Culture method for enhancing homing ability of mesenchymal stem cells in vitro

Abstract

The invention relates to a culture method for enhancing the homing ability of mesenchymal stem cells in vitro. The culture method comprises the following step: treating the mesenchymal stem cells by adopting tetramethylpyrazine. The method provided by the invention can significantly enhance the homing ability of the mesenchymal stem cells.

Description

Technical Field

[0001] The invention belongs to the technical field of stem cells, and in particular relates to an in vitro culture method for enhancing the homing ability of mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are primary and non-specific cells that can be isolated from various tissues. At present, mesenchymal stem cells are the key cell source for cell therapy and regenerative medicine. However, the key to the success of stem cell therapy is the homing of stem cells, which is also the key to the development and function of stem cell drugs. If the number of mesenchymal stem cells homing is small, the function will be limited. Therefore, it is urgent and necessary to increase the activity and number of mesenchymal stem cells in the body, stimulate and improve the efficiency of stem cell homing.

[0003] Chuanxiong (Xiong) is one of the most widely used and oldest traditional Chinese medicines in my country. As a commonly used medicinal material, Shennong's Herbal Classic lists it as a top grade. In the famous work Synopsis of the Golden Chamber by Zhang Zhongjing of the Eastern Han Dynasty, there are several prescriptions using Chuanxiong, such as Xionggui Jiaoai Decoction, Wenjing Decoction, Danggui Shaoyao San, Danggui San, Suanzaoren Decoction, etc. Tetramethylpyrazine (TMP) is an alkaloid and one of the active ingredients of Chuanxiong (Qióng). In recent decades, TMP has been shown to have a variety of pharmacological properties. It has been used to treat a variety of diseases with good therapeutic effects. At present, there are no reports on the culture method of using Chuanxiong to enhance the homing ability of mesenchymal stem cells. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides an in vitro culture method for enhancing the homing ability of mesenchymal stem cells.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] The present invention provides a culture method for enhancing the homing ability of mesenchymal stem cells in vitro, comprising the following steps: treating the mesenchymal stem cells with chuanxiongzine.

[0007] Furthermore, the concentration of chuanxiongzine is 0.60-0.70 mmol / L.

[0008] Furthermore, the concentration of chuanxiongzine was 0.64 mmol / L.

[0009] Further, the following steps may be included:

[0010] (1) inoculating human mesenchymal stem cells into a mesenchymal stem cell culture medium;

[0011] (2) When the confluence reaches 50-60%, the medium is changed and mesenchymal stem cell culture medium containing ligandroxithrin is added to culture the cells. The final concentration of ligandroxithrin is 0.64 mmol / L.

[0012] Furthermore, in step (2), the temperature of culturing cells is 37°C.

[0013] Further, in step (2), at 5% CO 2 Conditioned cells.

[0014] The beneficial effects of the present invention include: the present invention has found that the use of chuanxiongzine to treat mesenchymal stem cells can upregulate stromal cell-derived factor 1 (SDF-1) and chemokine receptor CXCR4, and downregulate chemokine receptor CXCR7 to promote stem cell migration and homing. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of human mesenchymal stem cell culture (100×).

[0016] Figure 2 Results of CD105 mesenchymal stem cells detected with different fluorescent dyes, where the left picture shows DAPI-stained cell nuclei and the right picture shows Fluorescence-stained cell membranes.

[0017] Figure 3 The results of the experiment were statistically compared after 5 days of intervention of mesenchymal stem cells with low, medium and high concentrations of drugs, and the RNA copy numbers of SDF-1, CXCR4 and CXCR7 in each group were measured by qPCR. *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0018] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0019] The present invention uses human mesenchymal stem cells taken from the umbilical cord of healthy parturients as the research object, and uses phenotypic markers that are characteristic of mesenchymal relationships to identify the cells as P1 generation cells. The standard drug of chuanxiongzine was selected as the intervention drug, and the gene copy number was detected by qPCR. The research results of the present invention show that the 0.64mmol / L chuanxiongzine group has a certain expression-promoting effect on the SDF-1 gene of the stem cells themselves, and has a significant expression-promoting effect on CXCR4, a key molecule for the homing of mesenchymal stem cells (p<0.01); it has a reducing effect on the expression of CXCR7, indicating that the use of chuanxiongzine to treat mesenchymal stem cells can enhance the homing ability of mesenchymal stem cells.

[0020] The present invention uses chuanxiongzine standard products, which are easy to standardize. Reusing high-precision absolute quantitative PCR technology, drawing detection standard curves, calculating cloned plasmid copy numbers, the expression level of stem cell homing genes can be accurately quantified, and the regulatory effect of chuanxiongzine on the cell homing of human mesenchymal stem cells can be accurately evaluated. Chuanxiongzine is easy to use clinically and is suitable for clinical promotion and application to improve the success rate of stem cell transplantation. The present invention uses traditional Chinese medicine standard products, which are high-purity extracts of traditional Chinese medicine with high uniformity and good stability. Some of its chemical or physical properties have been accurately determined, which provides a guarantee for studying the accurate drug effects of traditional Chinese medicine. Umbilical cord mesenchymal stem cells are widely available, easy to obtain, easy to separate and amplify in vitro, low immunogenicity, and also have paracrine and immunoregulatory functions, can promote hematopoiesis, and have immunoregulatory ability. They are widely used in clinical treatment. The human mesenchymal stem cells treated with chuanxiongzine provided by the present invention have very obvious clinical application value.

[0021] The experimental methods used in the following examples are all conventional methods in the art unless otherwise specified. The materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained through commercial channels or prepared by conventional methods by those skilled in the art. The solutions involved in the present invention are all prepared with water as the solvent unless otherwise specified.

[0022] In the embodiment, stem cell complete medium was purchased from Shanghai Chunshi Biotechnology Co., Ltd., with the catalog number CS-K853P. Mesenchymal stem cell medium was purchased from Shanghai Chunshi Biotechnology Co., Ltd., with the catalog number CS-K853X. Primary antibody was purchased from Proteintech, with the catalog number 10862-1-AP. Secondary antibody was an anti-primary antibody labeled with a fluorescent signal molecule, purchased from Proteintech, with the catalog number SA00006-2. Fluoromount-G was purchased from SouthernBiotech, with the catalog number 0100-01.

[0023] Chuanxiongzine drug: The selected Chuanxiongzine drug standard, namely Chuanxiongzine hydrochloride (purity ≥98% by liquid chromatography analysis, Chengdu Munster), has a molecular formula of C 8 H 12 N 2 HCl, the molecular structure is shown in Formula 1.

[0024]

[0025] The following is an introduction through specific embodiments.

[0026] Example 1 Cell collection and cell culture

[0027] Human mesenchymal stem cells were obtained from Shanghai Chunshi Biotechnology Co., Ltd. The human mesenchymal stem cell collection method includes the following steps: the umbilical cord is obtained from a healthy pregnant woman with her informed consent, and the umbilical cord is processed within 6 hours, and the umbilical cord is transferred to a biological safety cabinet, and the periphery of the umbilical cord and the inner cavity of the umbilical vein are fully rinsed with PBS buffer containing double antibodies (penicillin and streptomycin, the final concentration of penicillin is 1%, and the final concentration of streptomycin is 1%), and the tissue block is cut into a square with a side length of about 0.1 cm, and the supernatant is discarded after repeated washing, and placed in a culture dish containing complete stem cell culture medium, incubated at 37°C for 30-60 minutes, clamped into a culture bottle with tweezers, and inverted in 5% CO 2 Incubate in an incubator for 2 h, add 2 mL of complete stem cell culture medium to soak the tissue blocks but not to make them float, and place in a 5% CO 2 In the cell culture incubator, change the medium every 3 days. When the cells growing around the tissue block fuse into a sheet, remove the tissue block, digest the cells with trypsin, and re-plate the bottle.

[0028] Cell culture: Mesenchymal stem cell culture medium was used for culture at 37°C for 72 hours.

[0029] The results of human umbilical cord mesenchymal stem cell culture were observed under an optical microscope. Figure 1 As shown, from Figure 1 It can be seen that the cells are in good condition.

[0030] Example 2 Cell Identification

[0031] (1) Cell climbing: 3 glass slides were placed in a 24-well plate, 1 mL of mesenchymal stem cell culture medium was added to each well, and 2×10 human umbilical cord mesenchymal stem cells (cultured using the method of Example 1) were added. 4 pcs / well. 37℃, 5% CO 2 Incubate incubator for 2 h or overnight.

[0032] (2) Fixation: After the cells have climbed up the slide, remove the culture medium, wash once with PBS buffer, add paraformaldehyde solution (final concentration of paraformaldehyde is 4%, mass percentage) and fix at 4°C for 30 min. Wash three times with PBS buffer, 5 min each time. Alternatively, do not remove the PBS buffer for the last time and place at 4°C overnight.

[0033] (3) Membrane Permeabilization and Sealing: Remove moisture from the glass slide and place it on a culture dish support. Take 50 μL of membrane permeabilization and sealing solution and drop it on the waterproof film. Cover the cell side of the glass slide with sealing film for 2 h.

[0034] The membrane permeabilization blocking solution was prepared by the following method: Trition X-100 solution (Trition X-100 mass percentage is 0.5%) and PBS buffer solution were mixed at a volume ratio of 1:1 to obtain a mixed solution, and serum was added, and the volume percentage of serum to the mixed solution was 10%.

[0035] (4) Primary antibody incubation: After the membrane is broken and blocked, take 50 μL of the primary antibody solution and place it on the waterproof film (in a wet box). Cover the glass slide (the side with cells) with the sealing film and place it at 4°C (can be kept for up to one week).

[0036] The preparation method of the primary antibody solution comprises the following steps: diluting the primary antibody with PBS buffer at a ratio of 1:100 (volume ratio).

[0037] (5) Secondary antibody incubation: Incubate in secondary antibody solution (secondary antibody: PBS buffer = 1:500, volume ratio) at room temperature in the dark for 2 h, then wash with PBS buffer three times, 5 min each time, and stain with 4',6-diamidino-2-phenylindole (DAPI) solution for 5 min. In the DAPI solution, the volume ratio of DAPI to PBS buffer is 1:1000. Wash with PBS buffer three times, 5 min each time.

[0038] (6) Embedding: Place 1 drop of Fluoromount-G on each slide and cover the side with cells with sealing film. The cells were identified as P1 cells, confirming that they are the first generation of stem cells, indicating that the cells have the highest activity.

[0039] CD105 is a cell phenotype marker that can indicate that it is a mesenchymal stem cell. The results of immunofluorescence identification of human mesenchymal stem cell phenotype are as follows Figure 2 As shown. Figure 2 It can be seen that the cells used in the experiment are CD105 positive mesenchymal stem cells. The identified human mesenchymal stem cells were used in subsequent experiments.

[0040] Example 3

[0041] Verify the effect of chuanxiongzine on the homing ability of human mesenchymal stem cells. SDF-1, CXCR4 and CXCR7 are genes for evaluating stem cell homing. By analyzing their expression in stem cells before and after the drug, the changes in their homing ability can be determined. If the expression of SDF-1 and CXCR4 genes is upregulated and the expression of CXCR7 is downregulated, it means that the homing ability of stem cells has increased.

[0042] The experimental method includes the following steps: human mesenchymal stem cells are cultured in a 6-well cell culture plate, and a blank control, a low concentration group, a medium concentration group, and a high concentration group are set, each group having 3 wells. Human mesenchymal stem cells are inoculated with mesenchymal stem cell culture medium and cultured until the confluence is 50-60%, and the medium is changed, and then 1 mL of culture medium is added to each well, wherein the blank control group is added with mesenchymal stem cell culture medium, the low concentration group is added with mesenchymal stem cell culture medium containing chuanxiongzine (the final concentration of chuanxiongzine is 0.64mmol / L), the medium concentration group is added with mesenchymal stem cell culture medium containing chuanxiongzine (the final concentration of chuanxiongzine is 1.28mmol / L), and the high concentration group is added with mesenchymal stem cell culture medium containing chuanxiongzine (the final concentration of chuanxiongzine is 2.56mmol / L). At 37°C and 5% CO 2 The cells were cultured in a cell culture incubator for 5 days, the medium was changed every other day, and the cells were collected by centrifugation on the 5th day for absolute quantitative gene copy number qPCR detection.

[0043] The primer sequences used for analyzing SDF-1, CXCR4 and CXCR7 are shown in Table 1 , respectively. The primers were synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

[0044] Table 1 Absolute quantitative qPCR primers

[0045]

[0046] The absolute quantitative gene copy number qPCR detection method can refer to the literature "Li Li, Zhao Chengping, Li Hong, et al. Establishment of the absolute quantitative PCR standard curve method for plasmid preparation [J]. Journal of Agricultural Biotechnology, 2011, 19(06): 1157-1162." Each group was tested three times.

[0047] In the above detection method, the target genes of SDF-1, CXCR4 and CXCR7 are respectively inserted into the vector pLB-SimpleVector (available commercially), and three plasmids containing the genes of SDF-1, CXCR4 and CXCR7 are constructed, and the standard curve is drawn using the plasmids as the standard. The plasmids are constructed and synthesized by Suzhou Hongxun Biotechnology Co., Ltd. using the following method.

[0048] The gene information of SDF-1, CXCR4 and CXCR7 is shown in Table 2 .

[0049] Table 2 Inserted gene information

[0050]

[0051] The amount of carrier used was calculated according to the following formula. The calculation results are shown in Table 3.

[0052] Vector amount (ng) = 3*(PCR product length (bp) / 2974)*17.5

[0053] Table 3

[0054]

[0055] Add the components shown in Table 4 (which can be purchased commercially) and perform a ligation reaction. The ligation reaction conditions can be conventional experimental conditions in the art or performed according to the instructions of the reagents.

[0056] Table 4 Ligation reaction system

[0057]

[0058] Transformation, single clone selection and bacterial shaking include the following steps:

[0059] ① Take part of the ligation product and add it to 50-100 μL DH5α competent cells (competent cells should be taken out of the -70℃ refrigerator and placed on an ice bath. Add the ligation product when they are just thawed. The amount of ligation product added should not exceed one tenth of the volume of competent cells), flick to mix, and place on ice for 30 minutes;

[0060] ② Place the centrifuge tube at a constant temperature of 42℃ for 90 seconds. After taking out the tube, immediately place it in an ice bath for 2-3 minutes. Do not shake the centrifuge tube during this period.

[0061] ③ Add 250-500 μL of LB medium (without antibiotics) preheated at 37°C to the centrifuge tube and culture at 150 rpm and 37°C for 45 min to express the relevant resistance marker gene on the plasmid and resuscitate the bacteria;

[0062] ④ Mix the bacterial solution in the centrifuge tube, pipette 100 μL onto the LB solid agar medium containing ampicillin, and spread the cells evenly with a sterile elbow glass rod. After the surface of the plate is dry, invert the plate and culture at 37°C for 12-16 hours;

[0063] ⑤ In the clean bench, select a single colony visible to the naked eye and place it into LB medium containing ampicillin, one colony in one tube, and culture overnight at 150 rpm and 37°C with shaking (12-16 hours, preferably with turbid bacteria).

[0064] The bacterial solution cultured overnight with shaking was used to extract the plasmid using the TIANprep Rapid Mini Plasmid Kit. The cloned plasmid copy number was calculated according to the following formula, and the calculation results are shown in Table 5.

[0065] Plasmid copy number (copies / μL) = [plasmid concentration (ng / μL) × 10-9 ×6.02×10 23 ] / [Number of bases in cloned product × 660 (g / mol)

[0066] Table 5

[0067]

[0068] The detection standard curve was drawn according to the method described in the literature "Li Li, Zhao Chengping, Li Hong, et al. Establishment of a standard curve method for absolute quantitative PCR preparation by plasmid [J]. Journal of Agricultural Biotechnology, 2011, 19(06): 1157-1162."

[0069] The results of the absolute quantitative gene copy number qPCR test are shown in Table 6 and Figure 3 As shown in Table 6, it can be seen that the 0.64mmol / L chuanxiongzine group has a certain promoting effect on the expression of the SDF-1 gene of the stem cells themselves, and the 0.64mmol / L chuanxiongzine group has a significant promoting effect on the expression of CXCR4, a key molecule for the homing of mesenchymal stem cells (p<0.01); the 0.64mmol / L chuanxiongzine group has a reducing effect on the expression of CXCR7. This shows that chuanxiongzine has a promoting level on the expression of SDF-1 and CXCR4, molecules of the stem cell homing ability, and chuanxiongzine has an inhibitory level on the expression of CXCR7 molecules that can competitively inhibit the function of CXCR4.

[0070] Table 6 Absolute quantitative gene copy number (copies) qPCR detection results ( n=3)

[0071]

[0072] Figure 3 The results are the results of the statistical comparison of the RNA copy numbers of SDF-1, CXCR4, and CXCR7 in each group measured by qPCR after 5 days of intervention of mesenchymal stem cells with low, medium, and high concentrations of drugs. Among them, *p<0.05, **p<0.01, ***p<0.001. Figure 3 It can be seen that chuanxiongzine has a significant promoting and up-regulating effect on SDF-1 and CXCR4, the key molecules representing the homing ability of umbilical cord-derived mesenchymal stem cells; chuanxiongzine has a significant inhibitory effect on the CXCR7 molecule that has no significant effect on homing and can competitively inhibit the function of CXCR4.

[0073] In summary, chuanxiongzine has a significant expression-promoting effect on CXCR4, a key molecule in the homing of human mesenchymal stem cells, that is, chuanxiongzine is beneficial to the homing of mesenchymal stem cells; chuanxiongzine has drug concentration selectivity for the homing effect of mesenchymal stem cells. When chuanxiongzine is added to the culture medium at a concentration of 0.64mmol / L, the homing ability of stem cells can be significantly improved.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for enhancing the homing ability of mesenchymal stem cells in vitro, characterized in that: The following steps are involved: Mesenchymal stem cells were treated with chuanxiongzine.

2. The culture method according to claim 1, characterized in that: The concentration of chuanxiongzine is 0.60-0.7mmol / L.

3. The culture method according to claim 2, characterized in that: The concentration of chuanxiongzine was 0.64mmol / L.

4. The culture method according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) inoculating human mesenchymal stem cells into a mesenchymal stem cell culture medium; (2) When the confluence reaches 50-60%, the medium is changed and mesenchymal stem cell culture medium containing ligandrostenone is added to culture the cells. The final concentration of ligandrostenone is 0.64 mmol / L.

5. The culture method according to claim 4, characterized in that: In step (2), the temperature of the cultured cells is 37°C.

6. The culture method according to claim 4, characterized in that: In step (2), the cells were cultured under 5% CO2 conditions.

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