mesenchymal stem cells, which enables amniotic mesenchymal stem cells after multiple passages to still maintain high proliferation capacity. These studies not only confirm the effectiveness of active polypeptides in promoting the proliferation of mesenchymal stem cells, but also provide a strong basis for the development and application of new active polypeptides.

[0005] KRSR heparin-binding peptide is a kind of peptide molecule that can bind to heparin, which is widely present in the extracellular matrix and plays an important role in various biological processes. The binding of KRSR heparin-binding peptide to heparin may trigger a series of signal transduction processes, affecting the biological behavior of cells. For example, it may regulate cell proliferation, migration or apoptosis by activating or inhibiting specific signaling pathways.

[0006] RGD integrin binding peptide is a short peptide sequence derived from the extracellular matrix, which has the ability to specifically bind to integrin receptors. As an important receptor on the cell membrane, integrin regulates various functions of cells by activating signal transduction pathways. The binding of RGD peptide to integrin can trigger a series of signal transduction events, affecting cell proliferation, apoptosis and migration.

[0007] Based on the above background, the present application provides a novel active polypeptide to improve the proliferation ability of umbilical cord mesenchymal stem cells. SUMMARY

[0008] The first object of the present application is to provide a polypeptide for promoting the proliferation of mesenchymal stem cells, which can promote the proliferation of umbilical cord mesenchymal stem cells.

[0009] In order to achieve the above-mentioned object, the technical scheme adopted by the present application is:

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

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

[0012] In order to achieve the above-mentioned object, the technical scheme adopted by the present application is:

[0013] The application of a polypeptide for promoting the proliferation of mesenchymal stem cells is used for preparing a culture medium or a drug for promoting the proliferation of mesenchymal stem cells.

[0014] Further, the concentration of the polypeptide in the culture medium is 80-120ug / mL, calculated as the final concentration of the culture medium.

[0015] Further, the culture medium further comprises DMEM basic medium, 1-5% FBS.

[0016] Further, 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.

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

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

[0019] In order to achieve the above-mentioned object, the technical scheme adopted by the present application is:

[0020] A culture method for promoting mesenchymal stem cell proliferation, comprising the following steps:

[0021] The umbilical cord mesenchymal stem cells in the logarithmic growth phase are inoculated in the culture medium for proliferation culture, and the umbilical cord mesenchymal stem cells are obtained.

[0022] The culture medium comprises DMEM basic culture medium, 1-5% FBS and the polypeptide, and the concentration of the polypeptide is 80-120 μg / mL in the final concentration of the culture medium.

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

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

[0025] Further, the culture is carried out at 37 DEG C in a 5% CO2 environment, and the culture time is 4-6 days.

[0026] Compared with the prior art, the beneficial effects of the present application mainly lie in that:

[0027] The present application successfully prepares an active polypeptide (named as polypeptide 1, sequence KRSRGGSFFRGD) by using a solid-phase synthesis method, and the polypeptide can significantly promote the proliferation ability of umbilical cord mesenchymal stem cells. The experimental results show that when the concentration of the polypeptide 1 in the culture medium is 100 μg / mL, the adhesion rate of the umbilical cord mesenchymal stem cells can reach 94.16%, and at the same time, the relative proliferation rate is as high as 133.41%, that is, the polypeptide provided by the present application can not only promote the adhesion performance of the umbilical cord mesenchymal stem cells, but also significantly increase the proliferation speed.

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

[0029] In addition, in-depth research shows that the polypeptide 1 effectively promotes the proliferation of umbilical cord mesenchymal stem cells by up-regulating the expression of Nrf2 gene. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is the umbilical cord mesenchymal stem cell morphology diagram of example 2 of the present application;

[0031] Figure 2 is a result analysis diagram of the influence of Nrf2 protein expression under different culture conditions;

[0032] Figure 3 Figure 1 is a result analysis chart of the mRNA expression amount of Nrf2 under different culture conditions. DETAILED DESCRIPTION

[0033] The technical solutions of the present application are further described below in conjunction with the specific embodiments. However, those skilled in the art should understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if not specifically mentioned, are conventional products obtained through commercial channels.

[0034] 1. Example

[0035] Example 1

[0036] Example 1 provides a polypeptide for promoting the expression of Nrf2 gene. The present application prepares the polypeptide by solid phase synthesis method, and takes 0.25 mmol scale as an example to illustrate the synthesis process of the polypeptide. The specific process is as follows:

[0037] (1) Take Wang resin 0.25 g and place it into the reactor on the SYMPHONY type 12 channel polypeptide synthesizer, add solvent DMF to the reactor, so that the Wang resin is completely immersed in the solvent DMF, and swell for 30 min;

[0038] (2) Take 1 mmol of protected diaminocarboxylic acid (Fmoc-L-Lys(Fmoc)-OH) and place it in the reactor, so that the carboxyl group of the protected diaminocarboxylic acid is combined with the amino group of the Wang resin, then take 1 mmol of other protected amino acid monomers and place them in the bottle, according to the amino acid sequence in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, at 25°C, the target polypeptide amino acid sequence is automatically controlled by the computer program in the synthesizer from C terminal to N terminal in sequence, then deprotection, activation and connection are carried out, and then the next cycle is carried out, until all the amino acid monomers are completely connected, to obtain a polypeptide resin with side chain protection group.

[0039] (3) Deprotection and cutting of resin: the obtained polypeptide resin with side chain protection group is placed in a stoppered flask, and a cleavage reagent (consisting of 0.25 ml of water, 0.25 ml of ether, i.e. 1,2-dioxyethane, 1 mL of TIS, i.e. triisopropylsilane, 9.45 mL of trimethylolphenol) is added, and the reaction is carried out at 30°C under electromagnetic stirring for 2h, the reaction liquid is filtered, and the filtrate is collected; the resin is washed with trifluoroacetic acid, and the collected liquid and washing liquid are combined, then ether is added to produce a precipitate, which 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 basic medium containing 5% FBS respectively, to obtain polypeptide final concentrations of 50, 80, 100, 120, 150 μg / mL medium respectively.

[0053] The P3 generation of umbilical cord mesenchymal stem cells obtained in Example 2 were resuspended in the above-mentioned medium respectively, inoculated into T75 cell culture bottles, with a cell density of 1 x 10 4 individuals / mL, and cultured at 37°C in a 5% CO2 incubator; after 9h of adherent culture, the cells were digested using TrypLE enzyme and counted. The adherent rate = (number of adherent cells / total number of inoculated cells) x 100%, and the results are shown in Table 1.

[0054] Table 1

[0055]

[0056]

[0057] Table 1 is the result analysis of the adherent rate of umbilical cord mesenchymal stem cells after being cultured in different media. As can be seen from Table 1, compared with the medium added with polypeptides 2-3, the medium added with polypeptide 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 basic medium containing 5% FBS respectively, to obtain polypeptide final concentrations of 50, 80, 100, 120, 150 μg / mL medium respectively.

[0060] The P3 generation of umbilical cord mesenchymal stem cells of Example 2 were grown in DMEM medium (containing 5% FBS) to the logarithmic phase, digested and diluted into a cell suspension. The above-mentioned cell suspension was inoculated into the above-mentioned medium at a concentration of 2 x 10 4 individuals / mL respectively, and cultured at 37°C in a 5% CO2 cell incubator for 5d, with the medium being replaced every 2d during the culture. In addition, a control group was set up, and the medium consisted of DMEM basic medium + 5% FBS.

[0061] (1) After the end of the culture, the proliferation ability of the cells in each group was analyzed using the MTT method. The formula for calculating the relative proliferation rate is: experimental group / control group x 100%, and the results are shown in Table 2.

[0062] (2) After the culture is finished, the supernatant after centrifugation of each group cultured by using polypeptide with a final concentration of 100 μg / mL is collected, the supernatant is dialyzed by using a 50KD ultrafiltration membrane, the dialysate is collected by using a 100D ultrafiltration membrane, 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 is detected by using enzyme-linked immunoassay (ELISA kit), and the results are shown in Table 3.

[0063] Table 2

[0064]

[0065] Table 3

[0066]

[0067] Table 2 is the result analysis of the relative proliferation rate of umbilical cord mesenchymal stem cells after being cultured by using different culture media. As can be observed from Table 2, polypeptides 1-3 provided by the present application can all promote the proliferation of umbilical cord mesenchymal stem cells. However, compared with the culture medium added with polypeptides 2-3, the culture medium added with polypeptide 1 can better promote the proliferation of umbilical cord mesenchymal stem cells.

[0068] It is further observed that, with the increase of the amount of polypeptide used in the culture medium, the relative proliferation rate of umbilical cord mesenchymal stem cells gradually increases. When the concentration of polypeptide 1 in the culture medium is 100 μg / mL, the promoting effect on the proliferation ability of umbilical cord mesenchymal stem cells is best. When the concentration of polypeptide 1 in the culture medium is higher than 100 μg / mL, there is a downward trend in the proliferation ability of umbilical cord mesenchymal stem cells. Therefore, the present application preferably uses polypeptide 1 with a concentration of 100 μg / mL in the culture medium.

[0069] Table 3 is the result analysis of the cytokines secreted by umbilical cord mesenchymal stem cells after being cultured by using different culture media. As can be observed from Table 3, compared with the culture medium added with polypeptides 2-3, the culture medium added with polypeptide 1 can better promote the secretion of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) by umbilical cord mesenchymal stem cells, which has a far-reaching significance for the clinical application of umbilical cord mesenchymal stem cells.

[0070] The following experimental example uses polypeptide 1 with a concentration of 100 μg / mL in the culture medium to illustrate the technical effects that can be achieved by the polypeptide of the present application.

[0071] Experimental Example 4

[0072] The P3 generation umbilical cord mesenchymal stem cells of Example 2 are grown in DMEM culture medium (containing 5% FBS) to the logarithmic phase, digested and diluted into a cell suspension. The cell suspension is diluted to 2x104 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 to prepare culture medium for promoting the 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 the proliferation of mesenchymal stem cells 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 mesenchymal stem cells are umbilical cord mesenchymal stem cells.

6. A method for promoting the proliferation of mesenchymal stem cells, characterized in that: The following steps are involved: The umbilical cord mesenchymal stem cells in the logarithmic growth phase are inoculated into 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.

7. The method for promoting the proliferation of mesenchymal stem cells according to claim 6, wherein: The seeding density of the umbilical cord mesenchymal stem cells in the culture medium is 1-5×10 4 pieces / mL.

8. The method for promoting the proliferation of mesenchymal stem cells according to claim 6, wherein: The umbilical cord mesenchymal stem cells are P2-P5 generation cells.

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

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