中文摘要：

目的为腈水解酶2（NIT2）基因研究奠定基础。方法采用siRNA质粒转染技术制备NIT2基因沉默细胞模型,用筛网方法从大鼠肾脏中提取出肾小球用于培养原代系膜细胞,将NIT2靶序列构建pGU6/GFP/Neo质粒中（siNIT2质粒）,采用JETPRIME脂质体转染法将质粒转染入原代系膜细胞中,48h后荧光显微镜观察转染效率,利用SYBR GREEN 1定量PCR法检测NIT2基因表达,琼脂糖凝胶电泳观察PCR产物。结果荧光显微镜下转染阳性率可达50%,定量PCR结果示siNIT2质粒转染后NIT2 mRNA显著降低,琼脂糖电泳结果示PCR产物无明显杂带。结论 siRNA质粒转染法可抑制原代系膜细胞NIT2基因的表达;为NIT2基因功能研究奠定了基础。

英文摘要：

Objective To provide basis for the study of nitrilase 2（NIT2） gene.Methods NIT2 gene silencing cell model was prepared by siRNA plasmid transfection method,rat primary mesangial cells were cultured with glomeruli isolated from rat kidney by sieving.NIT2 targeting sequence was cloned into pGU6/GFP/Neo plasmid,JETPRIME was applied to transfect plasmid into cells,transfection efficiency was detected by fluorescence microscopy 48 hour later,SYBR GREEN 1 quantitative PCR was used to detect NIT2 gene expression and PCR products were analyzed by agarose gel electrophoresis.Results More than 50% positive cells were detected by fluorescence microscopy,the quantitative PCR showed that the NIT2 mRNA level decreased obviously after the transfection of siNIT2 plasmid,agarose gel electrophoresis showed that no non-specific bands in PCR products were found.Conclusions siRNA plasmid transfection can inhibit NIT2 gene expression in primary mesangial cells;this study provides basis for the study of nitrilase 2（NIT2） gene function.