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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.
In order to study the expression of neuron-specific genes in neurons and pancreatic islet cells by negative regulation of neuronal silencing factors (NRSF), NRSF expression was reduced by RNAi to further observe the expression of downstream genes regulated by NRSF.Methods: Promoter-luciferase (HIP-LUC) vector pcDNA3.1.RNAR sequence design software for the design of NRSF interference fragments and off-target control fragment, and then construct the lentiviral interference vector packaging produced lentivirus interference venom, with its Infection of HeLa cells with stable interference with NRSF cell lines, the interference effect of NRSF and its downstream gene expression were detected by RT-PCR, real-time quantitative PCR, Western blot, immunofluorescence staining and other methods to observe the impact of NRSF insulin promoter report Vector luciferase activity.We constructed a lentiviral vector with NRSF interference and successfully obtained HeLa cell line which stably interfered with NRSF and off-target control.The results of RT-PCR and real-time quantitative PCR showed that interference of NRSF interference fragment The efficiency was 56% (n = 6, P <0.01). Western blotting and immunofluorescence staining confirmed the NRSF (N = 3, P <0.01) .The results of RT-PCR indicated that the downstream gene began to be expressed after interfering with NRSF and the luciferase activity analysis showed that the activity of insulin promoter increased by 2.4 times (n = 3, The NRSF lentiviral vector was successfully constructed and the HeLa cell line stably interfered with NRSF was obtained.After interfering with NRSF, the downstream genes, especially the insulin gene, began to be expressed.This work will help us to further understand the role of NRSF in islet cell development and differentiation Regulation.