Effects And Mechanism On Chondrogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells Induced By Growth Differentiation Factor 5 In Combination With Icaritin

Objective:To investgate whether growth differentiation factor(GDF-5)and icaritin(ICT)can induce the Chondrogenic differentiation of Sprague Dawley rats bone marrow mesenchymal stem cells(r BMSCs),and then explore the role of Wnt/?-catenin signaling in cartilage differentiation of r BMSCs.Methods:(1)BMSCs were collected from Sprague Dawley rats.The morphology was observed by inverted microscope.Cells growth curve was assaied by CCK-8.(2)BMSCs at passage 3 were divided into 6 groups: BMSCs group,BMSCs+ICT group,BMSCs+GDF-5 group,BMSCs+GDF-5+ICT group,BMSCs+GDF-5+ICT+SB216763 group,BMSCs+GDF-5+ICT+XAV-939 group and induced for 14 days.The Morphology change was observed by inverted microscope.(3)After 14 days of induction,Alcian Blue stain showed the changes of proteoglycans.(4)After 14 days of induction,RT-PCR was used to detecte the m RNA expression of Aggrecan,COL2 and Sox9;the protein expression levels of COL2 were detected by Western blot.After 14 days of induction,the expression of several key components of Wnt/?-catenin signaling including Dvl1,GSK-3?,?-catenin,at m RNA levels was determined by RT-PCR assay.The expression of ?-catenin at protein levels was measured by Western blot assay.Results:(1)BMSCs at passage 3 predominantly consisted of homogeneous fibroblast-like and spindle-shaped cells.(2)After 14 days of induction,the BMSCs group show no proteoglycan staining;compared with the BMSCs group,BMSCs+GDF-5 group,BMSCs+GDF-5+ICT group and GDF-5+ICT+SB216763 group showed deeper proteoglycan staining;compared with the BMSCs+GDF-5+ICT group,BMSCs+GDF-5+ICT group showed less proteoglycan staining.(3)The results of cartilage differentiation marker genes detected by RT-PCR and western blot showed that compared with the BMSCs group,BMSCs+GDF-5group,BMSCs+GDF-5+ICT group cartilage differentiation marker genes Aggrecan,COL2,Sox9 and the protein expression levels of COL2 were increased gradually(P<0.05);compared with the BMSCs+GDF-5 group,BMSCs+GDF-5+ICT group cartilage differentiation marker genes Aggrecan,COL2,Sox9 and the protein expression levels of COL2 were also increased.(4)The results of several key components of Wnt/?-catenin signaling determined by RT-PCR demonstrated that compared with the BMSCs group,the down-regulation m RNA expression of GSK-3?(P<0.05)and the up-regulation m RNA expression of Dvl1 and ?-catenin in BMSCs+GDF-5 group and BMSCs+ICT+GDF-5 group(P<0.05).The results of western blot analysis suggested that the expression of ?-catenin was also increased in BMSCs+GDF-5 group and BMSCs+ICT+GDF-5 group;compared with the BMSCs+GDF-5 group,BMSCs+GDF-5+ICT group showed Wnt/?-catenin signaling pathways related gene?-catenin m RNA and protein expression levels were gradually increased,while Gsk3? m RNA expression levels were gradually decreased;compared with the BMSCs+GDF-5+ICT group,BMSCs+GDF-5+ICT+SB216763 group showed cartilage differentiation marker genes Aggrecan,COL2,Sox9 and the protein expression levels of COL2,Wnt/?-catenin signaling pathways related gene ?-catenin m RNA and protein expression levels were gradually increased,while Gsk3? m RNA expression levels were gradually decreased;On the contrary,BMSCs+GDF-5+ICT+XAV-939 group cartilage differentiation marker genes and Dvl1 m RNA expression levels were decreased;m RNA and protein expression levels of COL2 and?-catenin were also decreased while Gsk3? m RNA expression levels were significantly increased.Conclusion:(1)GDF-5 in combination with icaritin can induce differentiation of rat BMSCs into cartilage,Icaritin can promotes chondrogenic differentiation of BMSCs inducedby GDF-5.(2)Wnt/?-catenin signaling plays a postive role in chondrogenic differentiation of r BMSCs.