中文摘要：

为了降低生物人工肝（bioartificial liver system）中肝细胞胆汁酸的分泌,构建了胆固醇7α羟化酶慢病毒RNA干涉载体,并转染人肝脏细胞（L-02）。根据绿色荧光蛋白的表达评估转染效率后进行流式分选,获得高表达慢病毒干涉载体的细胞,并以野生型L-02细胞和仅转染pSicoR空载体的L-02细胞作对照,观察肝细胞胆固醇7α羟化酶的表达以及培养上清中总胆汁酸含量。利用半定量PCR、实时荧光定量PCR及Western-blot等实验方法检测了转染细胞中基因的干涉效果,结果显示：与对照组相比,在mRNA水平,转染慢病毒siRNA载体的L-02细胞,其胆固醇7α羟化酶基因的表达量仅为野生型L-02细胞表达量的31.2%,为转染pSicoR空载体的L-02细胞的34.1%,干涉效率分别为68.8%和65.9%,均具有显著差异（P〈0.05）;Western-blot结果显示胆固醇7α羟化酶在蛋白质水平表达也明显受到抑制,表明转染慢病毒siRNA下调了肝细胞中胆固醇7α羟化酶基因的表达,减少了胆汁酸的分泌。以上研究结果表明,利用RNAi技术可以获得低表达胆固醇7α羟化酶基因的肝细胞,并有效降低肝细胞中胆汁酸的分泌,为临床上生物人工肝的构建及应用奠定基础。

英文摘要：

To reduce expression of CYP7A1 in L-02 Human Hepatic cells, three small interference RNA expression vectors of CYP7A1 were constructed and transfect into L-02 cells via lentiviral system. The efficiency of virus transfection was identified by expression of green fluorescence protein （GFP） analyzed by fluorescence microscope, then the high GFP expression L-02 cells were sorted by fluorescence-activated cell sorting （FACS） according to strong GFP expression. Analysis of efficiency of RNA interference on CYP7A1 was detected by Real time-PCR and Western-blot. Compared with that in untransfected L-02 cells or control lentivirus transfected L-02 cells, the expression of CYP7A1 at mRNA level in L-02 cells transfected with pSicoR-CYP7AI-1 lentivirus is down regulated up to 31.2% and 34.1%. Using Western blot, the CYP7A1 expression at protein level was also detected to be inhibited after pSicoR-CYP7A1 transfection. And, the secretion of CYP7A1 was obviously suppressed after pSicoR-CYP7A1 transfection. Take together, the results suggested that expression and secretion of CYP7A1 could be down regulated by lentivirus plasmids pSicoR-CYP7A1 transfection in L-02 cells, which may be useful to reduce secretion of bile acids in human hepatic cells and so that may play an important role in bioartificial liver clinical usage.