SIRT1通过β-catenin信号调控干细胞成软骨与成骨分化

SIRT1 regulates osteogenic and chondrogenic differentiation of stem cells by β-catenin signal

目的探讨SIRT1对干细胞成软骨、成骨分化的调控作用及机制。方法 C3H10T1/2感染腺病毒Ad-SIRT1后于普通培养基培养,通过荧光显微镜了解腺病感染情况,采用Western blot验证目的蛋白是否高表达;在不同时间点,通过Western blot、阿尔新蓝染色、碱性磷酸酶染色检测干细胞成骨、成软骨分化关键转录调控因子及相关分化标志物的表达情况;使用XAV-939抑制β-catenin信号后,再利用Western blot检测成骨、成软骨分化标志物表达情况。结果 C3H10T1/2感染腺病毒Ad-SIRT1后,SIRT1获得高表达;在诱导分化后第5、7、9天,Ad-SIRT1组成软骨分化的标志物Ⅱ型胶原、蛋白多糖,以及成骨分化的标志物Ⅰ型胶原、骨钙素较Ad-GFP组明显增加(P<0.05);阿尔新蓝染色结果发现Ad-SIRT1组软骨细胞外基质分泌较Ad-GFP组明显增加;碱性磷酸酶染色结果发现Ad-SIRT1组部分细胞着色为阳性,而Ad-GFP组无明显着色的细胞;同时发现诱导分化后的第3、5天,Ad-SIRT1组中成软骨、成骨分化关键转录调控因子Sox9、Runx2的表达较Ad-GFP组明显增加(P<0.05);Ad-SIRT1组与Ad-GFP组比较,β-catenin乙酰化水平明显降低(P<0.05),而抑制β-catenin信号后,与Ad-SIRT1组比较,Ad-SIRT1+XAV-939组干细胞成软骨及成骨分化标志物表达明显降低(P<0.05)。结论 SIRT1能够通过β-catenin信号促进干细胞的成软骨及成骨分化。

ObjectiveTo explore the regulative role of SIRT1 in chondrogenic and osteogenic differentiation of stem cells and investigate its underlying mechanism.MethodsPlasmid Ad-SIRT1 was used to infect C3H10T1/2 cells to express SIRT1 exogenously,and its expression were detected by fluorescence microscopy and Western blotting.The chondrogenic and osteogenic differentiation key transcription regulators and differentiation markers were detected by Western blotting,Alcian Blue staining and Alkaline Phosphatase staining at different time points.The differentiation markers were also detected by Western blotting after β-catenin inhibited by XAV-939.ResultsSIRT1 was overexpressed in C3H10T1/2 cells after Ad-SIRT1 infection.The Ad-SIRT1 infected cells exhibited significantly elevated protein levels of the chondrogenic(typeⅡcollagen and aggrecan)and osteogenic markers(typeⅠcollagen and osteocalcin)when compared to Ad-GFP group in 5,7 and 9 d after differentiation induction(P<0.05).The C3H10T1/2 cells after Ad-SIRT1 infection showed enhanced secretion of cartilaginous matrix by Alcian Blue staining,and more cells were positive to alkaline phosphatase staining,but no such stained cells were observed in the cells infected with Ad-GFP.In 3 and 5 d after differentiation,the chondrogenic and osteogenic key transcription regulators Sox9 and Runx2 were significantly increased at the protein level in Ad-SIRT1 infected cells than the Ad-GFP infected cells(P<0.05).The level of acetylatedβ-catenin was also significantly decreased(P<0.05),and XAV-939 treatment decreased above chondrogenic and osteogenic differentiation markers(P<0.05).ConclusionSIRT1 can promote chondrogenic and osteogenic differentiation by β-catenin signal.