| Objective: An effective model for obtaining and identifying human synovial mesenchymal stem cells is initially established,the optimal concentration for culturing the synovial mesenchymal stem cells containing the Chinese medicine serum for Bushen Huoxue is explored,and the proliferation effect of the serum of Bushen Huoxue on the synovial mesenchymal stem cells is observed,To clarify the effect of serum containing Chinese herbs of Bushen Huoxue on chondrogenic differentiation of synovial mesenchymal stem cells in patients with knee osteoarthritis,so as to further provide an effective basic theory basis for the treatment of KOA with the method of Bushen Huoxue and the experimental basis for the clinical treatment of KOA with the combination of serum containing Chinese herbs and cell tissue transplantation technology ?Methods: Experiment 1: Acquisition and identification of synovial mesenchymal stem cells: The synovial tissue of a patient diagnosed with knee osteoarthritis and undergoing total knee replacement was isolated,digested and subcultured.The biological cell morphology was observed under the microscope,and surface markers were detected by flow cytometry,and it was identified as synovial mesenchymal stem cells.Experiment 2: Selection of the optimal serum concentration for the cultivation of synovial mesenchymal stem cells: The rats were intragastrically administered with Chinese medicine for Bushen Huoxue and normal saline,respectively,for 7days before blood collection from the heart.After treatment,serum containing Chinese medicine for Bushen Huoxue and blank serum were obtained.The cells were cultured in the medium containing two different concentrations of serum for 72 h and the cell proliferation was detected by CCK8 method to obtain the optimal concentration of serum.Experiment 3: Bushen Huoxue Chinese medicine serum promotes the chondrogenic differentiation of synovial mesenchymal stem cells.1.Experimental grouping: According to the different induction and culture treatments,it was divided into three groups:blank serum group,inducer group and Chinese medicine inducer group(10% serum containing Chinese medicine+chondrogenic induction medium).2.At 0h,24 h,48h and 72 h,the proliferation and growth curves were drawn by MTS assay in each group.3.After 21 days of culture,it is stained with Alixine Blue to detect cartilage formation;4.After 21 d of induction culture,the gene expression levels of COL?,ACAN and SOX9 were measured by QPCR.5.After21 d of induction culture,the protein levels of COL ?,ACAN and SOX9 were measured by WB.Results: 1.After the synovial tissue of patients with knee osteoarthritis was separated,digested and cultured,the adherent growth of cells could be observed under the microscope,which was spindle-shaped.The cells had wide body,strong refractive power,aggregation and clonal growth,and the cells were densely arranged,in the shape of vortex and cord,and they fused with each other.The identification by flow cytometry showed that the expression of cell surface markers CD44,CD105 and CD90 were positive,while the expression of CD34 and CD45 was negative.The cell isolated and obtain are identified as synovial mesenchymal stem cells.2.The proliferation of cultured cells in blank serum and serum containing Chinese medicine with different concentrations was detected by CCK8 method for 72 h,and the optimal concentration for cultured cells was selected as 10%.3.By MTS,the OD490 values of the three groups were analyzed and the proliferation growth curve of the group containing Chinese medicine inducer was drawn.With the prolongation of time,the OD490 values of the three groups were significantly increased.After the synovial mesenchymal stem cells were cultured for 48 h in the three groups,the OD490 value in the blank serum group was higher than that in the inducer group and the group containing Chinese medicine inducer.4.The observation under the microscope of Alixine Blue staining by SMSCs showed that there were extensive blue regions in the induction group containing Chinese medicine,and there were a large number of positive cells,which were more than those in the blank serum group and the inducer group.5.After 21 d of cell culture,the relative gene expression levels of COL ?,ACAN and SOX9 were detected by QPCR.The relative gene expression levels of Col ?,ACAN and SOx 9 in the Chinese medicine inducer group were the highest,followed by the inducer group and the blank serum group.6.WB test was performed after 21 d of cell culture.The relative protein expression levels of COL ? and SOX9 in the group containing Chinese medicine inducer were the highest,followed by the inducer group and the blank serum group.The expression levels of ACAN relative proteins in the inducer group and the group containing Chinese medicine inducer were comparable and higher than that in the blank serum group.Conclusion: Chinese medicinal serum for Bushen Huoxue can effectively promote chondrogenic differentiation of synovial mesenchymal stem cells in patients with knee osteoarthritis. |
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