小鼠纤维介素基因shRNA表达质粒的构建及体外RNA干扰

目的探讨纤维介素蛋白（fg12）的双链RNA（dsRNA）在体外对fg12凝血酶原酶基因表达的干扰作用及其规律。方法构建能产生鼠fg12（mfg12）的发夹状双链RNA（shRNA）的载体p-mfg12shRNA。将其与mfg12-EGFP融合基因表达质粒pEGFP—mfg12共转染（用量比为1：5）到中国仓鼠卵巢细胞（CHO细胞）作为实验组。另仅转染pEGFP—mfg12或共转染无关序列shRNA表达质粒和pEGFP—mfg12（用量比为1：5）作为对照组。转染24，48、72h后。观察mfg12-EGFP融合蛋白在CHO细胞中的表达情况，并通过流式细胞仪检测荧光细胞阳性率。分别将p—mfg12shRNA和mfg12cDNA表达质粒pcDNA3．1-mfg12共转染（用量比为1：5）到CHO细胞和人宫颈癌细胞（Hela细胞），作为试验组，另转染pcDNA3．1-mfg12或共转染无关序列shRNA表达质粒和pcDNA3．1-mfg12（用量比为1：5）或不转染任何质粒作为对照组。通过逆转录聚合酶链反应和免疫组织化学检测mfg12在CHO和Hela这两种细胞株中表达的情况。结果实验组较对照组的绿色荧光强度明显减弱。荧光细胞数明显减少。在CHO和Hela两种细胞株。均显示mfg12shRNA显著抑制mfg12mRNA和蛋白质的表达。结论本研究所构建的mfg12shRNA表达质粒p-mfg12shRNA可在体外高效特异地抑制mfg12的表达，为进一步的体内实验奠定了基础。

Construction of the p-mfgl2shRNA and its effect on mfg12 expression in vitro

Objective To construct the siRNA plasmid for mfgl2 gene, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/zero transplantation and fetal loss syndrome, and to investigate its inhibitory effects on mfgl2 expression in vitro. Methods A plasmid p-mfgl2shRNA complimentary to the sequerence responsible for the functional domain of mouse fgl2 (mfgl2) was constructed. The pcDNA3.1 mfgl2 expression construct was able to show a satisfactory fgl2 protein expression. The plasmid expression pEGFP and a construct expressing irrelevant shRNA with a random combination of the p-mfgl2shRNA sequence were used as controls. A pEGFP-mfgl2 expressing mfgl2-EGFP fusion protein was also constructed for screening of the effect of p-mfgl2shRNA on the mfgl2 expression. Results Cotransfection of p-mfgl2shRNA with pEGFP-mfgl2 decreased green fluorescent cells and the lightness of fluorescence within the cells at the 24 h, 48 h and 72 h post-transfection when compared with that in the control groups which were solely transfected with pEGFP-mfgl2. Furthermore the mfgl2 expression was significantly reduced when the pcDNA3.1 mfgl2 expression construct was cotransfected with p-mfgl2shRNA both at mRNA level by RT-PCR and protein level by RT-PCR, immunohis-tochemistry staining and FACS in both CHO cell and Hela cell lines. Conclusion The study demonstrated that the construct of p-mfgl2shRNA successfully interfered in the mfgl2 expression in vitro. It provides a basis for a further investigation of effect in