本文選題：DPP6 + 動作電位��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：心室纖顫(ventricular fibrillation)是引起心搏驟停(cardiac arrest)和心臟猝死(sudden cardiac death)最常見的原因,通過對病人的病因?qū)W、基因?qū)W研究和動物模型研究發(fā)現(xiàn)其發(fā)生常常是由于心臟離子通道的異常而出現(xiàn)。特發(fā)性室顫是遺傳性室顫的一種類型,由于其病因未知和臨床癥狀隱蔽而具有極高的致死率。前期有研究提示一種家族特發(fā)性室顫的發(fā)生與二肽基肽酶樣蛋白(Dipeptidyl peptidase-like protein,DPP)的亞型6(DPP6,又稱DPPX)的表達(dá)升高密切相關(guān),DPP6的異常高表達(dá)可引起家族特發(fā)性室顫的病理機(jī)制目前未知。DPP6是一種公認(rèn)的Kv4通道調(diào)節(jié)亞基,而后者是介導(dǎo)心臟中瞬間外向鉀電流(Ito)的主要通道,對正常心律的維持起重要作用,但目前對DPP6在心臟中的作用尤其是對心律失常發(fā)生的影響很少有報道,目前認(rèn)為DPP6異常導(dǎo)致心臟(尤其是浦肯野纖維)Ito電流增大是導(dǎo)致室顫的一個主要原因。雖然DPP6是Kv4通道的調(diào)節(jié)亞基,但有研究在神經(jīng)系統(tǒng)發(fā)現(xiàn)DPP6除影響A型鉀電流外,對鈉通道也有影響,并且發(fā)現(xiàn)在沒有Kv4通道表達(dá)的區(qū)域也有DPP6的表達(dá)。由此我們提出一個重要的問題,DPP6是否對心臟其它的離子通道也具有調(diào)節(jié)作用進(jìn)而影響室顫的發(fā)生。為回答此科學(xué)問題,本研究將在豚鼠心室肌細(xì)胞成功過表達(dá)DPP6后,通過觀測過表達(dá)DPP6對豚鼠心室肌細(xì)胞動作電位以及相關(guān)離子通道(如鈉通道、L型鈣通道等)的影響來發(fā)現(xiàn)其可能的細(xì)胞電生理學(xué)機(jī)制,此研究將為揭示DPP6在某種特發(fā)性室顫的病因、病理機(jī)制、疾病的預(yù)防和治療提供重要的實驗基礎(chǔ),具有重要的基礎(chǔ)研究和臨床意義。目的:(1)評價腺病毒介導(dǎo)的DPP6過表達(dá)載體在豚鼠心室肌細(xì)胞的過表達(dá)效果(2)過表達(dá)DPP6對豚鼠心室肌細(xì)胞的動作電位的影響。(3)過表達(dá)DPP6對豚鼠心室肌離子通道(鈉通道、L型鈣通道、IKr、IKs)的影響及機(jī)制。方法:(1)利用熒光顯微鏡觀測腺病毒攜帶的綠色熒光蛋白,實時定量PCR(qPCR)技術(shù),蛋白免疫印跡技術(shù)來評價腺病毒介導(dǎo)的DPP6過表達(dá)載體在豚鼠心室肌細(xì)胞的過表達(dá)效率;(2)應(yīng)用全細(xì)胞電流鉗技術(shù)記錄培養(yǎng)后的豚鼠心室肌細(xì)胞的動作電位,觀察過表達(dá)DPP6對心室肌細(xì)胞動作電位的影響;(3)利用電壓鉗技術(shù)檢測在豚鼠心室肌細(xì)胞過表達(dá)DPP6對鈉通道、鈣通道以及IKr、IKs電流的影響及其機(jī)制。結(jié)果:(1)感染DPP6過表達(dá)腺病毒的豚鼠心室肌細(xì)胞培養(yǎng)48小時后,用熒光顯微鏡可觀測到心室肌細(xì)胞有綠色熒光蛋白的表達(dá);通過qPCR技術(shù)檢測發(fā)現(xiàn)DPP6 mRNA的表達(dá)水平明顯提高;利用蛋白免疫印跡技術(shù)發(fā)現(xiàn)DPP6蛋白表達(dá)明顯提高;(2)過表達(dá)DPP6的豚鼠心室肌細(xì)胞與對照相比,動作電位時程顯著縮短,0相除極速率加快;(3)過表達(dá)DPP6的豚鼠心室肌細(xì)胞鈉電流密度顯著增加,激活曲線和失活曲線向超極化方向移動,且激活曲線移動更顯著,復(fù)活曲線無顯著變化;L型鈣電流密度顯著降低,激活曲線向去極化方向移動,失活曲線無變化;IKr和IKs無明顯變化。結(jié)論:(1)我們前期構(gòu)建的腺病毒介導(dǎo)的DPP6過表達(dá)載體能夠成功感染豚鼠心室肌細(xì)胞,可用于相關(guān)研究;(2)過表達(dá)DPP6致豚鼠心室肌細(xì)胞動作電位時程顯著縮短,0相除極速率加快,可能與過表達(dá)DPP6增加心室肌細(xì)胞鈉電流,減小鈣電流有關(guān),以上結(jié)果提示DPP6對心肌電活動的影響可能具有一定的病理意義。
[Abstract]:Ventricular fibrillation (ventricular fibrillation) is caused by cardiac arrest (cardiac arrest) and sudden cardiac death (sudden cardiac death) the most common cause, the etiology of the patient, genetic studies and animal model studies have found that the occurrence is often caused by abnormal cardiac ion channels. Idiopathic ventricular fibrillation is a type of genetic ventricular fibrillation, the mortality rate of the unknown etiology and clinical symptoms hidden but has high. Early studies suggest that a family of idiopathic ventricular fibrillation and two dipeptidyl peptidase like protein (Dipeptidyl peptidase-like, protein, DPP) subtype 6 (DPP6, also known as DPPX) elevated expression closely related to the abnormal expression of DPP6 may be the pathological mechanism of familial idiopathic ventricular fibrillation induced by.DPP6 is currently unknown regulatory subunit of a putative Kv4 channel, the latter is mediated by transient outward potassium current (Ito) in the heart of the main pass On the road, play an important role in maintaining normal rhythm, but the DPP6 function in the heart especially few reports on the influence of arrhythmia, it is believed that DPP6 leads to abnormal heart (especially Purkinje fibers) is the result of a Ito current increases mainly due to ventricular fibrillation. Although DPP6 is Kv4 channel the regulatory subunit, but studies have found that DPP6 removal effect of A type potassium current in the nervous system, also have an impact on the sodium channel, and the expression of Kv4 in the absence of channel expression are also DPP6. So we put forward an important question, whether DPP6 on the cardiac ion channels also play a role in the regulation of the other influence of the incidence of ventricular fibrillation. In order to answer this scientific problem, this study in guinea pig ventricular myocytes successfully after overexpression of DPP6, through the observation of overexpression of DPP6 on action potential of guinea pig ventricular myocytes and related ion channels (such as sodium. Road, etc.) of L type calcium channel effect to identify the possible electrophysiological mechanism, this study will reveal the etiology, DPP6 in a kind of idiopathic ventricular fibrillation pathological mechanism, provide an important experimental basis for the prevention and treatment of disease, and has important clinical significance in basic research. Objective: (1) evaluation of adenovirus mediated expression of DPP6 vector in the effect of expression in ventricular myocytes of guinea pig (2) effect of DPP6 overexpression on action potential in guinea pig ventricular myocytes. (3) the over expression of DPP6 in guinea pig ventricular myocytes ion channel (sodium channel, L type calcium channel, IKr, IKs) effect and the mechanism. Methods: (1) carrying green fluorescent protein by fluorescence microscope observation of adenovirus, real-time quantitative PCR (qPCR), Western blotting to evaluate the adenovirus mediated expression of DPP6 vector in the expression efficiency of guinea pig ventricular myocytes; (2) using the whole cell current clamp technique Action potentials in guinea pig ventricular myocytes cultured, observed the effect of DPP6 expression on ventricular myocyte action potential; (3) detected by voltage clamp technique in guinea pig ventricular myocytes overexpressing DPP6 on sodium channel, calcium channel and IKr, the current of IKs and its mechanism. Results: (1) training 48 hours of infection in guinea pig ventricular myocytes DPP6 expression after adenovirus, using fluorescence microscopy observed the expression of ventricular myocytes with green fluorescence protein; detected by qPCR found that the expression level of DPP6 mRNA increased significantly; by protein blot technique that DPP6 protein expression increased significantly; (2) the expression of DPP6 in guinea pigs the ventricular muscle cells compared with the control group, significantly shorten the action potential, 0 phase speed rate; (3) expression in guinea pig ventricular myocytes DPP6 density increased significantly, the curves of activation and inactivation to hyperpolarization The direction of movement, and the activation curve movement is more significant, the curve had no significant change; L type calcium current density was significantly decreased, the activation curve in the depolarizing direction, the inactivation curve changes; IKr and IKs did not change significantly. Conclusion: (1) adenovirus vector we previously constructed DPP6 expression vector can guide successful infection in guinea pig ventricular myocytes, can be used in related research; (2) overexpression process significantly reduced DPP6 induced action potential of guinea pig ventricular myocytes, 0 phase speed rate, and overexpression of DPP6 increased ventricular myocytes, decrease calcium current, the above results suggest that the effect of DPP6 on myocardial electrical activity may have pathological meaning.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R541.75

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王娜;華樂;楊甫;王玨;王志華;王瓊;董梅;王川;;腺病毒介導(dǎo)的DPP6過表達(dá)載體的構(gòu)建及其在大鼠心肌細(xì)胞內(nèi)表達(dá)的鑒定[J];暨南大學(xué)學(xué)報(自然科學(xué)與醫(yī)學(xué)版);2015年06期

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本文編號：1737346