RNA干扰法建立人NADH-细胞色素b5还原酶缺陷的细胞表型

Establishmeat of a cellular model with human NADH-cytochrome b5 reductase deficiency via RNA interference

目的探索经载体介导的RNA干扰法建立人NADH-细胞色素b5还原酶（EC1．6．2．2，NADH-cytochrome b5 reductase，65R）缺陷细胞系的可行性。方法设计并构建b5R的两种小干扰RNA（small interference RNA，siRNA）表达载体，脂质体转染法瞬时转染BEL-7402细胞，半定量逆转录-PCR分析重组载体的干扰效应；G418筛选两种表达载体稳定转染的BEL-7402细胞，并通过稳定转染的细胞克隆在b5R mRNA水平，酶活性，酶含量等方面干扰效果的鉴定，获得b5R缺陷的细胞克隆；噻唑蓝法测定b5R缺陷细胞的生长曲线。结果获得两个靶向b5R基因的siRNA重组表达载体pSib5R-1和pSib5R-2，其中pSig5R-2瞬时转染对BEL-7402细胞b5RmRNA的抑制率达68．3％。获得18个稳定转染的细胞克隆，pSib5R-2转染的克隆中有两个克隆b5RmRNA抑制率达48．2％和56．2％，酶活性抑制率为54．6％和63．5％，酶含量也有明显降低。b5R酶缺陷没有改变细胞的生长速度。结论b5R基因的表达可被载体介导的RNA干扰有效抑制，成功建立了b5R缺陷的细胞表型，为进一步研究b5R基因的功能以及探讨Ⅱ型隐性先天性高缺血红蛋白症的发生机制提供了实验依据和材料。

Objective To establish a cell line with human NADH-cytechrome b5 reductase （b5R） deficiency via RNA interference （RNAi）. Methods Two siRNA expressing vectors targeting the b5R mRNA were designed and constructed. Hepatecellular carcinoma BEL-7402 cells were transiently transfected with the two reeombinants by lipofectamineTM 2000, and semi-quantitative RT-PCR was carried out to analyze the suppression of bSR mRNA; BEL-7402 cells stably transfected with the two siRNA expressing vectors were selected in the media with G418. By analyses of the mRNA, enzymatic activity and protein level of b5R, several cell clones with deficiency of b5R were established. The cell growth curve of BEL-7402 cells with b5R deficiency was detected by M＇IT assay. Results Two siRNA expressing vectors targeting bSR mRNA were obtained, namely pSibSR-1 and pSibSR-2. When BEL-7402 cells were transfected tran- siently with pSibSR-2, the expression of bSR mRNA was significantly suppressed with a suppression ratio of 68.3%, indicating that pSib5R-2 could trigger the degradation of b5R mRNA effectively. Eighteen clones stably integrated exogenous plasmids were obtained. In two clones from pSibSR-2 transfection, the expression of bSR mRNA was suppressed by up to 48.2% and 56.2%, and the enzymatic activity was inhibited by up to 54.6% and 63.5%, respectively. The protein levels also decreased significantly. The defect of b5R did not change the cell growth rate. Conclusion The expression of bSR in BEL-7402 could be suppressed by vector-based RNA interference effectively. We established a cellular model with defect of b5R successfully, which can be used as a tool in investigation of the biological function of b5R and molecular mechanism of type II recessive congenital methemoglobinemia.