沉默结缔组织生长因子基因对肝星状细胞生长和细胞周期的影响

Effects of connective tissue growth factor gene-silencing on cell growth and cell cycle of hepatic stellate cells

目的·探讨沉默结缔组织生长因子(connective tissue growth factor,Ctgf)基因对大鼠肝星状细胞HSCT6的生长、细胞周期及其转化生长因子-β1(transforming growth factor-β1,TGF-β1)、Smad3和Smad7表达的影响。方法·采用RNA干扰技术构建Ctgf基因的pCDH/Ctgf-shRNA重组慢病毒载体。该重组载体经病毒包装后获得高感染力的pCDH/Ctgf-shRNA病毒颗粒用于感染HSCT6细胞。荧光显微镜观测被感染HSCT6细胞的绿色荧光蛋白(green fluorescent protein,GFP)的表达情况;CCK-8试剂盒检测被感染HSCT6细胞的生长变化;流式细胞术检测被感染HSCT6细胞的周期变化。Real-time PCR和Western blotting分别检测pCDH/CtgfshRNA病毒对HSCT6细胞的Ctgf基因的沉默效应及对TGF-β1、Smad3和Smad7表达的影响。结果·成功构建Ctgf基因的重组病毒载体pCDH/Ctgf-shRNA。荧光显微镜观测表明,被感染重组病毒的HSCT6细胞显著表达GFP。CCK-8检测结果证实,与对照细胞相比,沉默Ctgf基因的HSCT6细胞生长明显受抑,72 h开始两者差异具有统计学意义(P<0.05)。流式细胞术检测表明,沉默Ctgf基因的HSCT6细胞可被阻滞于S期。Real-time PCR和Western blotting检测表明,pCDH/Ctgf-shRNA病毒能有效沉默HSCT6细胞的Ctgf基因,下调TGF-β1、Smad3基因及其蛋白的表达,上调Smad7基因及其蛋白的表达;与对照比较,差异皆有统计学意义(均P<0.05)。结论·沉默Ctgf基因能抑制HSCT6细胞生长,使其TGF-β1、Smad3的表达下调,同时上调其Smad7的表达;这种生长受抑可能与其TGF-β1/Smads(Smad3/Smad7)信号通路受阻紧密相关。

Objective·To investigate the effects of silencing connective tissue growth factor(Ctgf)gene on the growth,cell cycle and the expression of TGF-β1,Smad3 and Smad7 of rat hepatic stellate cell line HSCT6.Methods·The recombinant lentivirus vector pCDH/Ctgf-shRNA of Ctgf gene was constructed by RNA interference.The recombinant vector was packaged to obtain highly infectious pCDH/Ctgf-shRNA lentivirus particles for HSCT6 infection.The expression of green fluorescent protein(GFP)in the transfected HSCT6 cells was observed under fluorescence microscope.The effects of Ctgf-shRNA lentivirus on the growth of HSCT6 cells were tested by CCK-8.The effects of Ctgf-shRNA lentivirus on the cell cycle of HSCT6 cells were analyzed by flow cytometry(FCM).The effects of Ctgf-shRNA lentivirus on the expression of mRNA of Ctgf,Tgf-β1,Smad3 and Smad7,and their proteins in HSCT6 cells were detected by real-time PCR and Western blotting,respectively.Results·The lentiviral vector pCDH/Ctgf-shRNA has been constructed successfully.The HSCT6 cells transfected by Ctgf-shRNA lentivirus significantly expressed GFP under fluorescence microscope.The results of CCK-8 confirmed that the growth of HSCT6 cells transfected by Ctgf-shRNA lentivirus was slower than that of controls and the differences were statistically significant after being cultured for 72 h(P<0.05).The results of FCM revealed that the growth of HSCT6 cells transfected by Ctgf-shRNA lentivirus was blocked in the S phase of cell cycle.The results of real-time PCR and Western blotting showed that the Ctgf-shRNA lentivirus effectively silenced Ctgf gene,down-regulated the expression of genes and encoding proteins of TGF-β1,and Smad3 of HSCT6 and up-regulated the expression of genes and encoding proteins of Smad7 of HSCT6 cells.The differences between transfected cells and controls were statistically significant(P<0.05).Conclusion·Silencing Ctgf gene can effectively inhibit the growth of HSCT6 cells,down-regulate the expression of TGF-β1 and Smad3 and up-regulate the expression of Smad7.The inhibition of the growth of HSCT6 cells may be closely related to interference of the TGF-β1/Smads(Smad3 and Smad7)signaling pathway.