A culture preparation and culture method for improving stem cell homing ability

Abstract

The present invention relates to a culture preparation and a culture method for improving the homing ability of stem cells, and belongs to the field of stem cell technology. The culture preparation is composed of stromal cell-derived factor 1α (SDF‑1α), cypermethrin and basal culture medium. Through the action of cypermethrin, the promoting effect of low-concentration SDF‑1α on the homing ability of stem cells (especially human bone marrow mesenchymal stem cells) can be enhanced. Mechanism studies have found that cypermethrin pretreatment can increase the sensitivity of stem cells to SDF‑1α, enhance the activation of the SDF‑1α / CXCR4 signaling pathway, and upregulate the expression of related proteins, thereby promoting the migration and homing of stem cells. In general, the present invention utilizes the effect of cypermethrin, a natural product of a plant, to avoid the adverse reactions that may be caused by the use of high-concentration SDF‑1α, reduce the cost and risk of stem cell therapy, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cells, and in particular relates to a culture preparation and a culture method for improving the homing ability of stem cells. Background Art

[0002] Stem cell homing, also known as directed migration or homing of stem cells, refers to the process by which stem cells selectively migrate to specific damaged or diseased tissues through the blood circulation system in the body. This phenomenon is extremely important in the field of stem cell therapy because it is directly related to the efficiency and effectiveness of stem cell therapy. The improvement of stem cell homing ability means that they can locate to tissues that need repair more quickly and play a therapeutic role there, such as promoting wound healing, tissue regeneration or serving as a carrier for drug delivery.

[0003] Among the many factors that affect stem cell homing, the signaling axis composed of stromal cell-derived factor 1α (SDF-1α) and its receptor CXCR4 plays a core role. The SDF-1α / CXCR4 signaling axis not only regulates the proliferation, movement and growth of stem cells, but also directly affects the homing behavior of stem cells. Although the SDF-1 / CXCR4 signaling axis has potential application value in stem cell homing, it still faces some challenges and limitations in practical applications. First, the production, purification and application of SDF-1α involve high costs, which limits its application in extensive clinical practice. Secondly, according to the literature: Effects of stromal cell-derived factor-1α on the migration of mesenchymal stem cells, high concentrations of SDF-1α will inhibit the migration of mesenchymal stem cells, while low concentrations of SDF-1α have limited effects on promoting the migration of mesenchymal stem cells. Therefore, how to make low concentrations of SDF-1α play a better role in promoting cell migration to improve the homing ability of stem cells is a problem that needs to be solved at present. Summary of the invention

[0004] The object of the present invention is to provide a culture preparation and a culture method for improving the homing ability of stem cells, so as to better exert the stem cell homing promoting effect of SDF-1α.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Firstly, the present invention provides a culture preparation for improving the homing ability of stem cells, characterized in that the culture preparation consists of stromal cell-derived factor 1α, imperatae and a basal culture medium.

[0007] Preferably, the stem cells are human bone marrow mesenchymal stem cells.

[0008] Preferably, the human bone marrow mesenchymal stem cells are P3 generation human bone marrow mesenchymal stem cells.

[0009] Preferably, in the culture preparation, the concentration of stromal cell-derived factor-1α is in the range of 25-100 ng / mL.

[0010] Preferably, in the culture preparation, the concentration range of stromal cell-derived factor-1α is 100 ng / mL.

[0011] Preferably, in the culture preparation, the concentration of Imperataein is in the range of 50-100 μmol / L.

[0012] Preferably, in the culture preparation, the concentration of Imperata cylindrica is 100 μmol / L.

[0013] Preferably, the basic culture medium is DMEM culture medium containing 10% fetal bovine serum.

[0014] Secondly, the present invention provides a culture method for improving the homing ability of stem cells, the culture method comprising the following steps:

[0015] (1) inoculating stem cells into a culture vessel, using a basal medium containing 50-100 μmol / L imperataein, and pre-treating the stem cells according to conventional cell culture conditions;

[0016] (2) After the pretreatment step is completed, the pretreated stem cells are transferred to a basal culture medium containing 25-100 ng / mL stromal cell-derived factor-1α and continue to be cultured according to conventional cell culture conditions to enhance the homing ability of the stem cells.

[0017] Preferably, the stem cells are human bone marrow mesenchymal stem cells.

[0018] Preferably, the human bone marrow mesenchymal stem cells are P3 generation human bone marrow mesenchymal stem cells.

[0019] Preferably, the basic culture medium is DMEM culture medium containing 10% fetal bovine serum, the pretreatment time is 24 hours, the continued culture time is 24 hours, and the conventional cell culture conditions are 37° C. and 5% CO 2 .

[0020] Secondly, the present invention provides the use of Imperatae Indole in enhancing the migration and homing of stem cells induced by stromal cell-derived factor-1α.

[0021] Preferably, the stem cells are human bone marrow mesenchymal stem cells, and the imperataein promotes stem cell migration and homing by increasing the sensitivity of bone marrow mesenchymal stem cells to stromal cell-derived factor-1α, thereby enhancing the activation of the SDF-1α / CXCR4 signaling pathway.

[0022] The beneficial effects of the present invention are:

[0023] The present invention proposes a novel culture preparation and culture method, which can effectively improve the migration and homing ability of human bone marrow mesenchymal stem cells. The culture preparation is composed of SDF-1α, Imperata cylindrica and basal culture medium, and the stimulating effect of SDF-1α on stem cell homing is enhanced by the action of Imperata cylindrica.

[0024] Mechanistic studies have found that pretreatment with Imperata cylindrica can increase the sensitivity of stem cells to SDF-1α and enhance the activation of the SDF-1α / CXCR4 signaling pathway, thereby improving the migration and homing ability of stem cells. This provides a theoretical basis for the combined application of Imperata cylindrica and SDF-1α in stem cell therapy.

[0025] The present invention utilizes the effect of the plant natural product Imperataein, avoids the adverse reactions that may be caused by using high concentrations of SDF-1α, reduces the cost and risk of stem cell therapy, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The experimental results of the effects of different concentrations of SDF-1α on the migration of human bone marrow mesenchymal stem cells;

[0027] Figure 2 The results show the difference in migration of human bone marrow mesenchymal stem cells after pretreatment with Imperata cylindrica and then treatment with SDF-1α.

[0028] Figure 3 The experimental results show the effects of different cylindroclase pretreatments on the migration of human bone marrow mesenchymal stem cells.

[0029] Figure 4 The results show the difference in protein expression of SDF-1α / CXCR4 signaling axis in human bone marrow mesenchymal stem cells after pretreatment with Imperata cylindrica and then treated with SDF-1α. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0031] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0032] Example 1: Verification of the effect of different concentrations of SDF-1α on the migration of human bone marrow mesenchymal stem cells

[0033] Since there are differences in the concentrations of SDF-1α used in different literatures, the present invention firstly detects the optimal concentration of SDF-1α for promoting the homing of P3 human bone marrow mesenchymal stem cells.

[0034] Experimental methods:

[0035] Bone marrow mesenchymal stem cells at passage P3 were digested with trypsin, counted, and the cell density was adjusted to 1×10 using serum-free DMEM medium. 5 cells / mL;

[0036] SDF-1α (purchased from Peprotech) was prepared into 12.5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL solutions using DMEM medium containing 10% FBS;

[0037] The Transwell chamber was placed in a 24-well plate, 200 μL of cell suspension was added to the upper chamber of the chamber, 600 μL of the above-mentioned SDF-1α solution of different concentrations was added to the lower chamber of the chamber, and the control group was DMEM medium without SDF-1α added;

[0038] After treatment, the 24-well plate was placed in a 37°C, 5% CO2 incubator for 24 h;

[0039] Take the 24-well plate out of the incubator, remove the Transwell chamber, discard the culture medium in the upper chamber, wash twice with PBS, and use a cotton swab to wipe off the cells on the upper membrane;

[0040] The chamber was fixed in 4% paraformaldehyde for 20 min, washed twice with PBS, stained with crystal violet for 20 min, washed three times with PBS, observed and photographed under an inverted phase contrast microscope, and 5 fields of view were randomly selected for counting and statistics.

[0041] Experimental results:

[0042] The experimental results are shown in Figure 1 In the results, it can be seen that when P3 human bone marrow mesenchymal stem cells were treated with 12.5ng / mL SDF-1α, although a slight increase in the number of cell migration was observed, this increase did not reach statistical significance;

[0043] When the concentration of SDF-1α was increased to 25ng / mL, 50ng / mL and 100ng / mL, we observed a significant increase in the number of cell migration. This phenomenon is statistically significant, which means that SDF-1α has a promoting effect on cell homing at these concentrations. However, when the concentration reached 200ng / mL, the number of cell migration decreased. Therefore, the present invention determines that for P3 generation bone marrow mesenchymal stem cells, the promoting effect of 100ng / mL of SDF-1α is the most significant.

[0044] Example 2: Verification of the effect of different concentrations of Imperatae cylindrica on enhancing the homing ability of human bone marrow mesenchymal stem cells induced by SDF-1α

[0045] The CAS number of cylindrin is 17904-55-1, and its molecular formula is C 31 H 52 O. In previous studies, it was found that Imperata cylindrica can be used in epidermal stem cells to promote the proliferation of epidermal stem cells and reduce the senescence of aged epidermal stem cells. However, there has been no report on the application of Imperata cylindrica in bone marrow mesenchymal stem cells, nor has there been any report on the use of Imperata cylindrica for enhancing the homing ability of stem cells.

[0046] Experimental methods:

[0047] Bone marrow mesenchymal stem cells at generation P3 were seeded into 6-well culture plates. After the cells were completely attached to the wall, the culture medium was removed and replaced with DMEM culture medium containing 0, 25, 50, or 100 μmol / L Imperataein for pretreatment for 24 h.

[0048] After treatment, the bone marrow mesenchymal stem cells were digested with trypsin, counted, and the cell density was adjusted to 1×10 using serum-free DMEM medium. 5 cells / mL;

[0049] Place the Transwell chamber in a 24-well plate and proceed as follows:

[0050] Control group: 200 μL of cell suspension treated with 0 μmol / L Imperataein was added to the upper chamber, and DMEM medium containing 10% FBS was added to the lower chamber;

[0051] Drug treatment group 1: 200 μL of cell suspension treated with 0 μmol / L Imperataein was added to the upper chamber, and 100 ng / mL SDF-1α solution was added to the lower chamber;

[0052] Drug treatment group 2: 200 μL of cell suspension treated with 25 μmol / L Imperataein was added to the upper chamber, and 100 ng / mL SDF-1α solution was added to the lower chamber;

[0053] Drug treatment group 3: 200 μL of cell suspension treated with 50 μmol / L Imperataein was added to the upper chamber, and 100 ng / mL SDF-1α solution was added to the lower chamber;

[0054] Drug treatment group 4: 200 μL of cell suspension treated with 100 μmol / L Imperataein was added to the upper chamber, and 100 ng / mL SDF-1α solution was added to the lower chamber;

[0055] After treatment, the 24-well plate was placed in a 37°C, 5% CO2 incubator for 24 h;

[0056] Take the 24-well plate out of the incubator, remove the Transwell chamber, discard the culture medium in the upper chamber, wash twice with PBS, and use a cotton swab to wipe off the cells on the upper membrane;

[0057] The chamber was fixed in 4% paraformaldehyde for 20 min, washed twice with PBS, stained with crystal violet for 20 min, washed three times with PBS, observed and photographed under an inverted phase contrast microscope, and 5 fields of view were randomly selected for counting and statistics.

[0058] Experimental results:

[0059] The experimental results are shown in Figure 2 It can be seen from the observation that the number of cell migration in drug treatment group 1, drug treatment group 2, drug treatment group 3 and drug treatment group 4 added with SDF-1α is higher than that in the control group, which is consistent with the results of Example 1;

[0060] Further analysis showed that when cells were pretreated with 25 μmol / L of Imperata cylindricain and then treated with SDF-1α, the number of cell migration in drug-treated group 2 did not increase significantly compared with drug-treated group 1 without Imperata cylindricain pretreatment. This finding suggests that 25 μmol / L Imperata cylindricain pretreatment may not enhance the effect of SDF-1α on the migration ability of bone marrow mesenchymal stem cells (BM-MSCs);

[0061] However, when the concentration of pre-treatment cypermethrin was increased to 50 μmol / L and 100 μmol / L, respectively, and then treated with SDF-1α, the number of cell migration in drug treatment group 3 and drug treatment group 4 increased significantly compared with cells without any pre-treatment. This result shows that pre-treatment with a higher concentration of cypermethrin can significantly enhance the effect of SDF-1α on the migration ability of BM-MSCs.

[0062] In summary, the experimental results show that the effect of cypermethrin pretreatment on SDF-1α in promoting BM-MSCs migration is dose-dependent, and 50μmol / L and 100μmol / L cypermethrin pretreatment can significantly enhance the effect of SDF-1α. These findings provide valuable data support for the combined application of cypermethrin and SDF-1α in stem cell therapy.

[0063] Example 3: Verification of the effect of different concentrations of Imperatae on the migration of human bone marrow mesenchymal stem cells

[0064] In order to detect whether Imperatae Indole can enhance the effect of SDF-1α in promoting migration by directly promoting the migration of human bone marrow mesenchymal stem cells, the present invention detected the effect of Imperatae Indole directly on the migration of human bone marrow mesenchymal stem cells.

[0065] Experimental methods:

[0066] Bone marrow mesenchymal stem cells at generation P3 were seeded into 6-well culture plates. After the cells were completely attached to the wall, the culture medium was removed and replaced with DMEM culture medium containing 0, 25, 50, or 100 μmol / L Imperataein for pretreatment for 24 h.

[0067] After treatment, the bone marrow mesenchymal stem cells were digested with trypsin, counted, and the cell density was adjusted to 1×10 using serum-free DMEM medium. 5 cells / mL;

[0068] Place the Transwell chamber in a 24-well plate and proceed as follows:

[0069] Control group: 200 μL of cell suspension treated with 0 μmol / L Imperataein was added to the upper chamber, and DMEM medium containing 10% FBS was added to the lower chamber;

[0070] 50 μmol / L Imperataein group: 200 μL of cell suspension treated with 50 μmol / L Imperataein was added to the upper chamber, and DMEM medium containing 10% FBS was added to the lower chamber;

[0071] 100 μmol / L Imperataein group: 200 μL of cell suspension treated with 100 μmol / L Imperataein was added to the upper chamber, and DMEM medium containing 10% FBS was added to the lower chamber;

[0072] After treatment, the 24-well plate was placed in a 37°C, 5% CO2 incubator for 24 h;

[0073] Take the 24-well plate out of the incubator, remove the Transwell chamber, discard the culture medium in the upper chamber, wash twice with PBS, and use a cotton swab to wipe off the cells on the upper membrane;

[0074] The chamber was fixed in 4% paraformaldehyde for 20 min, washed twice with PBS, stained with crystal violet for 20 min, washed three times with PBS, observed and photographed under an inverted phase contrast microscope, and 5 fields of view were randomly selected for counting and statistics.

[0075] Experimental results:

[0076] The experimental results are shown in Figure 3 It can be seen from the observation that compared with the control group, there is no significant difference in the number of migration of bone marrow mesenchymal stem cells treated with 50μmol / L and 100μmol / L Imperatae Inu. This shows that Imperatae Inu cannot directly promote the migration ability of bone marrow mesenchymal stem cells. Therefore, Imperatae Inu does not enhance the cell migration promoting effect of SDF-1α by improving the migration ability of bone marrow mesenchymal stem cells.

[0077] Example 4: Verification of the difference in SDF-1 and CXCR4 expression between human bone marrow mesenchymal stem cells pretreated and untreated with Imperatae cylindrica after stimulation with SDF-1α

[0078] Since SDF-1α mainly promotes cell migration and homing by stimulating the expression of SDF-1 and CXCR4 in bone marrow mesenchymal stem cells, this example will explore whether the pretreatment with Imperatae cylindrica promotes the expression of SDF-1 and CXCR4 by increasing the sensitivity of bone marrow mesenchymal stem cells to SDF-1α, thereby improving the migration ability of cells.

[0079] Experimental methods:

[0080] Bone marrow mesenchymal stem cells at generation P3 were seeded into 6-well culture plates. After the cells were completely attached to the wall, the culture medium was removed and replaced with DMEM culture medium containing 0, 50, or 100 μmol / L Imperataein for pretreatment for 24 h.

[0081] After the preprocessing is completed, proceed as follows:

[0082] Control group: After pretreatment with DMEM medium containing 0 μmol / L Imperataein, DMEM medium without SDF-1α was added and cultured for 24 h;

[0083] Drug treatment group 1: After pretreatment with DMEM medium containing 0 μmol / L Imperataein, DMEM medium containing 100 ng / mL SDF-1α was added and cultured for 24 h;

[0084] Drug treatment group 2: After pretreatment with DMEM medium containing 25 μmol / L Imperataein, DMEM medium containing 100 ng / mL SDF-1α was added and cultured for 24 h;

[0085] Drug treatment group 3: After pretreatment with DMEM medium containing 50 μmol / L Imperataein, DMEM medium containing 100 ng / mL SDF-1α was added and cultured for 24 h;

[0086] After the culture was completed, the culture medium was removed, 80 μL of protein lysis solution was added to each well, the cells were scraped off with a cell scraper, the lysis solution containing the cells was transferred to an EP tube, and the cells were disrupted with an ultrasonic cell disruptor;

[0087] Centrifuge at 4°C, 12,000 rpm for 20 min, and transfer the supernatant to a new EP tube;

[0088] The protein concentration was determined using the BCA method, and 5× SDS loading buffer was added to keep the concentration of each group of protein samples consistent;

[0089] Assemble the gel stand, prepare 12% lower gel and 5% upper gel, assemble the electrophoresis device and start electrophoresis;

[0090] The upper concentrated gel was electrophoresed at a constant voltage of 80 V for 30 min, and the lower separation gel was electrophoresed at a constant voltage of 120 V for about 60 min. The electrophoresis was stopped when the bromophenol blue band moved to about 1 cm from the bottom of the separation gel.

[0091] Place the gel in the electroporation clip and make a transfer clip according to the "sandwich" model. All bubbles need to be removed during the process. Install the electroporation device and place it in an ice box. Pour the transfer solution and use a constant current of 250mA. After 90 minutes of electroporation, the transfer is completed.

[0092] After electroporation, the PVDF membrane was taken out and placed in 5% skim milk powder and sealed on a shaker for 1 h;

[0093] According to the corresponding protein size, SDF-1α, CXCR4 and β-actin were incubated overnight at 4°C on a shaker;

[0094] The primary antibody was aspirated, the membrane was washed three times with PBS, 5 min each time, and incubated with the secondary antibody at room temperature for 1.5 h;

[0095] The secondary antibody was discarded, the membrane was washed three times with PBS, each time for 5 min, and then developed and exposed.

[0096] The experimental results are shown in Figure 4 In the experiment, it can be seen that compared with the untreated control group, the drug treatment group 1 treated with SDF-1α alone showed a significant upregulation of SDF-1α and CXCR4 protein expression, which is consistent with the existing cognition and confirms that SDF-1α can stimulate the activation of the SDF-1α / CXCR4 signaling pathway in BMSCs;

[0097] Further observation revealed that the protein expression levels of SDF-1α and CXCR4 in drug-treated group 2 and drug-treated group 3 pretreated with cypermethrin were significantly higher than those in drug-treated group 1 without cypermethrin pretreatment. This finding indicates that cypermethrin pretreatment significantly enhanced the sensitivity of BMSCs to SDF-1α, leading to an increase in the expression levels of cell homing-related proteins SDF-1α and CXCR4, thereby increasing the migration and homing ability of bone marrow mesenchymal stem cells.

[0098] In summary, cypermethrin pretreatment increased the sensitivity of BMSCs to SDF-1α and enhanced the activity of the SDF-1α / CXCR4 signaling pathway, which may enhance the homing ability of BMSCs and provide a new strategy for cell therapy using BMSCs.

Claims

1. A culture preparation for improving stem cell homing ability, characterized in that: The culture preparation consists of stromal cell-derived factor 1α, imperataein and basal culture medium; The stem cells are human bone marrow mesenchymal stem cells; In the culture preparation, the concentration range of stromal cell-derived factor 1α is 25-100 ng / mL; In the culture preparation, the concentration range of Imperataein is 50-100 μmol / L.

2. A culture preparation for improving stem cell homing ability according to claim 1, characterized in that: The basic culture medium is DMEM culture medium containing 10% fetal bovine serum.

3. A method for improving the homing ability of human bone marrow mesenchymal stem cells, characterized in that: The culture method comprises the following steps: (1) Inoculate stem cells into a culture vessel, use a basal medium containing 50-100 μmol / L imperataein, and pretreat the stem cells according to conventional cell culture conditions; (2) After the pretreatment step is completed, these pretreated stem cells are transferred to a basal culture medium containing 25-100 ng / mL stromal cell-derived factor-1α and continue to be cultured according to conventional cell culture conditions to enhance the homing ability of the stem cells.

4. A method for cultivating human bone marrow mesenchymal stem cells for improving their homing ability according to claim 3, characterized in that: The basic culture medium is a DMEM culture medium containing 10% fetal bovine serum, the pretreatment time is 24 hours, the continued culture time is 24 hours, and the conventional cell culture conditions are 37° C. and 5% CO 2 .

Images