中文摘要：

为研究神经元限制性沉默因子（NRSF）负调控神经元及胰岛细胞中神经特异性基因的表达，拟通过RNAi的方法降低NRSF表达以进一步观察其调控的下游基因的表达情况．构建含人胰岛素启动子．荧光素酶（HIP-LUC）的pcDNA3．1报告载体．通过RNAi序列设计软件进行NRSF干涉片段及脱靶对照片段的设计，然后构建慢病毒干涉载体．包装产生慢病毒干涉毒液，用其感染HeLa细胞获得稳定干涉NRSF的细胞株，利用RT-PCR、实时定量PCR、蛋白质免疫印迹、免疫荧光染色等方法检测NRSF的干涉效果及其下游基因的表达情况，观察干涉NRSF后胰岛素启动子报告载体荧光素酶活性的变化．构建具有NRSF干涉效果的慢病毒干涉载体成功，并获得了稳定干涉NRSF及脱靶对照的HeLa细胞株，RT-PCR及实时定量PCR检测结果表明，NRSF干涉片段的干涉效率为56％（n=6，P〈0．01），蛋白质免疫印迹、免疫荧光染色方法检测证实干涉后NRSF蛋白表达水平明显降低．RT-PCR实验表明干涉NRSF后下游基因开始表达，荧光素酶活性分析表明，干涉NRSF后胰岛素启动子活性增强了2．4倍（n=3，P〈0．01）．上述结果表明，成功构建NRSF的慢病毒干涉载体并获得稳定干涉NRSF的HeLa细胞株，干涉NRSF后，其下游基因特别是胰岛素基因开始表达．这一研究工作有助于我们进一步了解NRSF在胰岛细胞发育分化中的调控作用．

英文摘要：

The transcriptional repressor RE1 silencer transcription factor （NRSF/REST） is an important factor that restricts some neuronal traits in neurons. It has been confirmed that the insulin gene was regulated by NRSF via NRSE. Here, the relationship between NRSF and insulin gene was investigated by means of RNA interference （RNAi）. The expression of N-RSF was down-regulated by lentiviral RNA interference vector, and the interference efficiency is 56% （n = 6, P 〈 0.01）. When the expression of NRSF was down-regulated, genes regulated by NRSF, such as SCG10, BDNF, pax4 and insulin, began to express in HeLa cells. The human insulin promoter activity was up-regulated 2.4-fold after RNA interference （n = 3, P 〈 0.01）, confirmed by dual-luciferase assay.