RNA干扰沉默E637K负显性突变基因效应的研究

发布时间：2018-01-27 05:44

本文关键词： 长QT综合征快激活延迟整流性钾离子电流小干扰RNA 基因治疗　出处：《宁波大学》2012年硕士论文　论文类型：学位论文

【摘要】：背景与目的：长QT综合征(LQTS)是导致儿童和青少年猝死的主要病因，作为遗传性疾病，目前的治疗方法均具有局限性，且不能根治，个体化基因治疗是最有效的根治方法之一。在我国主要以由hERG基因突变引起的LQT2多见，开展hERG基因的研究，对LQTS特异性的治疗，心脏性猝死的防治有重要的意义。本实验旨在研究以呈负显性机制的E673K-hERG突变基因为靶点，探讨RNA干扰（RNA interference, RNAi）沉默E673K-hERG突变基因的效果及作用机制，从而实现基因表达的主动调控，为LQTS特异、安全和有效治疗提供了新策略和新途径。材料与方法：1.将WT-hERG和（或）E637K-hERG质粒以及pRK5-GFP转染至HEK293细胞中，构建WT-hERG、E637K-hERG和WT/E637K-hERG细胞模型。2.用siRNA对WT-hERG、E637K-hERG和WT/E637K-hERG细胞模型进行干预。3.采用全细胞膜片钳技术检测siRNA干扰不同细胞模型前后通道Ikr电流的变化。结果：在荧光倒置显微镜下观察，，瞬时转染后大约45%-60%HEK293细胞有绿色荧光蛋白表达，表明细胞模型构建成功。全细胞膜片钳技术记录Ikr电流如下： 1. siRNA对hERG通道Ikr电流幅度的影响：siRNA干扰WT/E637K-hERG后，hERG通道Ikr的激活电流和尾电流的电流幅度都较干扰前明显增大，分别增加了68.71%和65.92%；而siRNA干扰WT-hERG前后Ikr激活电流和尾电流的电流幅度没有明显变化。siRNA干扰E637K-hERG前后，均未检测到Ikr电流。 2.siRNA对hERG通道Ikr电流特性的影响： siRNA干扰WT/E637K-hERG后， Ikr激活电流的最大半激活电压（V1/2）和稳态失活电流的最大半失活电压（V1/2）与干扰前比较，分别向正向移动了9.62mV和17.41mV；但siRNA干扰WT-hERG前后激活电流的（V1/2）和稳态失活电流的（V1/2）与干扰前比较没有明显的变化。虽然siRNA干扰WT/E637K-hERG和WT-hERG前后Ikr电流的失活时间常数没有明显的差异，但siRNA干扰WT/E637K-hERG后减慢了通道Ikr电流失活速度。siRNA干扰WT/E637K-hERG和WT-hERG前后Ikr电流的失活恢复和去失活恢复时间常数没有明显影响。结论：本实验首次发现了siRNA有效地沉默了E637K-hERG，使WT/E637K-hERG通道Ikr电流幅度增大，并且纠正了WT/E637K-hERG通道Ikr电流的特性，使尾电流及稳态失活的的电向正向移动,并且减慢通道失活的速度，但对WT hERG通道电流没有明显的作用。实现了基因的主动调控，为LQTS特异、有效治疗方法提供了实验基础和理论依据。
[Abstract]:Background & objective: long QT syndrome (LQTS) is the main cause of sudden death in children and adolescents. As a hereditary disease, the current treatment methods are limited and can not be cured. Individualized gene therapy is one of the most effective methods of radical cure. In China, the LQT2 caused by hERG gene mutation is the most common. The study of hERG gene is carried out, and the specific treatment of LQTS is carried out. The aim of this study was to study the E673K-hERG mutation gene as the target. To investigate the effect and mechanism of silencing E673K-hERG mutation gene by RNA interference RNAi, so as to realize the active regulation of gene expression. It provides a new strategy and approach for the specific, safe and effective treatment of LQTS. Materials and methods: 1. WT-hERG and / or E637K-hERG plasmid and pRK5-GFP were transfected into HEK293 cells to construct WT-hERG. E637K-hERG and WT/E637K-hERG cell model. 2. WT-hERG with siRNA. E637K-hERG and WT/E637K-hERG cell model intervention .3.Whole-cell patch clamp technique was used to detect the Ikr currents in the channels before and after siRNA interference in different cell models. Change. Results: observed under fluorescence inverted microscope, green fluorescent protein was expressed in about 45-60H HEK293 cells after transient transfection. The results showed that the cell model was successfully constructed. The Ikr current was recorded by whole-cell patch clamp technique as follows: 1. The effect of siRNA on the amplitude of Ikr current in hERG channel after WT/E637K-hERG interference. The amplitude of activated current and tail current of hERG channel Ikr were increased by 68.71% and 65.92, respectively, compared with those before interference. However, the amplitude of Ikr activated current and tail current did not change significantly before and after siRNA interference with WT-hERG. SiRNA interfered with E637K-hERG before and after E637K-hERG. No Ikr current was detected. 2. The effect of siRNA on Ikr current characteristics of hERG channel: after siRNA interferes with WT/E637K-hERG. The maximum half-activation voltage V1 / 2 of Ikr activation current and the maximum half-inactivation voltage V1 / 2 of steady-state inactivated current were compared with those before interference. 9.62mV and 17.41mV were moved forward respectively. But siRNA interferes with V1 / 2 of activated current before and after WT-hERG) and V1 / 2 of steady-state inactivated current). There was no significant change in the Ikr current inactivation time constant before and after siRNA interference with WT/E637K-hERG and WT-hERG, although there was no significant difference in the inactivation time constant of Ikr current before and after siRNA interference. But siRNA interfered with WT/E637K-hERG and slowed down the inactivation rate of channel Ikr current. SiRNA interfered with WT/E637K-hERG and Ikr before and after WT-hERG. The inactivation recovery and deactivation recovery time constants of the current have no significant effect. Conclusion: siRNA effectively silenced E637K-hERG and increased the amplitude of Ikr current in WT/E637K-hERG channel for the first time. The characteristics of Ikr current in the WT/E637K-hERG channel are corrected, the tail current and the steady-state inactivated electric current are moved forward, and the inactivation speed of the channel is slowed down. But it has no obvious effect on the current of WT hERG channel. It realizes the active regulation of gene and provides the experimental basis and theoretical basis for the specific and effective treatment of LQTS.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.4

【参考文献】