Wnt7a在BMSCs成骨分化中的作用和机制

Function and mechanisms of Wnt7a in bone mesenchymal stem cells osteogenic differentiation

目的 探索Wnt7a在骨髓间充质干细胞（BMSCs）成骨分化中的作用和机制.方法 分离和培养人BMSCs,使用lipo2000在BMSCs中转染干扰Wnt7a和mock寡核苷酸序列,3d后通过Westen Blot 分析寡核苷酸干扰序列siRNA-Wnt7a的干扰效果.诱导培养10d后通过Westen Blot分析BMSCs细胞沉默Wnt7a对骨标志蛋白osteocalcin和骨调控蛋白Runx2表达的影响.诱导培养2周后通过Alizarin S Red染色检测在BMSCs中沉默Wnt7a对骨诱导的影响.结果 在BMSCs中转染寡核苷酸干扰序列Wnt7a和mock 对照序列,3d后Western Blot结果显示寡核苷酸干扰序列Wnt7a可明显降低BMSCs中Wnt7a的表达.诱导培养10d后通过Western Blot试验发现沉默Wnt7a后骨分化标志蛋白osteocalcin表达降低;同时检测到诱导培养10d后,BMSCs沉默Wnt7a组Runx2表达降低.诱导培养2周后通过Alizarin S Red染色显示骨形成受阻.结论 Wnt7a在BMSCs成骨分化中通过激活骨调控蛋白Runx2促进骨分化标志蛋白osteocalcin,在BMSCs成骨形成中扮演着重要角色.

Objective To explore the function and mechanisms of Wnt7a in bone msenchymal stem cells （BMSCs） osteogenic differentiation.Methods BMSCs were isolated and cultured.After transfection of siRNA-Wnt7a and mock in BMSCs and induction by osteogenic medium for 3 d,Western Blot were employed to detect the silencing effect of siRNA-Wnt7a.Then the effect of silencing Wnt7a on the expression of osteocyte marker osteocalcin and osteogenesis regulator protein Runx2 were detected by Western Blot after culture by osteogenic medium for 10 d.Finally,Alizarin S Red staining was used to detect the effect of silencing siRNA-Wnt7a on osteogenesis after culture by osteogenic medium for 2 weeks.Results The silencing effect of siRNA-Wnt7a was confirmed by Western Blot after transfection of oligocleotides siRNA-Wnt7a and mock into mesechymal stem cells.The silencing Wnt7a decreased the expression of osteocalcin and Runx2 after culture by osteogenic medium for 10 d.Alizarin S red staining results showed that silencing Wnt7a blocked bone differentiation.Conclusions The results demonstrated that Wnt7a could promot osteocyte marker osteocalcin expression by upregulating osteogenesis regulator protein Runx2 expression,which played a key role in BMSCs osteogenic differentiation.