siRNA表达载体介导靶向NADH-细胞色素b5还原酶基因的RNA干扰实验研究

Experimental study on siRNA expressing vector-based RNA interference targeting human NADH-cytochrome b5 reductase

目的探索经载体介导的RNA干扰抑制人NADH-细胞色素b5还原酶(b5R)基因表达的可行性。方法应用网络在线软件设计并合成两条针对b5R mRNA的RNA干扰的DNA模板片段,分别将其克隆至经过改建、带有neo基因的pSilencer1.0-U6载体上,构建成siRNA真核表达载体;脂质体转染法将上述重组质粒转入人肺成纤维细胞和BEL-7402肝癌细胞株。通过半定量RT-PCR和荧光定量RT-PCR分析,检测所构建的重组siRNA真核表达载体对b5R基因表达的干扰效应。结果获得两个能在细胞内生成靶向b5R基因siRNA的重组载体pSib5R-1和pSib5R-2;其中pSib5R-2对成纤维细胞b5RmRNA的抑制率达81·7%,对BEL-7402细胞b5RmRNA的抑制率达60%。结论两种细胞b5R基因的表达均可被载体介导的RNA干扰有效抑制;成功构建了针对b5R基因的siRNA表达载体,为建立稳定表达siRNA的、b5R酶缺陷的细胞模型提供了实验基础。

Objective.. To explore the inhibition of expression of NADH cytochrome b5 reductase （b5R） via vector - based RNA interference （RNAi）. Methods： Two RNA interference DNA templates targeting b5R mRNA were designed via online software and synthesized. By ligation, the fragments were inserted into modified pSileucer1. 0 - U6 with a neo gene to construct the recombinant siRNA expressing plasmids. The identified recombinants were introduced in to human lung fibroblast and BEL -7402 cells with lipofectamin ^TM2000. Semi - quantitative and real - time fluorescence quantitative RT - PCR were carried out to analyze the suppression of b5R mRNA. Results： Two recombinant plasmids pSib5R - 1 and pSib5R -2, which direct the yields of siRNAs targeting b5R in cells, were constructed, when human lung fibroblast and BEL -7402 cells transfected with pSib5R - 1, the expression of b5R mRNA showed no obvious alteration as compared with controls. While transfected with pSib5R- 2, the expression of b5R mRNA was significantly suppressed with a suppression ratio of 81.7% and 60% in human lung fibroblast and BEL-7402 cells, respectively, indicating that pSib5 R -2 could trigger the RNAi of b5R gene effectively. Conclusion： The expression of b5R in both human lung fibroblast and BEL- 7402 could be suppressed by vector- based RNA interference successfully, we constructed the siRNA expressing vectors targe- ting b5R gene, which provided experimental base for establishment of cell lines with stable suppression of b5R gene.