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目的以Smad3基因为研究对象,筛选出该基因的有效siRNA干扰载体,并探讨下调表达Smad3基因对多肽N-乙酰氨基半乳糖转移酶(ppGalNAcTs)家族mRNA水平表达的影响。方法将事先构建的3个带有荧光标记的干扰载体分别转染人肝星状细胞株(HSC)中,通过RT-PCR、Western blot方法分别从mRNA水平及蛋白水平检测干扰效率。并通过转染有效的Smad3基因的siRNA表达载体,检测下调Smad3以后对糖基转移酶ppGalNAcTs家族mRNA表达的影响情况。结果成功筛选到Smad3基因的有效siRNA干扰载体,检测出转染了Smad3的siRNA表达载体的肝星状细胞中Smad3的mRNA和蛋白表达都受到了抑制,而糖基转移酶ppGalNAcTs的mRNA表达也随之发生了一些变化。结论成功筛选到一个Smad3基因的siRNA干扰载体,继而可转染人肝星状细胞,并抑制其中Smad3蛋白的表达,为研究siRNA方法在肝纤维化中的治疗作用提供了实验基础和理论支持。
Objective To screen Smad3 gene as an effective siRNA interference vector and investigate the effect of down-regulating Smad3 expression on the expression of ppGalNAcTs mRNA. Methods Three fluorescent plasmids were constructed and transfected into human hepatic stellate cell line (HSC) respectively. The interference efficiency was detected by RT-PCR and Western blot respectively from mRNA and protein levels. The expression of glycosyltransferase ppGalNAcTs family mRNA was detected by transfecting Smad3 gene siRNA expression vector. Results Smad3 gene was successfully screened by siRNA. The mRNA and protein expression of Smad3 were inhibited in HSCs transfected with Smad3 siRNA, and the mRNA expression of ppGalNAcTs Some changes have taken place. Conclusion The siRNA targeting vector of Smad3 gene was successfully screened and transfected into human hepatic stellate cells and inhibited the expression of Smad3 protein. This study provides the experimental basis and theoretical support for studying the therapeutic effect of siRNA in hepatic fibrosis.