A polypeptide for promoting the proliferation of human umbilical cord mesenchymal stem cells and its application in in vitro culture of human umbilical cord mesenchymal stem cells

Abstract

The present invention discloses a polypeptide that promotes the proliferation of human umbilical cord mesenchymal stem cells and its application in the in vitro culture of human umbilical cord mesenchymal stem cells. Mesenchymal stem cells have all the characteristics of stem cells, have the potential for self-renewal and multidirectional differentiation, and can differentiate into a variety of mesenchymal tissues, such as bone, cartilage, fat, etc. How to make human umbilical cord mesenchymal stem cells proliferate rapidly in vitro so as to quickly obtain a sufficient number of seed cells in a short time is of great significance. The present invention provides a polypeptide whose sequence is shown in Sequence NO.1, which has an obvious effect of promoting the proliferation of human umbilical cord mesenchymal stem cells in vitro, and will not change the phenotype of human umbilical cord mesenchymal stem cells, and can be used for the efficient in vitro amplification of human umbilical cord mesenchymal stem cells.

Description

Technical Field

[0001] The present invention belongs to the field of stem cells and relates to in vitro culture of stem cells, and specifically relates to a polypeptide for promoting the proliferation of human umbilical cord mesenchymal stem cells and an application of the polypeptide in in vitro culture of human umbilical cord mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells are a type of stem cells with immunomodulatory and regenerative potentials. They are mainly derived from the mesoderm and widely found in tissues such as bone marrow, fat, umbilical cord, placenta, and deciduous teeth. In 2006, the International Society for Cell & Gene Therapy (ISCT) defined the minimum standards for mesenchymal stem cells, including cell culture characteristics, surface marker expression, and differentiation potential. The discarded umbilical cord Wharton's jelly tissue of newborns contains abundant mesenchymal stem cells, which have the advantages of wide sources, little ethical controversy, and strong expansion capacity. They are considered to be an important source of allogeneic therapy. At present, there are about 40 umbilical cord mesenchymal stem cell products that have been registered as drugs and obtained clinical approval in China. The indications for treatment include graft-versus-host disease, knee osteoarthritis, rheumatoid arthritis, acute respiratory distress syndrome, lupus nephritis, and acute-on-chronic liver failure, which far exceed mesenchymal stem cell products from other tissue sources.

[0003] Human umbilical cord mesenchymal stem cells (hUC-MSCs) are widely used in clinical practice. They have low immunogenicity and can be expanded on a large scale, making them a universal allogeneic therapeutic product. Although the mesenchymal stem cell products currently available internationally are mainly derived from bone marrow and adipose tissue, umbilical cord tissue has unique advantages in inflammation and immune regulation, and has good application prospects in the treatment of diseases such as systemic lupus erythematosus, diabetes, and rheumatoid arthritis.

[0004] If human umbilical cord mesenchymal stem cells are to be widely used, the first problem to be solved is how to make them proliferate rapidly in vitro so as to obtain a sufficient number of seed cells in a short period of time. However, the proliferation rate of conventional culture needs to be improved.

[0005] In order to solve the above problems, the present invention is proposed. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the present invention aims to provide a polypeptide for promoting the proliferation of human umbilical cord mesenchymal stem cells and the application of the polypeptide in the in vitro culture of human umbilical cord mesenchymal stem cells.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] A polypeptide for promoting the proliferation of human umbilical cord mesenchymal stem cells in vitro, the sequence of which is shown in Sequence NO.1.

[0009] Use of the polypeptide shown in Sequence 1 above in promoting the proliferation of human umbilical cord mesenchymal stem cells in vitro.

[0010] Use of the polypeptide shown in Sequence 1 above in preparing a culture medium for promoting the proliferation of human umbilical cord mesenchymal stem cells in vitro.

[0011] Preferably, the culture medium is prepared by adding an effective concentration of the polypeptide of claim 1 to a basic culture medium.

[0012] More preferably, the culture medium further contains human platelet lysate.

[0013] Beneficial effects:

[0014] The present invention provides a polypeptide having a sequence as shown in Sequence NO.1, which has an obvious effect of promoting the proliferation of human umbilical cord mesenchymal stem cells in vitro without changing the phenotype of human umbilical cord mesenchymal stem cells, and can be used to prepare a culture medium for promoting the proliferation of human umbilical cord mesenchymal stem cells in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The HPLC chromatogram and chromatographic peak integrated area of ​​peptide 1;

[0016] Figure 2 is the mass spectrometry detection image of peptide 1;

[0017] Figure 3 This is a morphological picture of human umbilical cord mesenchymal stem cells observed under an inverted microscope;

[0018] Figure 4 This is the result of Alizarin red staining of human umbilical cord mesenchymal stem cells after osteogenic differentiation;

[0019] Figure 5 This is the Oil Red O staining result of human umbilical cord mesenchymal stem cells after adipogenic differentiation;

[0020] Figure 6 Alcian blue staining results of human umbilical cord mesenchymal stem cells after induced differentiation into chondrogenesis. DETAILED DESCRIPTION

[0021] The following is a detailed description of the substantive content of the present invention in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. In the following embodiments, the instruments and reagents not specifically explained are all commercially available conventional instruments and reagents.

[0022] Peptide 1 sequence: KNSTEYATG (Sequence NO.1).

[0023] 1. Peptide Synthesis

[0024] Peptide 1 was commissioned to be synthesized by Synpeptide Co., Ltd. (http: / / www.synpeptide.com) and synthesized from the C-terminus to the N-terminus according to conventional solid phase synthesis method, which specifically includes the following steps:

[0025] 1. Weigh 3 g of RINK resin (substitution degree 0.3 mmol / g) into a 150 mL reactor and soak it with 50 mL of dichloromethane (DCM).

[0026] 2. After 2 hours, wash the resin with 3 times the volume of N,N-dimethylacetamide (DMF) and then drain it. Repeat this four times and drain the resin for later use.

[0027] 3. Add a certain amount of 20% piperidine (piperidine / DMF) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF 3 times the volume of the resin and then drain it.

[0028] 4. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100°C for 1 minute). If the resin has color, it means that the deprotection is successful.

[0029] 5. Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (HOBT) into a 50 mL centrifuge tube, add 20 mL of DMF to dissolve them, then add 3 mL of N,N-diisopropylcarbodiimide (DIC) and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30 °C for reaction.

[0030] 6. After 2 hours, cap the resin with a certain amount of acetic anhydride (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, then wash it four times with DMF 3 times the volume of the resin, and drain it for later use.

[0031] 7. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it with DMF four times and then drain it.

[0032] 8. Take a small amount of resin and test it with the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100°C for 1 min). If the resin has color, it means that the deprotection is successful.

[0033] 9. Weigh an appropriate amount of the second amino acid and HOBT into a 50 mL centrifuge tube, add 25 mL of DMF to dissolve them, then add 2.5 mL of DIC and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30°C for reaction.

[0034] 10. After 1 hour, take a small amount of resin for testing using the ninhydrin method (two drops of test A and test B, react at 100°C for 1 minute). If the resin is colorless, the reaction is complete; if the resin has color, it means that the condensation is incomplete and the reaction should continue.

[0035] 11. After the reaction is complete, wash the resin four times with DMF, then drain, add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor, and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF, then drain to check whether the protection is removed.

[0036] 12. Follow steps 9-11 to connect the following amino acids.

[0037] 13. After the last amino acid is attached, remove the protection, wash with DMF four times, and then drain the resin with methanol. Then use 95 cutting solution (trifluoroacetic acid: 1,2-ethanedithiol: triisopropylsilane: water = 95:2:2:1) to cut the polypeptide from the resin (add 10mL cutting solution per gram of resin), and centrifuge and precipitate four times with ice ether (cutting solution: ether = 1:9). Finally, separate and purify with HPLC, and then freeze-dry to obtain.

[0038] 2. HPLC detection

[0039] 1. Chromatographic conditions

[0040] Mobile phase A: 0.1% Trifluoroacetic in 100% Water;

[0041] Mobile phase B: 0.1% Trifluoroacetic in 100% Acetonrtrile;

[0042] Flow rate: 1 mL / min;

[0043] Wavelength: 220nm;

[0044] Chromatographic column: SHIMADZU Inertsil ODS-SP (4.6*250mm*5um);

[0045] Injection volume: 30 μL;

[0046] Elution gradient: 0.00-25.00 min, 1%-50% B; 25.00-25.01 min, 50%-100% B; 25.01-30.00 min, 100%-100% B; 30.01 min, Stop.

[0047] 2. Test results

[0048] HPLC chromatogram and chromatographic peak integrated area and other information such as Figure 1 As shown, the purity of polypeptide 1 was 99.03%.

[0049] 3. Mass spectrometry detection

[0050] Mass spectrometry detection chart Figure 2 As shown, the [M+H]H+ molecular weight is 970.93, which is consistent with the theoretical molecular weight of peptide 1 of 969.992 (the theoretical molecular weight is calculated using the peptide molecular weight calculator of Hefei Guopeptide Biotechnology Co., Ltd.).

[0051] Example 1: Polypeptide 1 promotes the proliferation of human umbilical cord mesenchymal stem cells in vitro

[0052] 1. Experimental Materials

[0053] DMEM / F12 culture medium was a product of Sigma-Aldrich.

[0054] Human platelet lysate (HPL) was purchased from Beijing Bio-Innovation Technology Co., Ltd.

[0055] Human umbilical cord mesenchymal stem cell osteogenic differentiation culture kit, human umbilical cord mesenchymal stem cell adipogenic differentiation culture kit, and human umbilical cord mesenchymal stem cell chondrogenic differentiation culture kit were all purchased from Guangzhou Saiye Biotechnology Co., Ltd.

[0056] 2. Experimental Methods

[0057] 1. Culture and passage of umbilical cord mesenchymal stem cells

[0058] hUC-MSCs were isolated and cultured from umbilical cord tissue using conventional methods in the art. The operation method is as follows: the umbilical cord of a full-term healthy newborn was taken, and repeatedly rinsed with PBS buffer containing gentamicin sulfate until there was no blood color under a sterile environment to separate the Wharton's jelly of the umbilical cord. It was cut into about 2-3 mm with ophthalmic scissors. 3The tissue blocks of different sizes were placed in a sterile cup containing PBS buffer containing gentamicin sulfate and washed three times. The washed Wharton's gel tissue blocks were transferred to a cell culture bottle, and DMEM / F12 culture medium containing 10% fetal bovine serum and 1% double antibody was added, and cultured in a constant temperature incubator (37°C, 5% CO2, saturated humidity). After 1 week of culture, fibroblast-like cells were seen crawling out from around the tissue blocks. After trypsin digestion and passage, pure hUC-MSCs were obtained, which was P0. The passage was performed at a ratio of 1:2, and the passaged hUC-MSCs were obtained for subsequent experiments.

[0059] 2. Identification of human umbilical cord mesenchymal stem cells

[0060] (1) Morphological observation

[0061] Human umbilical cord mesenchymal stem cells with good growth status at the P3 generation were taken to observe the cell morphology under an inverted microscope.

[0062] (2) Determination of multidirectional differentiation ability

[0063] Human umbilical cord mesenchymal stem cells in good growth state at P3 were cultured for 2 weeks using the osteogenic differentiation culture kit, fixed with 4% paraformaldehyde, stained with alizarin red, and observed under a microscope.

[0064] Human umbilical cord mesenchymal stem cells in good growth state at P3 were cultured for 2 weeks using the adipogenic differentiation culture kit, fixed with 4% paraformaldehyde, stained with Oil Red O, and observed under a microscope.

[0065] Human umbilical cord mesenchymal stem cells in good growth state at P3 were cultured for 3 weeks using a chondrogenic differentiation culture kit, fixed with 4% paraformaldehyde, stained with Alcian blue, and observed under a microscope.

[0066] 3. Detection of proliferation ability of human umbilical cord mesenchymal stem cells

[0067] The proliferation ability of P3 generation human umbilical cord mesenchymal stem cells with good growth status was tested. A cell suspension was prepared with complete culture medium (DMEM / F12 culture medium containing 4% human platelet lysate), and 23 μL of cell suspension was mixed with 23 μL AO / PI and counted. The cells were divided into conventional group, polypeptide group I, polypeptide group II, and polypeptide group III. Two 15 mL centrifuge tubes were taken from each group, and 2×10 5 The treatment methods for different groups are as follows:

[0068] Conventional group: add 5 mL of complete culture medium and mix thoroughly by pipetting;

[0069] Peptide I group: add 5 mL of complete culture medium containing 5 μM peptide 1 and mix thoroughly by pipetting;

[0070] Peptide II group: add 5 mL of complete culture medium containing 10 μM peptide 1 and mix thoroughly by pipetting;

[0071] Peptide III group: add 5 mL of complete culture medium containing 20 μM peptide 1 and mix thoroughly by pipetting;

[0072] The cell suspension of each group was transferred into a T25 culture flask and then placed in a constant temperature incubator (37°C, 5% CO2, saturated humidity) for culture. After cell culture, the cell morphology and growth were continuously observed and recorded. After 72 hours of culture, the cells were digested and collected, and the cell number was determined using a cell counter, and the cell phenotype of each group of umbilical cord mesenchymal stem cells was identified using a flow cytometer.

[0073] 4. Statistical processing

[0074] The experimental results were expressed as mean ± standard deviation. GraphPad Prism 9.0 software was used for statistical analysis. One-way analysis of variance was used, and a significance level of P < 0.05 was considered statistically significant.

[0075] 3. Experimental Results

[0076] P3 human umbilical cord mesenchymal stem cells were observed under an inverted microscope. Figure 3 As shown in the figure, the cells are spindle-shaped, uniform in size, arranged in polarity, and the colonies are vortex-shaped, which is consistent with the morphological characteristics of human umbilical cord mesenchymal stem cells. Figure 4 (Alizarin red staining), Figure 5 (Oil Red O staining), Figure 6 As shown in (Alcian blue staining), the staining was positive, which is consistent with the osteogenic, adipogenic and chondrogenic differentiation characteristics of human umbilical cord mesenchymal stem cells.

[0077] The cell number and phenotypic identification results of human umbilical cord mesenchymal stem cells in each group after 72 hours of culture are shown in Table 1 (positive represents expression rate ≥ 95%, negative represents expression rate ≤ 2%).

[0078] Table 1

[0079]

[0080] The results in Table 1 show that different concentrations of polypeptide 1 can effectively promote the proliferation of human umbilical cord mesenchymal stem cells and show obvious dose dependence. The cell phenotypes of each group are consistent with the phenotype of human umbilical cord mesenchymal stem cells.

[0081] The above experimental results show that the polypeptide with the sequence shown in Sequence NO.1 has a significant effect of promoting the proliferation of human umbilical cord mesenchymal stem cells in vitro, and does not change the phenotype of human umbilical cord mesenchymal stem cells, and can be used to prepare a culture medium for promoting the proliferation of human umbilical cord mesenchymal stem cells in vitro.

[0082] Example 2: Human umbilical cord mesenchymal stem cell culture medium

[0083] A human umbilical cord mesenchymal stem cell culture medium is provided, which uses DMEM / F12 culture medium as a basic culture medium, adds 4% human platelet lysate, and further adds 5-20 μM of a polypeptide with a sequence as shown in Sequence NO.1.

[0084] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. A polypeptide for promoting the proliferation of human umbilical cord mesenchymal stem cells in vitro, the sequence of which is shown in Sequence NO.

1.

2. Use of the polypeptide according to claim 1 in preparing a culture medium for promoting the in vitro proliferation of human umbilical cord mesenchymal stem cells.

3. The use according to claim 2, characterized in that: The culture medium is prepared by adding an effective concentration of the polypeptide according to claim 1 to a basic culture medium.

4. The use according to claim 3, characterized in that: The culture medium also contains human platelet lysate.

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