The Promotion Effect Of Pannexin3 On The Chondrogenesis Of Bmscs By Lentivirus Transfection

Objection:The purpose of this study was to explore the influences on chondrogenesis in Pannxin3 transfected BMSCs.Methods:The bone marrow stem cells were extracted from SD mouse and cultured in vitro,P3 cells was chondrogenected,western blot was used to detecte the Pannexin3 protein level at 0d,3d,7d,14 d,21d.and then we do the process again,at this time the cells was devided into 3 groups: the Panx3 group:transfecting the Panenxin3 gene;the Mock group:transfecting the Mock gene; the control group:treated with nothing.Observing the cells by fluorescence microscope after 48 h,and evaluating the efficiency of transfection.inverted microscope was used to observe the growth of the cells;The proliferation of BMSCs was confirmed by MTT at 0h,24 h,48h,72 h.And the cells that cultured for 14 d was choosen to tobuidine blue stain to identify the type of the cells.RT-PCR was used to detecte the expression of the specific marker of chondrocytes:Collagen-type II and Aggrecan;Western-blot was used again to detecte the expression of Pannexin3 and.β-catenin;experimental data obtained with(x±S),Application of statistical package with SPSS19.0 statistical analysis,Data were statistically analyzed using a one-way analysis of variance, P<0.05 determined statistical.Results:1.The growth of BMSCs:after cultured for 48 h,only very little adherent cells,after culturing for 96 h,the shape of the cells was transforming into elongate,and arranged radially,the P3 cells can overspread the culture plate for more than 90%,and arranged spirally;2.The expression of Panx3 gradualy increased in the process of chondrogenesis of BMSCs,and reach its highest level at day 14(P<0.05),then decreased after day 14(P<0.05);3.the proliferation rate of the Panx3 group has decreased compared with the other two groups.3.The morphology of BMSCs after chondrogenesis procession:the cells was contractive,and transformed into elliptise,some of them was dead,rest of the cells aggregated;4.The Panx3 group and the Mock group cells was apeared remarkeble fluorescent,the transfection rate was aboat 85%;5.The MTT results suggest that the cells number was increased with time in all 3 group,but at 24 h,the Panx3 group cells’ OD value smaller than Mock gourp and Culture group cells(P<0.05),at 48 h and 72 h,the OD value siggnificantly smaller than the other 2 groups(P<0.05);6.After 14 days of chondrogenesis process,all 3groups has stained blue by toluidine blue;7.The expression of COLII and Aggrecan has largely increased compared with the other two groups.8.The expression of Pannexin3 was increased compared with the other two groups,but the expression ofβ-catenin was decreased(P<0.05).Conclusion:1.In the chondrogenetic differentiation process of SD-mouse’s BMSCs,the expression of Pannexin3 protein is increased since day 3,and a sharp increase at day 7,then reach its highest level at day 14;.2.Pannexin3 may inhibite the proliferation process;3.Pannexin3 can improve chondrogenetic differentiation;4.The decreasing level of β-catenin expression by Pannexin3 maybe one of the mechanism that improve the chondrogenetic differentiation.