摘要
目的构建Smad2特异性siRNA表达克隆,探讨其在肝星状细胞(HSCs)中是否能够有效地抑制Smad2的表达。方法利用生物软件进行Smad2mRNA二级结构模拟,选择合适的靶位点,使用pBSKU6载体表达Smad2特异性siRNA,在体外转染HSC鄄T6细胞株,应用RT鄄PCR和Western鄄blot技术检测Smad2mRNA和蛋白的表达情况。结果成功构建siRNA表达克隆,转染后Smad2在基因和蛋白的表达水平均受到了明显抑制。在构建成功的4个克隆中,CR4抑制mRNA表达最为显著,达90%以上;抑制蛋白表达率也达到80%左右。同时CR1和CR4能有效地下调HSC分泌Ⅲ型胶原。结论在体外实验中,载体表达的特异性siRNA可有效地抑制HSCs中Smad2的表达,并可对激活的HSCs分泌细胞外基质产生有益的影响。
Objective To construct a siRNA vector-based system, which could down-regulate the expression of Smad2 in the hepatic stellate cells (HSC)-T6 cell line. Methods Four target sequences of Smad2 mRNA were chosen by aid of computer designing. The PBSKU6 vector, which could express Smad2-target-siRNA, were established. The mRNA expression and protein synthesis in the HSC-T6 cell line were tested by RT-PCR and western blot technology. Collagen Ⅲ was also measured in the culture media effectively. Results Both mRNA and protein levels of Smad2 were effectively down-regulated. Collagen Ⅲ in the cell culture medium of HSC-T6 was reduced as well. Conclusions Smad2 targeted siRNA could reduce Smad2 expression in the HSC-T6 cell line and protected HSCs from the disadvantageous influences of TGF-β.
出处
《外科理论与实践》
2004年第4期295-297,300,共4页
Journal of Surgery Concepts & Practice
关键词
RNA干扰
肝星状细胞
转化生长因子
肝纤维化
RNA interference
Hepatic stellate cells
Transforming growth factor
Liver fibrosis
