Experimental Study on Chondrogenic Differentiation of Synovial Mesenchymal Stem Cells by Traditional Chinese Medicine of Tonifying Kidney and Activating Blood
Abstract: Objective: To explore the effect of traditional Chinese medicine of tonifying kidney and activating blood on chondrogenic differentiation of synovial mesenchymal stem cells(SMSCs). Methods:One patient with knee osteoarthritis and total knee arthroplasty was selected. The synovial tissue isolated during the operation was digested and cultured, and the expression of surface markers of adherent cells was detected by flow cytometry. At the same time,20 SPF male SD rats were randomly divided into traditional Chinese medicine serum group and blank serum group. The traditional Chinese medicine serum group was perfused with traditional Chinese medicine decoction of tonifying kidney and activating blood,and the blank serum group was perfused with the same amount of 0.9% spdoium chloride solution. After 7 days,blood was taken from the heart to obtain traditional Chinese medicine serum and blank serum. SMSCs were cultured in different concentrations of traditional Chinese medicine serum and blank serum for 24, 48 and 72 hours, CCK- 8 was tested for its cell activity and toxicity,and the appropriate culture concentration serum was obtained. Through different induction treatments,they were divided into the following three groups: the blank group was cultured in complete medium containing appropriate concentration of blank serum, the inducer group was cultured in chondrogenic induction differentiation medium containing appropriate concentration of blank serum,and the traditional Chinese medicine inducer group was cultured in chondrogenic induction differentiation medium containing appropriate concentration of traditional Chinese medicine serum for 21 days.The chondrogenic differentiation was observed by alcian blue staining in each group, and the Western blot detection and realtime quantitative gene amplification fluorescence detection system(qPCR) data of chondrogenic differentiation indexes typeⅡ collagen(ColⅡ),aggrecan(AGG) and SOX9 were counted and analyzed. Results:The positive rates of CD90 and CD105 were 99.08% and 92.88% respectively, and the positive rates of CD31 and CD34 were 0.07% and 0.03% respectively. Within 72 hours, the absorbance value of 10% concentration intervention group increased and decreased gradually with the extension of time. At 72 h,the absorbance value of 10% serum containing traditional Chinese medicine intervention SMSCs was significantly higher than that of 0% concentration group(P<0.05),and the absorbance value of 30% serum containing traditional Chinese medicine intervention SMSCs was lower than that of 10% concentration group(P<0.05). Therefore,10% serum concentration was selected as the appropriate concentration for follow- up experiment. After 21 days of grouping induction culture,alixin blue staining showed that the blue of the traditional Chinese medicine induction group was extensive and aggregated,and the positive staining was significantly higher than that of the blank group and the inducer group. qPCR showed that the gene expression of Col Ⅱ , AGG and SOX9 in the traditional Chinese medicine inducer group was significantly higher than that in the inducer group(P<0.05), and they were higher than those in the blank group(P<0.05). Western blot showed that the levels of Col Ⅱ and SOX9 protein in the traditional Chinese medicine inducer group were significantly higher than those in the inducer group(P<0.05). The levels of AGG protein in the traditional Chinese medicine inducer group and the inducer group were the same(P>0.05), and higher than those in the blank group(P<0.05). Conclusion:Traditional Chinese medicine for tonifying kidney and activating blood can effectively promote the chondrogenic differentiation of SMSCs,and its mechanism is related to the up regulation of the expression of Col Ⅱ,AGG and SOX9.