Mesenchymal stem cell screening method and application thereof

Abstract

The invention relates to a screening method of mesenchymal stem cells and application thereof, SOX9 gene in hUC-MSCs is used as an index for screening chondrogenic differentiation capability of mesenchymal stem cells, a cell selection standard is provided for basic research and clinical transformation of treating arthritis by stem cells, and it is proved that the screening method has the advantages that the screening method is simple, and the screening efficiency is high. The mesenchymal stem cells with high expression of SOX9 can significantly improve the treatment effect of the mesenchymal stem cells on arthritis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a screening method of mesenchymal stem cells and application thereof, in particular to a screening method of mesenchymal stem cells with strong chondrogenic differentiation capacity and application thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are adult stem cells derived from early developmental mesoderm, which have self-replication capacity and multi-directional differentiation potential, and widely exist in bone marrow, fat, umbilical cord, placenta and dental pulp and the like. A large number of studies show that MSCs can differentiate into osteoblasts, chondroblasts and adipocytes, so that MSCs have good tissue repair and regeneration capacity.

[0003] Up to now, there are more than 1500 clinical studies on MSCs treatment of related diseases registered in the American clinical trial database (www.clincaltrial.gov), but the treatment effects are uneven. We analyze these clinical studies and find that the tissue sources (such as umbilical cord, placenta, dental pulp, fat, amniotic membrane and the like), donor sources (different in gender, age and genetic background and the like), cell passages (such as P1, P2, P3, P5, P8 and the like) of mesenchymal stem cells used in these clinical studies are different. Based on this, we study human umbilical cord mesenchymal stem cells (hUC-MSCs) from different donor sources and find that although hUC-MSCs from different donor sources have similar cell morphology and cell surface markers, they have great functional differences in cell viability, three-lineage differentiation potential and immune regulation capacity. It is also due to this functional difference of mesenchymal stem cells that leads to great differences in clinical treatment effects, and finally hinders its wide clinical application.

[0004] Therefore, based on the molecular mechanism of disease onset, it is an important problem to be solved in the current field to establish a screening standard of hUC-MSCs with good treatment value, so as to improve the dose equivalence between patients and thus improve the treatment effect. SUMMARY

[0005] In order to solve the above technical problems in the background art, the application provides a screening method of mesenchymal stem cells and application thereof, which provides a cell selection standard for basic research and clinical transformation of stem cell treatment of arthritis (OA), and it is proved that mesenchymal stem cells with high expression of SOX9 can significantly improve the treatment effect on OA.

[0006] The technical solution of the application is that the application is a screening method of mesenchymal stem cells, and the special feature is that the hUC-MSCs are screened by the following steps: SOX9Genes as screening for mesenchymal stem cells for chondrogenic differentiation capacity of the index.

[0007] Further, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells, and the method comprises the following steps: 1) several umbilical cord tissues from healthy donors are screened, sterile, mycoplasma and virus detection is carried out, after passing the qualification, primary human umbilical cord mesenchymal stem cells are separated by tissue block suspension method, and are subcultured to P3 generation by using serum-free medium; 2) the P3 generation of human umbilical cord mesenchymal stem cells is detected, and after passing the detection, it is put into a temporary main cell bank; 3) the P3 generation of human umbilical cord mesenchymal stem cells is collected, Trizol is added, and it is gently and repeatedly blown and mixed; RNA is extracted, and after passing the quality control, transcriptome sequencing is carried out; 4) the sequencing data is analyzed by using bioinformatics technology, and the different gene expression matrix of human umbilical cord mesenchymal stem cells is obtained; according to the gene expression matrix, the expression amount of all human umbilical cord mesenchymal stem cell strains SOX9 Gene expression; 5) the chondrogenic capacity of different donor-derived hUC-MSCs in step 4) is quantitatively analyzed, that is, the diameter of chondrocytes is measured; 6) the gene expression amount in different donor-derived hUC-MSCs in step 3) SOX9 Correlation analysis is carried out between the gene expression amount and the corresponding chondrocyte diameter size in step 5); 7) the gene in hUC-MSCs SOX9 As a screening index for the strength of chondrogenic capacity, that is, the biomarker of mesenchymal stem cell chondrogenic capacity.

[0008] Further, the specific steps of step 1) are as follows: 1.1) under sterile conditions, the umbilical cord is collected, and the length of the umbilical cord between the two ligation parts is not less than 20 cm, which is placed in the umbilical cord storage and transportation bottle prepared in advance, and is transported to the cell culture room through the cold chain, and the primary cell separation operation of umbilical cord mesenchymal stem cells is carried out; 1.2) the umbilical cord is placed in a 15 cm culture dish with appropriate physiological saline, the two end ligation parts are removed, and it is cut into 2-3 cm small pieces, and then transferred to a 250 mL wide-mouth bottle with 75% alcohol for disinfection for 3 min; 1.3) the disinfected umbilical cord is transferred to a 250 mL wide-mouth bottle with physiological saline for washing 2-3 times to remove the residual blood on the surface; 1.4) Transfer the umbilical cord to a 15 cm culture dish with physiological saline, use forceps to peel off the umbilical cord along the umbilical vein, remove one umbilical vein and two umbilical arteries, and separate the Wharton's jelly into a 15 mL centrifuge tube with saline. Wash the Wharton's jelly 2-3 times to remove residual blood, and cut it into 1 mm 3 left and right size tissue blocks; 1.5) Inoculate the tissue blocks into the culture bottle with pre-added serum-free medium, and culture at 37℃ in a 5% incubator; 1.6) Perform the first half-volume medium exchange on the 7th day, and the second half-volume medium exchange on the 10th day. When the cell confluence reaches 60%-70%, harvest the primary human umbilical cord mesenchymal stem cells; 1.7) After counting the primary human umbilical cord mesenchymal stem cells, inoculate them into a T175 culture bottle at 9000 cells / cm 2 in a 5% CO2 incubator at 37℃. When the cell confluence reaches 80-90%, perform digestion and subculture until P3 human umbilical cord mesenchymal stem cells are obtained.

[0009] Further, the detection in step 2) includes sterile detection, mycoplasma, endotoxin, virus, morphology, surface marker, tri-lineage differentiation ability, telomere activity, STR, soft agar cloning, and immunomodulatory ability.

[0010] Further, in step 3), the ordinary transcriptome sequencing technology is used to quantitatively analyze the P3 generation SOX9 (fpkm) gene expression levels of human umbilical cord mesenchymal stem cells from different donors, and the specific steps are as follows: 3.1) When the human umbilical cord mesenchymal stem cells reach 80-90% confluence, remove the culture medium, wash twice with physiological saline, add TrypLE, digest for 2 min, add the stop solution, and repeatedly blow the cells; transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min; 3.2) Remove the supernatant, add Trizol, and gently blow repeatedly to mix; 3.3) Extract RNA, and after quality control, perform transcriptome sequencing; 3.4) Use bioinformatics technology to analyze the sequencing data to obtain different gene expression matrices; 3.5) According to the gene expression matrix, calculate the gene expression levels of human umbilical cord mesenchymal stem cells from different donors. SOX9

[0011] Further, the specific steps of step 4) are as follows: 4.1) Prepare the chondrogenic induction medium; 4.2) Collect and inoculate the cells; ​4.3) Chondrogenic induction differentiation culture: the chondrogenic induction differentiation culture medium is changed every 48 hours, and when the cells are aggregated into a group, the gun head is lightly picked to make it suspended culture. Continuous culture for 3-4 weeks;

[0012] 4.4) Staining and sectioning: aspirate the chondrogenic induction differentiation medium in the centrifuge tube, wash with PBS for 2 times, add 1 mL of 4% paraformaldehyde solution to each well for fixation for 60 min; aspirate the neutral formaldehyde solution, wash with PBS for 2 times, add Alcian blue staining solution to the centrifuge tube for 30 min; wash away the Alcian blue staining solution, wash with PBS for 2-3 times, embed the tissue with an embedding frame, and place it in a-80°C refrigerator overnight. The next day, perform frozen sectioning, add glycerol to the section, and then observe it under a microscope.

[0013] Further, the specific steps of step 4.1) are as follows: 4.1.1) The human mesenchymal stem cell chondrogenic induction basic medium and Alcian blue staining solution in the mesenchymal stem cell chondrogenic induction differentiation medium kit are stored at 2-8°C in the dark, and the components chondrogenic induction additive B, chondrogenic induction additive C, and chondrogenic induction additive D are stored at-20°C in the dark; all components must avoid repeated freezing and thawing and rewarming, each component has a shelf life of 1 year at the required temperature, the prepared premix is stored at 2-8°C, and the shelf life is 1 month, the complete culture medium is prepared and used immediately, and is stored at 2-8°C for no more than 72 h; 4.1.2) Premix preparation: thaw additive B and additive C at room temperature, after thawing, add A liquid, mix thoroughly, and configure the chondrogenic differentiation culture medium premix; thaw the additive, centrifuge at 1000 g for a short time to concentrate the solution at the bottom of the tube; after adding the solution in the tube to A liquid, wash the solution bottle twice with A liquid, and add the washing liquid to A liquid; 4.1.3) Induced complete culture medium: take 10 mL of premix and place it in a 15 mL centrifuge tube, add a straight additive D, mix thoroughly to make the induced complete culture medium, which is prepared and used immediately, and is placed at 2-8°C for no more than 72 h; 1 mL of premix needs to add 10 μL of additive D.

[0014] Further, the specific steps of step 4.2) are as follows: 4.2.1) Digest the cells: use an appropriate amount of TrypLE™ Select Enzyme to digest the cells to obtain a single cell suspension, centrifuge to collect the cells, resuspend the cells with cell complete culture medium, and count the number of cells; 4.2.2) Cell culture plate for osteogenic and adipogenic induction: coat with an appropriate amount of gelatin for 30 min in advance, then aspirate and air dry for use; 4.2.3) Seed the cells according to the following groups: 5×10 5Cells / tube + 2 mL chondrogenesis premix solution, centrifugation of cells, discard supernatant, culture with chondrogenesis complete culture solution; The application further provides a method for constructing a chondrogenic capacity quantitative standard in the screening method of the mesenchymal stem cell, and the method is characterized in that: the average value of the gene expression level of the FPMK value of the P3 generation cell strain of the human umbilical cord mesenchymal stem cell from different donors SOX9 is 16.5, and the average value of the FPMK is used as the quantitative standard of the chondrogenic capacity evaluation index of the human umbilical cord mesenchymal stem cell. SOX9

[0015] The application further provides an application of the mesenchymal stem cell to the treatment of arthritis.

[0016] The application provides a screening method and evaluation index for establishing high-quality human umbilical cord mesenchymal stem cell strains for the stem cell treatment of arthritis, so as to solve the problem of heterogeneity of the mesenchymal stem cell in the prior art, and establish a preclinical effectiveness evaluation system of the mesenchymal stem cell, wherein the average value of the SOX9 expression amount on the surface of the hUC-MSCs is used as the quantitative standard of the chondrogenic differentiation capacity of the high-quality human umbilical cord mesenchymal stem cell. SOX9 The higher the gene expression amount is, the stronger the chondrogenic differentiation capacity is. The cell selection standard is provided for the basic research and clinical transformation of the mesenchymal stem cell treatment of arthritis, so as to improve the treatment effect and rapidly promote the clinical application of the mesenchymal stem cell. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 FIG. 1 is a human umbilical cord mesenchymal stem cell identification result graph in the embodiment 1 of the application; Figure 2 FIG. 2 is a gene expression level fold line graph of the human umbilical cord mesenchymal stem cell from different donors in the embodiment 1 of the application; SOX9 Figure 3 FIG. 3 is a chondrogenic capacity situation graph of the human umbilical cord mesenchymal stem cell from the same donor in the embodiment 1 of the application; Figure 4 FIG. 4 is a correlation analysis graph of the expression level of the human umbilical cord mesenchymal stem cell and the chondrogenic capacity thereof in the embodiment 1 of the application; SOX9 Figure 5 FIG. 5 is a rat joint HE staining result graph in the embodiment 3 of the application; Figure 6 FIG. 6 is a detection result graph of inflammatory cytokines in the plasma of the rat in the embodiment 3 of the application. DETAILED DESCRIPTION

[0018] The application will be further described in detail below in combination with the drawings and specific embodiments: Embodiment 1 ​​​The application provides a screening method of mesenchymal stem cells, and the mesenchymal stem cells are human umbilical cord mesenchymal stem cells, and the method comprises the following steps: 1) screening and separating primary human umbilical cord mesenchymal stem cells from several human umbilical cords, and subculturing the primary human umbilical cord mesenchymal stem cells to P3 generation by using a serum-free culture medium. The specific operation is as follows:

[0019] 1.1) The research scheme is approved by the stem cell ethics committee of Xi'an High-tech Hospital; 1.2) screening healthy puerperae meeting the requirements of a biological sample library to collect samples, and signing an informed consent with the puerperae; 1.3) under a sterile condition, collecting the umbilical cord and the umbilical cord between two ligation positions with a length of not less than 20 cm, placing the umbilical cord in an umbilical cord storage and transportation bottle prepared in advance, transporting the umbilical cord to a cell culture room through a cold chain, and performing primary cell separation operation of umbilical cord mesenchymal stem cells; 1.4) placing the umbilical cord in a 15 cm culture dish added with an appropriate amount of normal saline, removing two ligation positions, cutting the umbilical cord into small pieces with a length of 2-3 cm, and then transferring the small pieces to a 250 mL wide-mouth bottle added with 75% alcohol for sterilization for 3 min; 1.5) transferring the sterilized umbilical cord to a 250 mL wide-mouth bottle added with normal saline for cleaning for 2-3 times to remove residual blood on the surface; 1.6) transferring the umbilical cord to a 15 cm culture dish added with normal saline, using forceps to peel the umbilical cord along the umbilical vein, removing one umbilical vein and two umbilical arteries, and separating Wharton's jelly into a 15 mL centrifuge tube added with normal saline, cleaning the Wharton's jelly for 2-3 times to remove residual blood, and cutting the Wharton's jelly into tissue blocks with a size of 1 mm 3 left and right; 1.7) inoculating the tissue blocks into a culture bottle added with a serum-free culture medium in advance, and culturing the tissue blocks in a 37℃, 5% incubator; 1.8) performing first half-volume medium replacement on the 7th day, performing second half-volume medium replacement on the 10th day, and harvesting the primary human umbilical cord mesenchymal stem cells when the cell fusion degree reaches 60%-70%; 1.9) after counting the primary human umbilical cord mesenchymal stem cells, inoculating the primary human umbilical cord mesenchymal stem cells into a T175 culture bottle according to 9000 cells / cm 2 , and culturing the primary human umbilical cord mesenchymal stem cells in a 37℃, 5% CO2 incubator until the cell fusion degree reaches 80-90%, and then performing digestion and subculture until P3 generation human umbilical cord mesenchymal stem cells are obtained; 2) performing related detection on the P3 generation human umbilical cord mesenchymal stem cells, and storing the P3 generation human umbilical cord mesenchymal stem cells in a cell bank after the detection is qualified, including but not limited to sterile detection, mycoplasma detection, endotoxin detection, virus detection, morphological detection, surface marker detection, three-line differentiation ability detection, telomere activity detection, STR detection, soft agar cloning detection and immune regulation ability detection. The results are as follows: Figure 1As shown, in terms of cell morphology, the hUC-MSCs obtained by us were small in cell volume, short spindle-shaped, and grew in a whirlpool shape, and the cell morphology was good. In terms of cell phenotype, the flow cytometry identification results showed that the hUC-MSCs obtained by us highly expressed CD44, CD73, CD90 and CD105, and did not express CD34, CD19, CD11b, CD45 and HLA-DR. In terms of differentiation ability, the results of three-system induction differentiation showed that after the hUC-MSCs obtained by us were induced into osteogenesis, and were stained with alizarin red, a large number of red-stained calcium nodules appeared; after being induced into adipogenesis, and being stained with oil red O, red-stained lipid droplets appeared; after being induced into chondrogenesis, and being stained with Alcian blue, a large number of blue-stained proteoglycans synthesized by chondrocytes appeared. The above results all meet the ISCT identification standard for MSCs, indicating that the separation and culture of hUC-MSCs are successful.

[0020] 3) Quantitative analysis of gene expression levels (FPKM) of different donor-derived human umbilical cord mesenchymal stem cells (P3 generation) by using ordinary transcriptome sequencing technology; the specific operation is as follows: SOX9 3.1) When the human umbilical cord mesenchymal stem cells are fused to 80-90%, remove the culture solution, wash twice with normal saline, add an appropriate amount of TrypLE, digest for 2 min, add a stop solution, and repeatedly blow the cells. Transfer an appropriate amount of cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min;

[0021] 3.2) Remove the supernatant, add an appropriate amount of Trizol, and gently blow repeatedly to mix; 3.3) Extract RNA, and after quality control, perform transcriptome sequencing; 3.4) Use bioinformatics technology to analyze the sequencing data to obtain different gene expression matrices; 3.5) According to the gene expression matrix, calculate the gene expression amount in different worker-derived human umbilical cord mesenchymal stem cells. SOX9

[0022] 4) According to the gene expression amount (FPKM value) in the sequencing results, the results are shown in SOX9 , 3 strains of low expression (UCMSCs09, UCMSCs10, UCMSCs34) and 3 strains of high expression (UCMSCs13, UCMSCs19, UCMSCs53) are selected respectively Figure 2 SOX9 SOX9 of Chondrogenic induction of human umbilical cord mesenchymal stem cell strains and evaluation of their chondrogenic ability. The specific operation is as follows:

[0023] 4.1) Chondrogenic induction medium preparation ​​​​​4.1.1 ) The human mesenchymal stem cell chondrogenic induction base medium and alizarin red staining solution in the mesenchymal stem cell chondrogenic induction medium kit (Guangzhou Saiye Biotechnology Co., Ltd., Catalog No.: HUXUC-9004) are stored at 2-8°C in the dark, and the components chondrogenic induction additive B, chondrogenic induction additive C, and chondrogenic induction additive D are stored at -20°C in the dark. All components must avoid repeated freezing and thawing and rewarming. The effective period of each component at the required temperature is 1 year. The prepared premix is stored at 2-8°C, and the effective period is 1 month. The complete culture medium is prepared and used immediately, and stored at 2-8°C for no more than 72 h.

[0024] 4.1.2) Preparation of premix: thaw additive B and additive C at room temperature, add A liquid after thawing, mix thoroughly, and configure chondrogenic differentiation culture liquid premix. (After thawing the additive, centrifuge 1000 g for a short time to concentrate the solution at the bottom of the tube. After adding the solution in the tube to A liquid, wash the solution bottle twice with A liquid, and add the wash liquid to A liquid.)

[0025] 4.1.3) Induced complete culture medium: take 10 mL of premix and add straight additive D to a 15 mL centrifuge tube, mix thoroughly to make induced complete culture medium. This liquid is prepared and used immediately, and stored at 2-8°C for no more than 72 h. The amount of additive D required for 1 mL of premix is 10 μL.

[0026] 4.2) Cell collection and inoculation 4.2.1) Digest the cells: use an appropriate amount of TrypLE™ Select Enzyme to digest the cells to obtain a single cell suspension. After centrifugal collection of the cells, resuspend the cells with cell complete culture medium, and count the number of cells; 4.2.2) Cell culture plates for osteogenic and adipogenic induction are coated with an appropriate amount of gelatin for 30 min. After the time is up, aspirate and air dry for use.

[0027] 4.2.3) Inoculate the cells as follows: 5 x 10 5 cells / tube (15 mL centrifuge tube) + 2 mL chondrogenic premix, centrifuge the cells, discard the supernatant, and culture with chondrogenic complete culture medium.

[0028] 4.3) Chondrogenic induction and differentiation culture: Change the chondrogenic induction and differentiation culture medium every 48 h, and use a gun head to lightly pick up the cells to make them suspend culture. Continue to culture for 3-4 weeks.

[0029] 4.4) Staining and sectioning: The chondrogenic induction medium in the centrifugal tube was aspirated, washed with PBS for 2 times, and 1 mL of 4% paraformaldehyde solution was added to each well for fixation for 60 min. The neutral formaldehyde solution was aspirated, washed with PBS for 2 times, and alcian blue staining solution was added to the centrifugal tube for 30 min. The alcian blue staining solution was washed away, washed with PBS for 2-3 times, embedded with embedding frame, and placed in a-80°C refrigerator overnight. The next day, frozen sections were cut, glycerol was added to the sections, and then the sections were observed under a microscope.

[0030] 5) Quantitative analysis of the chondrogenic ability of hUC-MSCs from different donors in step 4) was performed, i.e. the diameter of chondrocytes was measured. The results are shown in Figure 3 , and the hUC-MSCs from different donors all had certain chondrogenic ability.

[0031] 6) Correlation analysis was performed between the expression amount of SOX9 gene in hUC-MSCs from different donors in step 3) and the corresponding chondrocyte diameter in step 5). It was found that the expression amount of SOX9 gene in hUC-MSCs was proportional to the chondrocyte diameter, and the results are shown in Figure 4 .

[0032] 7) The expression amount of SOX9 gene in hUC-MSCs was used as a screening index for the strength of chondrogenic ability of mesenchymal stem cells, i.e. a biomarker for the chondrogenic ability of mesenchymal stem cells.

[0033] Example 2 This example 2 provides a method for constructing a quantitative standard for screening the chondrogenic ability of high-quality human umbilical cord mesenchymal stem cells, which comprises the following steps: 1) Human umbilical cord Wharton's jelly primary separation and culture of mesenchymal stem cells, subculture to P3 generation.

[0034] The specific operation is as follows: 1.1) This research scheme is approved by the stem cell ethics committee of Xi'an High-tech Hospital; 1.2) Screening healthy puerpera who meet the requirements of the biological sample library for sample collection, and signing informed consent with the puerpera; 1.3) Under sterile conditions, the umbilical cord with a length of not less than 20 cm between the two ligation sites is collected and placed in the prepared umbilical cord storage and transportation bottle, and then transported to the cell culture room through the cold chain for primary cell separation operation of umbilical cord mesenchymal stem cells; 1.4) The umbilical cord is placed in a 15 cm culture dish with appropriate physiological saline, the two end ligation sites are removed, and the cord is cut into 2-3 cm small pieces, and then transferred to a 250 mL wide-mouth bottle containing 75% alcohol for disinfection for 3 min; 1.5) After sterilization, the umbilical cord is transferred to a 250 mL wide-mouth bottle containing physiological saline for 2-3 washes to remove residual blood on the surface; 1.6) The umbilical cord is transferred to a 15 cm culture dish containing physiological saline, and the umbilical cord is peeled off along the umbilical vein using forceps to remove one umbilical vein and two umbilical arteries. The Wharton's jelly is separated into a 15 mL centrifuge tube containing saline, washed 2-3 times to remove residual blood, and cut into 1 mm 3 tissue blocks of equal size; 1.7) The tissue blocks are inoculated into culture bottles containing serum-free medium, and cultured at 37°C in a 5% CO2 incubator; 1.8) The first half-volume medium exchange is performed on the 7th day, and the second half-volume medium exchange is performed on the 10th day. When the cell confluence reaches 60-70%, the primary human umbilical cord mesenchymal stem cells are harvested; 1.9) After counting the primary human umbilical cord mesenchymal stem cells, they are inoculated into T175 culture bottles at a density of 9000 cells / cm 2 in a 5% CO2 incubator at 37°C. When the cell confluence reaches 80-90%, the cells are digested and passaged until P3 human umbilical cord mesenchymal stem cells are obtained; 2) P3 human umbilical cord mesenchymal stem cells are subjected to relevant tests, including but not limited to sterility testing, mycoplasma, endotoxin, virus, morphology, surface markers, tri-lineage differentiation ability, telomere activity, STR, soft agar cloning, and immunomodulatory ability.

[0035] 3) Quantitative analysis of gene expression levels in different donor-derived human umbilical cord mesenchymal stem cells (P3) is performed using ordinary transcriptome sequencing technology. The specific operation is as follows: SOX9

[0036] 3.1) When the human umbilical cord mesenchymal stem cells reach 80-90% confluence, remove the culture medium, wash twice with physiological saline, add an appropriate amount of TrypLE, digest for 2 minutes, add a stop solution, and repeatedly blow the cells. Transfer an appropriate amount of cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes;

[0037] 3.2) Remove the supernatant, add an appropriate amount of Trizol, and gently blow and mix; 3.3) Extract RNA, and after quality control, perform transcriptome sequencing; 3.4) Use bioinformatics technology to analyze the sequencing data and obtain different gene expression matrices; 3.5) Based on the gene expression matrix, calculate the gene expression levels of all human umbilical cord mesenchymal stem cell strains SOX9 .

[0038] ​4) The different donor-derived human umbilical cord mesenchymal stem cell (P3 generation) cell strains in step 3) SOX9 (FPMK value) gene expression level (as shown in Table 1), taking the average value, SOX9 The average value of gene FPMK = 16.5, which is the evaluation index of the chondrogenic ability of human umbilical cord mesenchymal stem cells SOX9 Quantitative standard.

[0039] Table 1 is the gene expression of different donor-derived human umbilical cord mesenchymal stem cells SOX9 Example 3 This embodiment 3 provides a kind of high quality human umbilical cord mesenchymal stem cells screened in example 1 is applied in the treatment of arthritis rat model.

[0040] 1) MSCs treatment arthritis rat model scheme: 1.1) Arthritis rat model establishment. In this study, Hulth method is used to model, after giving SD rats anesthesia, conventional shaving disinfection, taking bilateral knee joint, in the medial patella joint capsule is cut into joint. First, cut off the medial meniscus anterior horn, pull out and remove the front half of the medial meniscus, and then cut the anterior cruciate ligament with tissue scissors to verify success by anterior drawer test, then flush and suture the incision. After operation, free activity, postoperative penicillin continuous intramuscular injection for 3 days.

[0041] 1.2) MSCs treatment. After successful modeling, at 6 weeks after operation, arthritis model SD rats are randomly divided into 3 groups, i.e. normal saline group, low expression SOX9 hUC-MSCs cell (UC-MSCs09) group and high expression SOX9 hUC-MSCs cell (UC-MSCs19) group, 10 in each group. Joint cavity injection of hUC-MSCs treatment, 1 × 10 5 cells / each, 100 μL / each, once a week, for 5 weeks. After 1 week of treatment, 7% chloral hydrate was injected intraperitoneally to anesthetize and kill the rats to take synovial membrane, articular cartilage and serum for detection and observation.

[0042] 2) Detection of related indexes after MSCs treatment of arthritis model rats: 2.1) Macroscopic observation: immediately after each experimental rat was sacrificed by air embolism after anesthesia, the right hind limb knee joint tibial medial platform cartilage and synovial membrane were cleaned and disinfected. Scoring: ICRS scoring was used to evaluate cartilage repair, with a total score of 12 points. The higher the score, the better the cartilage repair effect, and the lower the score, the worse the cartilage repair effect.

[0043] ​2.2) Behavioral observation: All experimental rats were evaluated for knee joint at three time points, before modeling, 6 weeks after modeling (before treatment), and 1 week after the completion of all treatment interventions, using the Lequesne knee joint score, including pain response, gait, joint range of motion, and joint swelling of the experimental rats.

[0044] 2.3) Safety evaluation: The general safety of administration was evaluated during the 6-week treatment. The general condition of the rats was observed, including mental state, activity, fur color, water and food intake, bowel movements, body weight gain, and the like. After the rats were sacrificed, the liver and kidney organs were isolated. After deparaffinization and embedding, the changes in the organs were observed by staining to evaluate the systemic toxicity of local injection.

[0045] 2.4) HE, picro-sirius red staining, and toluidine blue staining: After staining, the synovial membrane and cartilage tissue of the rats in each group were observed under a microscope, and the cartilage degeneration Mankin score was evaluated. The results are shown in Figure 5 Compared with the UC-MSCs09 cell treatment group, the synovial membrane and cartilage tissue of the rats in the UC-MSCs19 cell treatment group had better morphology and structure.

[0046] 2.5) Micro-CT: Micro-CT was used to detect the bone microstructure of the knee joint.

[0047] 2.6) Immunohistochemical detection: Expression levels of col2, aggrecan, and MMP13.

[0048] 2.7) Cytokine detection: The rat whole blood was centrifuged at 1000 g for 10 min, and the supernatant was taken as plasma and stored in a -80°C refrigerator for standby. The inflammatory factors IL-1β, IL-6, and IFN-γ were detected, and the kit was purchased from Shanghai Trading Co., Ltd. Abways. The specific operation steps are as follows:

[0049] 2.7.1) According to the instructions, 1 mL of standard sample / diluent was taken into the freeze-dried standard, dissolved at room temperature, and stood for about 15 min. After mixing, the concentration was sequentially diluted to 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, and 0 pg / mL; 2.7.2) The concentration of the concentrated enzyme binding substance was diluted to 1:100 using biotinylated antibody diluent; 2.7.3) The concentrated enzyme binding substance was diluted to 1:100 using enzyme binding diluent; 2.7.4) The serum of each group of mice was diluted 1:2, and the suspended matter was removed by centrifugation; 2.7.5) Take out the plate from the balanced sealed bag, and respectively put the standard samples of different concentrations and the diluted plasma of each group of mice into the corresponding holes, seal the reaction holes with sealing tape, and incubate in a 37℃ incubator for 90 min; 2.7.6) Duplicate the sample and blank, and then wash the plate 4 times. Try to shake off the liquid in the hole, add 350 μL of washing liquid to each hole, stand for about 30 seconds, shake off the liquid again, and pat dry on filter paper;

[0050] 2.7.7) Leave the blank hole, and add 100 μL of biotinylated antibody working solution to each hole, seal the reaction hole, and incubate at 37℃ for 1 hour; 2.7.8) Wash the plate 4 times, leave the blank hole, add 100 μL of enzyme conjugate working solution to each hole, seal the reaction hole, and incubate at 37℃ for 30 min; 2.7.9) Wash the plate 4 times, add 100 μL of chromogenic agent to each hole, incubate at 37℃ for 15 min, and add 100 μL of stop solution to each hole and mix well; 2.7.10) Measure the OD450 value and record it, manually draw a standard curve with the standard sample concentration as the horizontal coordinate and the OD value as the vertical coordinate, connect the coordinate points of each standard sample with a smooth line, and the concentration of the sample can be found on the standard curve according to the OD value.

[0051] The results are shown in Table 1. Figure 6 Compared with the UC-MSCs09 cell treatment group, the UC-MSCs19 cell treatment group has lower levels of IL-1β, IL-6 and IFN-γ inflammatory factors in the plasma, and has better treatment effect.

[0052] In addition to human umbilical cord mesenchymal stem cells, other types of mesenchymal stem cells can also be screened by the above method and used for the treatment of arthritis.

[0053] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto, and the protection scope of the present disclosure should be subject to the protection scope of the claims.

[0054] The technical content not specifically described in the above embodiments and the present disclosure is the same as the prior art.

[0055] The present disclosure is not limited to the above embodiments, and all the contents described in the present disclosure can be implemented and have the good effects described above.

Claims

1. A method of screening mesenchymal stem cells, characterized by: SOX9 gene in hUC-MSCs is used as an index for screening chondrogenic differentiation ability of mesenchymal stem cells.

2. The method for screening mesenchymal stem cells according to claim 1, characterized in that: The mesenchymal stem cells are human umbilical cord mesenchymal stem cells, and the method comprises the following steps: 1) A plurality of umbilical cord tissues from healthy donors are screened, and sterile, mycoplasma and virus detection is performed, after passing the detection, primary human umbilical cord mesenchymal stem cells are separated by tissue block suspension method, and are subcultured to P3 generation by using serum-free medium; 2) The human umbilical cord mesenchymal stem cells P3 generation are detected, and after passing the detection, are placed into a temporary main cell bank; 3) The human umbilical cord mesenchymal stem cells P3 generation are collected, Trizol is added, and are gently repeatedly blown and mixed; RNA is extracted, and after passing quality control, transcriptome sequencing is performed; 4) Bioinformatics technology is used to analyze the sequencing data to obtain different gene expression matrices of human umbilical cord mesenchymal stem cells; according to the gene expression matrix, the expression amount of SOX9 gene of all human umbilical cord mesenchymal stem cell strains is calculated; 5) The chondrogenic ability of hUC-MSCs from different donors in step 4) is quantitatively analyzed, that is, the diameter of chondrogenic cells is measured; 6) The expression amount of SOX9 gene in hUC-MSCs from different donors in step 3) is correlated with the diameter of corresponding chondrogenic cells in step 5); 7) SOX9 gene in hUC-MSCs is used as a screening index for the strength of chondrogenic ability of hUC-MSCs, that is, a biomarker of chondrogenic ability of mesenchymal stem cells.

3. The method for screening mesenchymal stem cells according to claim 2, characterized in that: The specific steps of step 1) are as follows: 1.1) Under sterile conditions, the umbilical cord is collected, and the length of the umbilical cord between the two ligation sites is not less than 20 cm, which is placed in the umbilical cord storage and transportation bottle prepared in advance, and is transported to the cell culture room through cold chain transportation for primary cell separation operation of umbilical cord mesenchymal stem cells; 1.2) The umbilical cord is placed in a 15 cm culture dish containing an appropriate amount of physiological saline, the two end ligation sites are removed, and the small pieces of 2-3 cm are cut and transferred to a 250 mL wide-mouth bottle containing 75% alcohol for disinfection for 3 min; 1.3) The disinfected umbilical cord is transferred to a 250 mL wide-mouth bottle containing physiological saline and washed 2-3 times to remove the residual blood on the surface; 1.4) Transfer the umbilical cord to a 15 cm Petri dish with physiological saline, use forceps to strip the umbilical cord along the umbilical vein, remove one umbilical vein and two umbilical arteries, separate the Wharton's jelly into a 15 mL centrifuge tube with saline, wash the Wharton's jelly 2-3 times to remove residual blood, cut into 1 mm x 1 mm tissue blocks; 3 left and right size; 1.5) The tissue block is inoculated into the culture bottle with serum-free medium added in advance, and is cultured in a 37℃, 5% incubator; 1.6) The first half amount of liquid is changed at the 7th day, and the second half amount of liquid is changed at the 10th day, and when the cell confluence reaches 60%-70%, the primary human umbilical cord mesenchymal stem cells are harvested; 1.7) After counting the primary human umbilical cord mesenchymal stem cells, 9000 cells / cm 2 are inoculated into a T175 culture flask and cultured in a 37°C, 5% CO2 incubator until the cells reach 80-90% confluence, then digested and passaged until P3 generation of human umbilical cord mesenchymal stem cells are obtained.

4. The method of screening mesenchymal stem cells for treatment of arthritis according to claim 3, wherein: The detection in step 2) includes sterile detection, mycoplasma, endotoxin, virus, morphology, surface marker, three-line differentiation ability, telomere activity, STR, soft agar cloning and immunoregulatory ability.

5. The method for screening mesenchymal stem cells according to claim 4, characterized in that: In step 3), the ordinary transcriptome sequencing technology is used to quantitatively analyze the SOX9 (fpkm) gene expression level of human umbilical cord mesenchymal stem cells P3 generation from different donors, and the specific steps are as follows: 3.1) When the human umbilical cord mesenchymal stem cells are 80-90% confluent, remove the culture solution, wash twice with normal saline, add TrypLE, digest for 2 min, add the termination solution, and repeatedly blow the cells; transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min; 3.2) remove the supernatant, add Trizol, and gently repeatedly blow and mix; 3.3) extract RNA, and after quality control, perform transcriptome sequencing; 3.4) analyze the sequencing data using bioinformatics technology to obtain different gene expression matrices; 3.5) calculate the SOX9 gene expression amount in different types of human umbilical cord mesenchymal stem cells according to the gene expression matrix.

6. The method for screening mesenchymal stem cells according to claim 5, characterized in that: The specific steps of step 4) are as follows: 4.1) configure the chondrogenic induction culture solution; 4.2) collect and inoculate the cells; 4.3) chondrogenic induction and differentiation culture: the chondrogenic induction and differentiation culture solution is changed every 48 h, and when the cells are aggregated into clusters, use a gun head to lightly pick them up to make them suspend in culture. Continuously culture for 3-4 weeks; 4.4) staining and sectioning: aspirate the chondrogenic induction and differentiation solution in the centrifuge tube, wash with PBS for 2 times, add 1 mL of 4% paraformaldehyde solution to each well for fixation for 60 min; aspirate the neutral formaldehyde solution, wash with PBS for 2 times, add Alcian blue staining solution to the centrifuge tube for 30 min; wash away the Alcian blue staining solution, wash with PBS for 2-3 times, embed the tissue in an embedding frame, and place it in a-80℃ refrigerator overnight. The next day, perform frozen sectioning, add glycerol to the section, and then observe it under a microscope.

7. The method for screening mesenchymal stem cells according to claim 6, characterized in that: The specific steps of step 4.1) are as follows: 4.1.1) the human mesenchymal stem cell chondrogenic induction and differentiation medium kit is stored at 2-8℃ in the dark, the components chondrogenic induction additive B, chondrogenic induction additive C, and chondrogenic induction additive D are stored at-20℃ in the dark; all components must avoid repeated freezing and thawing and rewarming, each component has a shelf life of 1 year at the required temperature, the prepared premix is stored at 2-8℃, and the shelf life is 1 month, the complete culture medium is prepared and used immediately, and is stored at 2-8℃ for no more than 72 h; 4.1.2) premix preparation: thaw additive B and additive C at room temperature, after thawing, add A liquid, mix thoroughly, and configure the chondrogenic differentiation culture solution premix; centrifuge the thawed additive at 1000 g for a short time to concentrate the solution at the bottom of the tube; after adding the solution in the tube to A liquid, aspirate A liquid to wash the solution bottle twice, and add the washing liquid to A liquid; 4.1.3) induction complete culture solution: take 10 mL of premix and place it in a 15 mL centrifuge tube, add a straight additive D, mix thoroughly to make the induction complete culture solution, which is prepared and used immediately, and is placed at 2-8℃ for no more than 72 h; 1 mL of premix needs to add 10 μL of additive D.

8. The screening method of mesenchymal stem cells according to claim 7, wherein the specific steps of step 4.2) are as follows: 4.2.1) Digestion of cells: the cells were digested with an appropriate amount of TrypLE™ Select Enzyme to obtain a single cell suspension, and after centrifugal collection of the cells, the cells were resuspended with complete cell culture solution, and the number of cells was counted; 4.2.2) The cell culture plates for osteogenic and adipogenic induction were precoated with an appropriate amount of gelatin for 30 min, and after the time was up, the gelatin was aspirated and air-dried for use; 4.2.3) Cells were grouped for cell seeding; 5 x 10 5 cells / tube + 2 mL chondrogenic premix, centrifuged, supernatant discarded, and cultured with chondrogenic complete medium.

9. A method for constructing a chondrogenic capacity quantification standard in a method for screening mesenchymal stem cells according to claim 1, characterized by: The average value of the gene expression level of the FPMK value of SOX9 of the P3 generation cell strain of different donor-derived human umbilical cord mesenchymal stem cells was taken as the quantification standard of the SOX9 evaluation index of the chondrogenic ability of human umbilical cord mesenchymal stem cells, and the average value of FPMK was 16.

5.

10. Use of mesenchymal stem cells, characterized in that: for the treatment of arthritis.

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