慢病毒介导的Stat3沉默对成牙骨质细胞分化、凋亡、自噬的影响

Effects of Stat3 Gene Silencing by Lentivirus-Mediated shRNA on Cementoblast Differentiation,Apoptosis and Autophagy

目的:构建Stat3-shRNA慢病毒表达载体,观察慢病毒介导的信号传导及转录激活因子3(Stat3)沉默对小鼠成牙骨质细胞OCCM-30分化、凋亡、自噬的影响及相关机制。方法:构建含有Stat3shRNA的慢病毒载体,包装慢病毒后感染OCCM-30细胞,使用嘌呤霉素筛选出稳定细胞株。采用real-time PCR和Western Blot法检测慢病毒对Stat3的抑制效果。将细胞分成空白对照组、阴性对照组和Stat3抑制组三组,采用Western Blot检测Atg-5,Beclin-1,cleaved caspase-3,survivin蛋白水平的变化,使用real-time PCR法检测BSP,OCN,osterix在mRNA水平的变化。结果:成功构建Stat3-shRNA慢病毒表达载体并用慢病毒感染OCCM-30细胞,有效降低了Stat3的mRNA和蛋白的表达量。在mRNA水平上,Stat3抑制组BSP,OCN,osterix表达量降低。由Western Blot结果可知,Stat3抑制组Atg-5,Beclin-1,survivin蛋白表达量明显降低,cleaved caspase-3蛋白表达量增加。结论:慢病毒介导的Stat3-shRNA可有效抑制OCCM-30细胞内Stat3基因表达,从而抑制细胞分化和自噬,诱导凋亡,其相关机制可能是通过抑制Stat3信号转导通路实现的。

Objective:To construct mouse Stat3-shRNA lentiviral vector,then to investigate the effects of signal transducers and activators transcription 3(Stat3)gene silencing by lentivirus mediated Stat3-shRNA on the differentiation,apoptosis and autophagy of an immortalized murine cementoblast cell line OCCM-30.Methods:Mouse Stat3-shRNA lentivirus was constructed and infected into the cementoblast cell lines OCCM-30,and puromycin was used to select stable expression cell lines.Real-time PCR and Western Blot were used to measure the interference effects.Then,Western Blot was used to detect Atg-5,Beclin-1,cleaved caspase-3,and survivin protein expressions.Real-time PCR was used to measure mRNA expressions of BSP,OCN,and Osterix.Results:Stat3-shRNA lentivirus was successfully constructed and infected OCCM-30 cells.Both of the mRNA and protein levels of Stat3 were down-regulated in infected group.The real-time PCR results showed that in the infected group,the mRNA expression levels of BSP,OCN,and Osterix were reduced.Western Blot analyses demonstrated that the expression levels of Atg-5,Beclin-1,survivin were decreased,while cleaved caspase-3 expression level was increased in infected group.Conclusion:Stat3-shRNA lentivirus could down-regulate the expression of Stat3 in OCCM-30,so as to inhibit cells differentiation and autophagy,and accelerate cells apoptosis.The relative mechanism may be due to the block of Stat3 signaling pathway.