本文關(guān)鍵詞： 長(zhǎng)QT綜合征 快激活延遲整流性鉀離子電流 小干擾RNA 基因治療　出處：《寧波大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景與目的：長(zhǎng)QT綜合征(LQTS)是導(dǎo)致兒童和青少年猝死的主要病因，作為遺傳性疾病，目前的治療方法均具有局限性，且不能根治，個(gè)體化基因治療是最有效的根治方法之一。在我國(guó)主要以由hERG基因突變引起的LQT2多見(jiàn)，開(kāi)展hERG基因的研究，對(duì)LQTS特異性的治療，心臟性猝死的防治有重要的意義。本實(shí)驗(yàn)旨在研究以呈負(fù)顯性機(jī)制的E673K-hERG突變基因?yàn)榘悬c(diǎn)，探討RNA干擾（RNA interference, RNAi）沉默E673K-hERG突變基因的效果及作用機(jī)制，從而實(shí)現(xiàn)基因表達(dá)的主動(dòng)調(diào)控，為L(zhǎng)QTS特異、安全和有效治療提供了新策略和新途徑。 材料與方法：1.將WT-hERG和（或）E637K-hERG質(zhì)粒以及pRK5-GFP轉(zhuǎn)染至HEK293細(xì)胞中，構(gòu)建WT-hERG、E637K-hERG和WT/E637K-hERG細(xì)胞模型。2.用siRNA對(duì)WT-hERG、E637K-hERG和WT/E637K-hERG細(xì)胞模型進(jìn)行干預(yù)。3.采用全細(xì)胞膜片鉗技術(shù)檢測(cè)siRNA干擾不同細(xì)胞模型前后通道Ikr電流的變化。 結(jié)果：在熒光倒置顯微鏡下觀察，，瞬時(shí)轉(zhuǎn)染后大約45%-60%HEK293細(xì)胞有綠色熒光蛋白表達(dá)，表明細(xì)胞模型構(gòu)建成功。全細(xì)胞膜片鉗技術(shù)記錄Ikr電流如下： 1. siRNA對(duì)hERG通道Ikr電流幅度的影響：siRNA干擾WT/E637K-hERG后，hERG通道Ikr的激活電流和尾電流的電流幅度都較干擾前明顯增大，分別增加了68.71%和65.92%；而siRNA干擾WT-hERG前后Ikr激活電流和尾電流的電流幅度沒(méi)有明顯變化。siRNA干擾E637K-hERG前后，均未檢測(cè)到Ikr電流。 2.siRNA對(duì)hERG通道Ikr電流特性的影響： siRNA干擾WT/E637K-hERG后， Ikr激活電流的最大半激活電壓（V1/2）和穩(wěn)態(tài)失活電流的最大半失活電壓（V1/2）與干擾前比較，分別向正向移動(dòng)了9.62mV和17.41mV；但siRNA干擾WT-hERG前后激活電流的（V1/2）和穩(wěn)態(tài)失活電流的（V1/2）與干擾前比較沒(méi)有明顯的變化。雖然siRNA干擾WT/E637K-hERG和WT-hERG前后Ikr電流的失活時(shí)間常數(shù)沒(méi)有明顯的差異，但siRNA干擾WT/E637K-hERG后減慢了通道Ikr電流失活速度。siRNA干擾WT/E637K-hERG和WT-hERG前后Ikr電流的失活恢復(fù)和去失活恢復(fù)時(shí)間常數(shù)沒(méi)有明顯影響。 結(jié)論：本實(shí)驗(yàn)首次發(fā)現(xiàn)了siRNA有效地沉默了E637K-hERG，使WT/E637K-hERG通道Ikr電流幅度增大，并且糾正了WT/E637K-hERG通道Ikr電流的特性，使尾電流及穩(wěn)態(tài)失活的的電向正向移動(dòng),并且減慢通道失活的速度，但對(duì)WT hERG通道電流沒(méi)有明顯的作用。實(shí)現(xiàn)了基因的主動(dòng)調(diào)控，為L(zhǎng)QTS特異、有效治療方法提供了實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。
[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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R725.4

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