摘要/Abstract
摘要： 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/Ctgf-shRNA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（PCtgf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。
关键词: 结缔组织生长因子, 肝星状细胞, 转化生长因子-&, beta, 1, Smad3, Smad7
Abstract:
Objective · To investigate the effects of silencing connective tissue growth factor (Ctgf) gene on the growth, cell cycle and the 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 constructedRNA interference. The recombinant vector was packaged to obtain highly infectious pCDH/Ctgf-shRNA lentivirus particles for HSCT6 infection. The 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 testedCCK-8. The effects of Ctgf-shRNA lentivirus on the cell cycle of HSCT6 cells were analyzedflow cytometry (FCM). The effects of Ctgf-shRNA lentivirus on the of mRNA of Ctgf, Tgf-β1, Smad3 and Smad7, and their proteins in HSCT6 cells were detectedreal-time PCR and Western blotting, respectively. Results · The lentiviral vector pCDH/Ctgf-shRNA has been constructed successfully. The HSCT6 cells transfectedCtgf-shRNA lentivirus significantly expressed GFP under fluorescence microscope. The results of CCK-8 confirmed that the growth of HSCT6 cells transfectedCtgf-shRNA lentivirus was slower than that of controls and the differences were statistically significant after being cultured for 72 h (P0.05). The results of FCM revealed that the growth of HSCT6 cells transfectedCtgf-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 of genes and encoding proteins of TGF-β1, and Smad3 of HSCT6 and up-regulated the of genes and encoding proteins of Smad7 of HSCT6 cells. The differences between transfected cells and controls were statistically significant (P0.05). Conclusion · Silencing Ctgf gene can effectively inhibit the growth of HSCT6 cells, down-regulate the of TGF-β1 and Smad3 and up-regulate the 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.
Key words: connective tissue growth factor (CTGF), hepatic stellate cell, transforming growth factor-β1 (TGF-β1), Smad3, Smad7
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