A medium for improving migration ability of mesenchymal stem cells

Abstract

The application provides a culture medium for improving migration ability of mesenchymal stem cells, and belongs to the technical field of stem cells.The culture medium provided by the application is a migration-enhancing culture medium, which takes natural compound sinigrin as a core effective component and takes DMEM culture medium as an auxiliary culture medium.In addition, the application provides a new method for synergistically enhancing migration ability of mesenchymal stem cells by using a combination of sinigrin and small interfering RNA (siRNA) for lnc-XPNPEP1-1.The migration ability of cells can be synergistically enhanced by first transfecting mesenchymal stem cells with siRNA and then treating the mesenchymal stem cells with the migration-enhancing culture medium containing sinigrin, and the effect is significantly better than the effect of using the culture medium containing sinigrin alone.The scheme of the application can effectively improve the migration efficiency of mesenchymal stem cells, thereby improving the treatment potential of the mesenchymal stem cells in the field of tissue repair and regenerative medicine, and has important application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of stem cells, and particularly relates to a culture medium for improving the migration ability of mesenchymal stem cells. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are a type of non-hematopoietic adult stem cells originating from mesoderm. They have a series of unique characteristics and functions in biology. One of the most remarkable features of MSCs is their strong self-renewal ability, which enables them to undergo multiple cell divisions under in vitro conditions without losing their characteristics and potential. In vitro, MSCs can be differentiated into specific types of cells such as osteoblasts, chondrocytes, and adipocytes under specific induction conditions. This multi-directional differentiation ability makes MSCs have extremely high application value in regenerative medicine and tissue engineering.

[0003] However, despite the great therapeutic potential of MSCs, there are still some technical and biological challenges to overcome before they can be applied to clinical treatment. Among them, improving the migration efficiency of MSCs is a key issue. In damaged tissues, MSCs need to be able to effectively migrate and colonize in order to exert their therapeutic effects. However, current studies have shown that the migration and colonization ability of MSCs in vivo is not ideal, which may limit their effectiveness in treatment. Low migration rate may result in insufficient cell population formation of MSCs at the injury site, thereby affecting the treatment effect. Therefore, how to effectively provide the migration efficiency of mesenchymal stem cells is a problem that needs to be solved at present.

[0004] Sinigrin is a major glucosinolate found in Brassicaceae plants, with CAS No.: 3952-98-5. It has antioxidant, anti-apoptotic, anti-tumor and anti-inflammatory effects. At the same time, studies have found that it also has anti-atherosclerotic effects. In previous research by the company, it was found that sinigrin had no obvious effect on the proliferation of epidermal stem cells, but research on the effect of sinigrin on the migration ability of mesenchymal stem cells has not been reported. SUMMARY

[0005] The purpose of the present application is to provide a culture medium for improving the migration ability of mesenchymal stem cells, thereby effectively enhancing the migration ability of mesenchymal stem cells and better exerting their therapeutic potential.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] Firstly, the present application provides a migration-enhancing culture medium for improving the migration ability of mesenchymal stem cells, wherein sinigrin is used as the core effective component for promoting the migration ability of mesenchymal stem cells.

[0008] Preferably, the migration-enhancing medium takes DMEM medium as an auxiliary component.

[0009] Preferably, in the migration-enhancing medium, the concentration of sinigrin ranges from 25 to 50 μmol / L; and the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0010] Secondly, the application provides application of sinigrin in preparation of a migration-enhancing medium for improving migration ability of umbilical cord mesenchymal stem cells.

[0011] Preferably, in the migration-enhancing medium, the concentration of sinigrin ranges from 25 to 50 μmol / L.

[0012] Secondly, the application provides a culture method for improving migration ability of mesenchymal stem cells, which comprises the following steps:

[0013] (1) a migration-enhancing medium containing sinigrin is prepared by using a conventional cell culture medium containing fetal bovine serum;

[0014] (2) mesenchymal stem cells are inoculated into a cell culture dish containing the migration-enhancing medium;

[0015] (3) the cell culture dish is placed into a cell culture box and cultured under conventional culture conditions for a certain time, so as to improve the migration ability of the mesenchymal stem cells.

[0016] Preferably, the conventional cell culture medium is DMEM medium, and the concentration of sinigrin in the migration-enhancing medium ranges from 25 to 50 μmol / L.

[0017] Preferably, the conventional culture conditions are 37℃ and 5% CO2 culture conditions, and the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0018] Secondly, the application provides a method for synergistically enhancing migration ability of mesenchymal stem cells by combining sinigrin and siRNA against lnc-XPNPEP1-1:1, which is characterized by comprising the following steps:

[0019] (1) mesenchymal stem cells are inoculated into a culture dish, and when the cells reach a suitable cell density, siRNA against lnc-XPNPEP1-1:1 is transfected into the mesenchymal stem cells;

[0020] (2) 24 hours after the transfection treatment, the culture medium is replaced with a migration-enhancing medium containing sinigrin, and the pretreatment is continued for another 24 hours, so that mesenchymal stem cells with synergistically enhanced migration ability are obtained.

[0021] Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells; the suitable cell density is 60-85% cell density; and the concentration of sinigrin in the migration-enhancing medium is 25-50 μmol / L.

[0022] Preferably, the sense strand of the siRNA is UAAUUUGGCAUUACAAUAG, and the antisense strand of the siRNA is CAUUGUAAUGCCAAAUUA.

[0023] Secondly, the present application provides a use of a combination of sinigrin and siRNA against lnc-XPNPEP1-1:1 for synergistically enhancing the migration ability of mesenchymal stem cells.

[0024] Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0025] Preferably, the sense strand of the siRNA is UAAUUUGGCAUUACAAUAG, and the antisense strand of the siRNA is CAUUGUAAUGCCAAAUUA.

[0026] Secondly, the present application provides a composition for synergistically promoting the migration ability of mesenchymal stem cells, which comprises a migration-enhancing medium containing sinigrin and siRNA against lnc-XPNPEP1-1:1.

[0027] Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, and the concentration of sinigrin in the migration-enhancing medium is 25-50 μmol / L.

[0028] Preferably, the sense strand of the siRNA is UAAUUUGGCAUUACAAUAG, and the antisense strand of the siRNA is CAUUGUAAUGCCAAAUUA.

[0029] The present application has the following beneficial effects:

[0030] The present application provides a migration-enhancing medium for improving the migration ability of mesenchymal stem cells, which takes sinigrin as the core effective component. It is first confirmed in the present application that sinigrin can improve the migration ability of mesenchymal stem cells, so as to better exert the therapeutic potential of mesenchymal stem cells.

[0031] Meanwhile, it is found in the present application that the combination of sinigrin and siRNA against lnc-XPNPEP1-1:1 can synergistically enhance the migration ability of umbilical cord mesenchymal stem cells. This strategy not only improves the effect of a single drug or intervention method, but also may more comprehensively improve the migration characteristics of mesenchymal stem cells through multiple pathways and multiple targets, thereby providing a new idea for improving the therapeutic effect. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Detection result graph of CCK-8 experiment of umbilical cord mesenchymal stem cells after treatment of sinigrin;

[0033] Figure 2 Detection result graph of cell scratch experiment of umbilical cord mesenchymal stem cells after treatment of sinigrin;

[0034] Figure 3 12h cell migration rate of umbilical cord mesenchymal stem cells after treatment of sinigrin;

[0035] Figure 4 24h cell migration rate of umbilical cord mesenchymal stem cells after treatment of sinigrin;

[0036] Figure 5 Western Blot experiment detection result of CXCR4 and SDF-1 protein expression in umbilical cord mesenchymal stem cells after treatment of sinigrin;

[0037] Figure 6 Transwell experiment detection result of influence of combination of sinigrin and siRNA of lnc-XPNPEP1-1:1 on migration of umbilical cord mesenchymal stem cells;

[0038] Figure 7 Transwell experiment statistical result of influence of combination of sinigrin and siRNA of lnc-XPNPEP1-1:1 on migration of umbilical cord mesenchymal stem cells;

[0039] In the figure, ** represents P<0.01, and *** represents P<0.001. DETAILED DESCRIPTION

[0040] The embodiments of the present application are used to better illustrate the present application, but are not the contents of the present application, and are limited to the embodiments. Therefore, the skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above content, and still belong to the protection scope of the present application.

[0041] Umbilical cord mesenchymal stem cells are the most commonly used cell type in stem cell therapy due to the advantages of simple collection in vitro, strong in vitro expansion capacity, and the like, so the present application selects umbilical cord mesenchymal stem cells to perform the experiments of the present application.

[0042] Example 1

[0043] Detect whether sinigrin will affect the proliferation of umbilical cord mesenchymal stem cells

[0044] (1) Take out the umbilical cord mesenchymal stem cells stored in liquid nitrogen, place them in a 37°C rapid thawing device, centrifuge to remove the cell freezing solution, resuspend the cells in new DMEM medium (containing 10% fetal bovine serum), and then evenly inoculate the cells into a cell culture dish and place it in a cell culture incubator (37°C, 5% CO2) for culture, so that the cells can adhere and start to grow;

[0045] (2) After the umbilical cord mesenchymal stem cells are cultured to the P3 generation, they are digested to prepare a cell suspension, which is inoculated into a 96-well cell culture plate, and then the 96-well plate is placed in a cell culture incubator and maintained under normal cell culture conditions;

[0046] (3) Use DMEM medium to dilute sinigrin to 25 μmol / L and 50 μmol / L solutions, and filter sterilize them;

[0047] (4) After the cells are fully attached, use a pipette to remove the culture medium in the 96-well plate, and add DMEM medium, 25 μmol / L sinigrin solution, and 50 μmol / L sinigrin solution to different wells, respectively;

[0048] (5) Place the 96-well plate in the cell culture incubator again and culture for 72 h, add 10 μL of CCK-8 to each well, and then place it in the cell culture incubator for 3 h of incubation. Then measure the absorbance of each well, calculate the average value and standard error.

[0049] According to the data shown in Figure 1 , we can observe that the absorbance measurement value of umbilical cord mesenchymal stem cells cultured with DMEM medium is 0.684 ± 0.065, the absorbance measurement value of umbilical cord mesenchymal stem cells cultured with 25 μmol / L sinigrin solution is 0.691 ± 0.052, and the absorbance measurement value of umbilical cord mesenchymal stem cells cultured with 50 μmol / L sinigrin solution is 0.686 ± 0.059. By comparing these data, we can find that whether it is 25 μmol / L or 50 μmol / L sinigrin solution, its effect on the proliferation of umbilical cord mesenchymal stem cells is not significant.

[0050] Example 2

[0051] Detecting whether sinigrin will affect the migration of umbilical cord mesenchymal stem cells

[0052] (1) After preparing the umbilical cord mesenchymal stem cells into a cell suspension, inoculate them into a 6-well cell culture plate until the confluence of the cells reaches 80%;

[0053] (2) Use a sterile 200 μL pipette tip to draw a clear, straight line on the cultured cell monolayer;

[0054] (3) Wash the cells gently with sterile PBS to remove cell debris and unattached cells from the scratch area;

[0055] (4) Add DMEM medium, 25 μmol / L sinigrin solution, and 50 μmol / L sinigrin solution to different wells, respectively, and then place the culture plate back into the cell incubator for further culture.

[0056] (5) At 0, 12, and 24 h, respectively, observe and record the cell migration into the scratch area using a microscope.

[0057] The images of the experimental results are shown in Figure 2 , while the statistical data of the cell migration rate are shown in Figure 3 and Figure 4 , corresponding to the observation time points of 12 h and 24 h, respectively. By analyzing these data, it can be found that, in the control group using only DMEM medium, the umbilical cord mesenchymal stem cells show a significantly enhanced cell migration rate compared to those cultured with 25 μmol / L and 50 μmol / L sinigrin. This phenomenon indicates that the treatment of sinigrin can effectively promote the migration ability of umbilical cord mesenchymal stem cells.

[0058] Upon further observation of the data, we noticed that the cell migration rate increases with the increase of sinigrin concentration, which reveals a positive correlation between the cell migration ability and the sinigrin concentration in a dose-dependent manner.

[0059] Example 3

[0060] CXCR4 (C-X-C motif chemokine receptor 4) and SDF-1 (stromal cell-derived factor 1) are key molecules for the migration and homing of umbilical cord mesenchymal stem cells, so the present application detects the regulation of sinigrin on CXCR4 and SDF-1 to further verify its regulation on the migration of umbilical cord mesenchymal stem cells.

[0061] 1. Cell treatment

[0062] (1) Prepare umbilical cord mesenchymal stem cells into a cell suspension, and then inoculate them into a 6-well cell culture plate until the confluence of the cells reaches 90%;

[0063] (2) Add DMEM medium, 25 μmol / L sinigrin solution, and 50 μmol / L sinigrin solution to different wells, respectively, and then place the culture plate back into the cell incubator for further culture for 24 h.

[0064] 2. Cell collection and protein extraction

[0065] (1) Take out the culture plate from the cell incubator, remove the culture medium, add 100 μL protein lysate to each well, and scrape the cells from the bottom of the culture plate using a cell scraper;

[0066] (2) Use a pipette to transfer the collected cells to pre-labeled EP tubes;

[0067] (3) Place the EP tubes on ice for 30 min, then place them in a centrifuge and centrifuge at 12000 rpm for 15 min at 4°C;

[0068] (4) After centrifugation, carefully pipette the supernatant into a new labeled EP tube to obtain the protein-containing sample.

[0069] 3. Protein electrophoresis and electrotransfer

[0070] (1) First, prepare 12% separation gel and 5% concentration gel, assemble the electrophoresis frame, add 10 μL protein sample to each well, and add protein Marker as a molecular weight reference;

[0071] (2) During electrophoresis, set the concentration gel constant voltage to 90V and the separation gel constant voltage to 120V. Stop the electrophoresis process when the bromophenol blue dye reaches the bottom of the gel;

[0072] (3) After removing the gel, assemble it in the transfer holder in the order of sponge pad, three layers of filter paper, PAGE gel, PVDF membrane, three layers of filter paper, and sponge pad. Place the transfer holder in the electrotransfer slot, add ice box for membrane transfer, and transfer for 1.5 hours;

[0073] (4) After membrane transfer, remove the PVDF membrane and place it in a TBST buffer containing 5% skim milk powder for 1 hour of blocking at room temperature on a shaker;

[0074] (5) After blocking, wash the membrane with TBST buffer 3 times for 10 minutes each. According to the expected protein band size and protein Marker display, cut the PVDF membrane to the appropriate size and incubate the CXCR4 and SDF-1, β-actin primary antibodies separately, and incubate overnight in a 4°C refrigerator;

[0075] (6) After primary antibody incubation, wash the membrane with TBST buffer 3 times for 10 minutes each, then incubate the secondary antibody for 1 hour at room temperature;

[0076] (7) After secondary antibody incubation, wash the membrane with TBST buffer 3 times for 10 minutes each, and finally develop and expose for protein band expression.

[0077] The experimental results are shown in Figure 5 , from Figure 5It can be seen that, consistent with the results of the cell scratch experiment, the expression levels of CXCR4 and SDF-1 in the umbilical cord mesenchymal stem cells treated by sinigrin solution are both significantly higher than those in the umbilical cord mesenchymal stem cells treated by DMEM medium alone. This result shows that the addition of sinigrin in the culture medium can effectively promote the expression of CXCR4 and SDF-1 in umbilical cord mesenchymal stem cells, thereby helping to further improve the migration ability of umbilical cord mesenchymal stem cells.

[0078] Example 4

[0079] In the previous research of the company, it was found that siRNA-2 of non-coding gene lnc-XPNPEP1-1:1 (application number: 202110822834.0, sense strand: UAAUUUGGCAUUACAAUAG, antisense strand: CUAUUGUAAUGCCAAAUUA) can effectively promote the migration ability of epidermal stem cells, so the present application attempts to use it for umbilical cord mesenchymal stem cells and combine it with sinigrin to explore whether it can better promote the migration of umbilical cord mesenchymal stem cells.

[0080] (1) After preparing umbilical cord mesenchymal stem cells into a cell suspension, inoculate into a 6-well cell culture plate until the confluence of the cells reaches 70%;

[0081] (2) Transfect siNC and siRNA-2 into umbilical cord mesenchymal stem cells according to the instructions of Lipofecatamine 2000;

[0082] (3) After 24 hours of transfection, digest the cells, and inoculate the siNC and siRNA-2 transfected cells into a new 6-well plate, and treat the cells according to the following treatment grouping:

[0083] Control group: inoculate cells that are transfected with siNC, and add DMEM medium for continuous culture;

[0084] siRNA group: inoculate cells that are transfected with siRNA-2, and add DMEM medium for continuous culture;

[0085] Sinigrin group: inoculate cells that are transfected with siNC, and add 50 μmol / L sinigrin solution for continuous culture;

[0086] siRNA+sinigrin group: inoculate cells that are transfected with siRNA-2, and add 50 μmol / L sinigrin solution for continuous culture;

[0087] (4) After 24 hours of continuous culture, digest the cells to prepare cell suspensions of each group of cells;

[0088] (5) Place the Transwell chamber in the 24-well plate, inoculate 200 μL of cell suspension of each group in the chamber, and the cell amount is 5 x 10 3 , add DMEM medium containing 10% fetal bovine serum to the lower layer of the chamber of the 24-well plate, and then place the 24-well plate in the cell culture box and process for 24 h;

[0089] (6) After the treatment is completed, use tweezers to take out the Transwell chamber, pour off the culture solution in the upper layer, wash with PBS, and gently scrape off the cells in the upper layer with a cotton swab. Place the Transwell chamber in 4% paraformaldehyde fixing solution and fix at room temperature for 30 min;

[0090] (7) Pour off the fixing solution, wash the chamber with PBS, and then stain with staining solution for 20 min;

[0091] (8) After staining, wash with PBS to remove excess staining solution, and place the Transwell chamber under a microscope for observation. Randomly select 5 fields of view and count the number of cells that have migrated to the lower layer.

[0092] The experimental results are shown in Figure 6 and Figure 7 , wherein the average number of cells that migrated in the control group was 102, the average number of cells that migrated in the siRNA group was 128, the average number of cells that migrated in the sinapine group was 167, and the average number of cells that migrated in the siRNA + sinapine group was 246;

[0093] The siRNA group increased by 25.49% compared to the control group, and the effect was poorer than that of epidermal stem cells, which may be related to the expression difference of lnc-XPNPEP1-1:1 in different cells;

[0094] The sinapine group increased by 63.73% compared to the control group, and the siRNA + sinapine group increased by 141.18% compared to the control group. It can be seen that the promotion effect of the combination of siRNA and sinapine is 1.58 times the effect of the two alone, 5.54 times the effect of siRNA alone, and 2.21 times. When siRNA of lnc-XPNPEP1-1:1 and sinapine are used together for umbilical cord mesenchymal stem cells, they can synergistically promote the migration of umbilical cord mesenchymal stem cells, thereby better promoting migration and improving treatment effect.

Claims

1. The application of myrosin in the preparation of a migration-enhancing culture medium that improves the migration ability of umbilical cord mesenchymal stem cells, characterized in that, The concentration of myrosin in the migration-enhancing medium ranges from 25 to 50 μmol / L.

2. A method for synergistically enhancing the migration ability of umbilical cord mesenchymal stem cells using a combination of myrosinase and siRNA targeting lnc-XPNPEP1-1:1, characterized in that... The method includes the following steps: (1) The umbilical cord mesenchymal stem cells were seeded into a culture dish. When the cells reached a suitable cell density, the siRNA targeting lnc-XPNPEP1-1:1 was transfected into the umbilical cord mesenchymal stem cells. (2) After transfection for 24 hours, the culture medium was replaced with migration enhancement medium containing 25-50 μmol / L myrosinase and pretreated for another 24 hours to obtain umbilical cord mesenchymal stem cells with synergistically enhanced cell migration ability.

3. The method according to claim 2, which uses a combination of myrosinone and siRNA targeting lnc-XPNPEP1-1:1 to synergistically enhance the migration ability of mesenchymal stem cells, is characterized in that... The optimal cell density is 60-85% cell density.

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