摘要
目的:研究肝星状细胞(HSC)中smad2特异性小干扰RNA(siRNA)对Ⅰ型胶原表达的抑制作用,探讨抗肝纤维化的基因治疗新方法。方法:设计合成靶向Smad2基因的siRNA,将筛选成功的siRNA瞬时转染入体外培养的肝星状细胞(HSC),并给予转化生长因子β(TGF-β)刺激,应用RT-PCR和Western blot技术检测对照组与实验组Ⅰ型胶原mRNA水平和蛋白水平表达差异,研究siRNA对Ⅰ型胶原表达的抑制作用。结果:siRNA能明显降低肝星状细胞中Smad2的RNA和蛋白的表达水平,证实筛选的siRNA有效,能特异性抑制Smad2的基因表达;TGF-β刺激肝星状细胞后,与对照组比较,siRNA转染组细胞外基质(ECM)成分Ⅰ型胶原的表达水平明显降低(P<0.05)。结论:siRNA能够抑制TGFβ对肝星状细胞的激活,阻断TGFβ-Smads传导通路,使Ⅰ型胶原分泌下调,有效抑制TGFβ诱导的肝纤维化。
Objective: To investigate the inhibitory effect of collagen I expression by Smad2 targeted siRNA in hepatic stellate cell, and to explore the new methods of Liver fibrosis gene therapy. Methods: Four target sequences of Smad2 mRNA were chosen by aid of computer designing. The lipidosome vector which expressed Smad2-target- siRNA, was established. The effective siRNA was screened then transfected into the hepatic stellate cells (HSC) in vitro culture, and give the transformation growth factor beta (TGF-β) stimulation. The mRNA expression and protein synthesis in the HSC cell line were tested by RT-PCR and western blot technology. Results: Both mRNA and protein levels of samd2 were effectively down-regulated. The screening siRNA effective was confirmed, and the expression of smad2 gene could be inhibited specifically. Collagen I in the cell culture medium of HSC was reduced as well. (p〈0. 05). Conclusions: Smad2 targeted siRNA could reduce the expression of Smad2 and collagen I in the HSC cell line and protecte HSC from the disadvantageous influences of TGF-β.
出处
《现代生物医学进展》
CAS
2011年第22期4328-4330,4361,共4页
Progress in Modern Biomedicine