The Experimental Study Of Cyclical Dynamic Compressive Loading Stimulate Chondrogenisis Of MSCs Transduced By The Lentiviral Vector Of SOX9 Gene Knockdown

Objective:To observe the effect of cyclical dynamic compressive loading on the chondrogenesis of MSCs and MSCs transduced by the lentiviral vector of SOX9 gene knockdown. Compare the difference effect.and analyze the importent significant of compressive loading and SOX9 on the chondrogenesis of MSCs. Methods:Mouse bone marrow mesenchymal stem cells were isolated and purified by adhesion method.After subculturing and proliferation,flow cytometry identify the cell.The sh RNA lentiviral vector with SOX9 gene silencing was constructed and identified.which was transfected into bone mesenehymal stem cells. The expression of target gene was detected by RT-PCR.MSCs(C), SOX9 gene silencing MSCs(si C).The cells were encapsulated in 2% alginate.pre-cultured( Mesenchymal Stem Cell Chondrogenesis Differentiation Medium:MCDM,7days)Accroding to the treatment differece,we divided our research into eight group: A group:C cell treated with cyclical dynamic compressive loading(DL) only.B group:C cell treated with DL and MCDM.C group:C cell treated with MCDM only. D,E,F group treated respectively with the same methods that using by A,B,C group,while the cell is si C.G,H group acted as control group,which treated with general Medium only(G group:C cell; H group:si group).The treatment all sustained 7 days. Cyclical dynamic compressive loading((2h/d, 20 Kpa, 50% at 0.5 Hz)) is produced by Flexcell-5000.RT-PCR were performed after the treatment.SOX9,ROCK1 and COL-Ⅱ were tested in RT-PCR experiment.COL-Ⅱprotein was tested using immunofluorescence technique. Results:RT-PCR test:the expression of SOX9 and COL-Ⅱm RNA : B group is respectively higher then C group and A group,C group is higher then G group,E group is respectively higher then F group and D group,D group is higher then H group(P<0.05),there is no difference between A group and G group,the difference between B group and E group is limited(P>0.05). The expression of ROCK1 m RNA:E group is higher then B group(P<0.05). Conclusion:cyclical dynamic compressive loading can accelerate the chondrogenesis of MSCs. Cyclical dynamic compressive loading can increase the expression of SOX9、COL-Ⅱ in the group of cell transduced by the lentiviral vector of SOX9 gene knockdown. Cyclical dynamic compressive loading can decrease the expression of ROCK1 m RNA.Cyclical dynamic compressive loading promote chondrogenisis of MSCs through the pathway correlated with SOX9.