發(fā)布時間：2018-01-16 23:25

  本文關(guān)鍵詞：AngⅡ?qū)π氖壹〖?xì)胞Ikr電流的調(diào)節(jié)及其信號傳導(dǎo)機(jī)制　出處：《河北醫(yī)科大學(xué)》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 血管緊張素II 離子通道 動作電位 心室 鉀通道 受體 蛋白激酶C 信號轉(zhuǎn)導(dǎo)

【摘要】： 心肌肥厚、心衰是冠心病、高血壓、心瓣膜病等多種心血管病的常見合并癥。心肌肥厚、心衰時發(fā)生病理性電生理重構(gòu)使得心臟的電生理不穩(wěn)定性增加,常常伴發(fā)心律失常,心衰病人約一半以上因惡性心律失常的發(fā)生而猝死(即心性猝死Sudden cardiac death, SCD)。腎素-血管緊張素系統(tǒng)(renin-angiotensin system, RAS)是由腎素、血管緊張素以及其受體構(gòu)成的重要體液系統(tǒng),在調(diào)節(jié)心血管系統(tǒng)的正常生理功能與高血壓、心肌肥大、充血性心力衰竭等諸多心血管的病理過程中具有重要作用。RAS不僅存在于體液系統(tǒng),而且在包括心臟、血管在內(nèi)的許多組織中也有RAS,在局部調(diào)節(jié)生理病理過程。血管緊張素II(Ang II)是其該系統(tǒng)發(fā)揮作用的主要體液因子,其受體有1型(AT1)和2型(AT2)兩種。近幾年大規(guī)模的臨床試驗(yàn)研究發(fā)現(xiàn),通過血管緊張轉(zhuǎn)化酶抑制劑(angiotensin-converting enzyme inhibitors,ACEI)抑制Ang II的合成或AT1受體阻斷劑可能因減少致命性心律失常的發(fā)生而明顯降低病人死亡率。實(shí)驗(yàn)表明,抑制RAS可有效治療缺血再灌誘發(fā)的心律失常。ACE抑制劑和AT1受體阻斷劑成為目前臨床高血壓、心衰病人治療的重要藥物。然而,這些藥物潛在抗心律失常的機(jī)制尚不清楚。 正常心臟電活動取決于各種離子有序的跨膜轉(zhuǎn)運(yùn),先天遺傳基因改變或后天獲得性因素(如心臟病理性變化、藥物等)均可引起離子通道功能紊亂,構(gòu)成了心律失常的物質(zhì)基礎(chǔ)。心肌肥厚、心衰時最突出的表現(xiàn)是心肌細(xì)胞復(fù)極化過程變慢,而導(dǎo)致動作電位的延長,為細(xì)胞產(chǎn)生后除極、尖端扭轉(zhuǎn)性室速等心律失常創(chuàng)造了前提條件。心肌復(fù)極鉀電流存在明顯的種屬差別。大動物、人的復(fù)極化鉀電流主要是Iks和Ikr。目前認(rèn)為,人心肌中Ikr由HERG(human ether-a-go-go-related gene)基因編碼,HERG通道的激活引發(fā)動作電位3期復(fù)極的開始,對心肌的復(fù)極化有至關(guān)重要的作用。由于HERG基因突變或藥物誘導(dǎo)的Ikr阻斷減少心臟HERG電流,分別產(chǎn)生與7號常染色體有關(guān)的先天性LQTS和獲得性LQTS,這兩種LQTS的形成都與延遲心臟復(fù)極,延長體表心電圖的QT間期,有較高的尖端扭轉(zhuǎn)型室性心律失常和猝死的危險。最近的實(shí)驗(yàn)表明一些G蛋白偶聯(lián)受體如α-和β-腎上腺素受體通過細(xì)胞內(nèi)第二信使如cAMP,PKA和PKC調(diào)節(jié)HERG通道。然而,迄今為止Ang II對心室細(xì)胞復(fù)極過程起重要作用的Ikr的影響尚了解甚少。 故本研究利用膜片鉗技術(shù),在急性分離的豚鼠心室肌細(xì)胞和異源表達(dá)系統(tǒng)上觀察了Ang II對kr/HERG電流的影響,并分析了中介Ang II作用的細(xì)胞內(nèi)信號途經(jīng),為病理情況下心律失常發(fā)生的分子機(jī)制及解釋ACEI、AT1受體阻斷劑的藥效提供實(shí)驗(yàn)依據(jù)。 第一部分表達(dá)HERG蛋白穩(wěn)定細(xì)胞株的建立及其鑒定 目的:建立穩(wěn)定表達(dá)HERG通道蛋白的HEK293細(xì)胞株。 方法:用Lipofectin 2000轉(zhuǎn)染試劑將HERG基因轉(zhuǎn)染到HEK293細(xì)胞,經(jīng)G418篩選,用熒光顯微鏡,Western Blot和全細(xì)胞膜片鉗技術(shù)檢測HERG基因在HEK293細(xì)胞中的穩(wěn)定表達(dá)情況。 結(jié)果:G418篩選,培養(yǎng)4周陽性克隆形成。熒光顯微鏡,Western Blot和全細(xì)胞膜片鉗技術(shù)檢測,證實(shí)HERG基因得到了穩(wěn)定表達(dá)并記錄到了轉(zhuǎn)染成功的HERG電流,通過HERG通道對其特異性阻斷劑E-4031的IC50,進(jìn)一步表明在HEK細(xì)胞上正常HERG通道蛋白表達(dá)。 結(jié)論:建立了穩(wěn)定表達(dá)HERG基因的HEK293細(xì)胞株,表達(dá)的HERG蛋白能形成有功能的通道,產(chǎn)生HERG電流。 第二部分Ang II作用于AT1受體抑制心室肌細(xì)胞的快速延遲整流鉀(Ikr)電流 目的:觀察Ang II對豚鼠心室肌細(xì)胞Ikr的影響。 方法:在急性分離的豚鼠心室肌細(xì)胞及共表達(dá)HERG和人Ang II AT1受體基因的HEK293細(xì)胞上,全細(xì)胞膜片鉗技術(shù)記錄Ikr/HERG電流,觀察Ang II對電流影響。 結(jié)果:Ang II以濃度依賴的方式抑制Ikr,IC50是8.9 nM。Ang II (100 nM)延長心室肌細(xì)胞動作電位APD50和APD90,分別延長20%和16%。AT1受體阻斷劑losartan(1μM)完全取消AngII對Ikr電流的抑制作用。在共表達(dá)HERG和AT1受體基因的HEK293細(xì)胞上,Ang II也抑制HERG電流,減慢通道的激活、去活和再恢復(fù)。PKC抑制劑stausporine和Bis-1明顯減弱Ang II對Ikr電流的抑制作用。 結(jié)論:Ang II作用于AT1受體,經(jīng)過PKC途經(jīng)抑制心室肌細(xì)胞的Ikr電流。這對解釋病理情況下,如心肌肥厚心衰時,Ang II的升高導(dǎo)致心律失常的發(fā)生提供了一個可能的機(jī)制。 第三部分Ang II通過PKCε亞型抑制Ikr/HERG電流 目的:分析中介Ang II抑制Ikr/HERG電流的PKC亞型及其下游分子機(jī)制。 方法:在急性分離的豚鼠心室肌細(xì)胞及共表達(dá)HERG和人Ang II AT1受體基因的HEK293細(xì)胞上,觀察選擇性PKC抑制劑、激動劑及PKC亞型轉(zhuǎn)位抑制肽對Ang II作用的影響,分析中介其作用的PKC亞型;觀察HERG通道磷酸化位點(diǎn)突變對Ang II作用的影響,分析PKC下游的分子機(jī)制。 結(jié)果:在HEK293細(xì)胞上,應(yīng)用PKC抑制劑stausporine和Bis-1明顯減小了Ang II對HERG電流的抑制作用;長期孵育PMA下調(diào)PKC也明顯減小了Ang II對HERG電流的抑制作用。細(xì)胞內(nèi)鈣螯合劑濃度增加不影響Ang II抑制HERG電流。特異性亞型PKC抑制劑G?-6976和G?-6983不影響Ang II對HERG電流的抑制作用;PKCε亞型的轉(zhuǎn)位抑制劑明顯對抗Ang II對HERG電流的抑制作用。在豚鼠心室肌細(xì)胞上,PKCε亞型的轉(zhuǎn)位抑制劑明顯減小Ang II對Ikr的抑制作用。HERG通道的磷酸化位點(diǎn)突變后,幾乎取消了Ang II對HERG電流的抑制作用。 結(jié)論:(1)Ang II主要通過PKCε的激活抑制Ikr/HERG。(2)Ang II通過PKCε使HERG通道蛋白磷酸化,調(diào)節(jié)HERG通道的功能。 結(jié)論 1.本研究建立了表達(dá)HERG基因的穩(wěn)態(tài)HEK293細(xì)胞株,表達(dá)的蛋白能形成有功能的通道,產(chǎn)生HERG電流。 2. Ang II作用于AT1受體,經(jīng)過PKC途經(jīng)抑制心室肌細(xì)胞的Ikr電流。這對解釋病理情況下,如心肌肥厚心衰時,Ang II的升高導(dǎo)致心律失常的發(fā)生提供了一個可能的機(jī)制。 3. Ang II主要通過PKCε的激活抑制Ikr/HERG。 4. Ang II有可能通過PKCε使HERG通道磷酸化,調(diào)節(jié)HERG通道的功能。
[Abstract]:Cardiac hypertrophy, heart failure is a common complication of coronary heart disease, hypertension, heart disease and other cardiovascular disease. Pathological myocardial hypertrophy, electrophysiological remodeling increases cardiac electrophysiological instability is often associated with heart failure, arrhythmia, heart failure patients about more than half due to the occurrence of malignant arrhythmia and sudden death (i.e. sudden cardiac death Sudden cardiac death, SCD). The renin-angiotensin system (renin-angiotensin system RAS) is a renin angiotensin system consisting of an important body fluid and its receptor, in normal physiological function and high blood pressure, regulating cardiovascular hypertrophy, the pathological process of congestive heart failure and other cardiovascular plays an important role in not only.RAS in fluid system, but also in many tissues including heart, blood vessels, there are RAS, regulation of physiological and pathological processes in local angiotensin II (Ang. II) is the main body factors play its role in the system, its receptor type 1 (AT1) and type 2 (AT2) two. In recent years, large-scale clinical trials found by angiotensin converting enzyme inhibitors (angiotensin-converting enzyme, inhibitors, ACEI) inhibited Ang synthesis of II or AT1 receptor antagonist may because of the reduction of fatal arrhythmia and reduce patient mortality. Experimental results show that the inhibition of RAS arrhythmia.ACE inhibitors and AT1 receptor induced effective treatment of reperfusion blockers has become important clinical hypertension, drug therapy in patients with heart failure. However, the mechanism of these potential antiarrhythmic drugs is not clear.
Normal cardiac electrical activity depends on the transmembrane transport in various ions orderly, congenital genetic change or acquired factors (such as cardiac pathological changes, drug etc.) can cause ion channel dysfunction, constitute the material basis of arrhythmia. Myocardial hypertrophy, heart failure is the most prominent manifestation of myocardial repolarization process slowly, which led to the prolongation of the action potential, as the cells produced after depolarization, torsades de pointes arrhythmias such as creating preconditions. There is obvious difference between species. Large animal myocardial repolarization potassium current, repolarising potassium current one is mainly Iks and Ikr. currently believe that the human heart by HERG (Ikr human ether-a-go-go-related gene) gene encoding, to activate the HERG channel lead to action potential repolarization of phase 3, have crucial effect on myocardial repolarization. Due to mutations in the HERG gene or drug induced by blocking Ikr Reduce the cardiac HERG current, respectively, and 7 chromosome associated congenital and acquired LQTS LQTS, the two LQTS Chengdu and delayed cardiac repolarization electrocardiogram, prolonged QT interval, have a higher risk of torsade de pointes ventricular arrhythmias and sudden death. Recent experiments showed that G protein coupled receptors such as alpha and beta adrenergic receptors by intracellular second messengers such as cAMP, PKA and PKC regulate HERG channels. However, so far Ang II cells on ventricular repolarization process effect of Ikr is still poorly understood.
This study utilizes the patch clamp technique in isolated guinea pig ventricular myocytes and heterologous expression system on the effect of Ang II on kr/HERG current, and analyzes the role of II Ang mediated intracellular signal pathway for the molecular mechanism of pathogenesis of cases of arrhythmia and explain ACEI, AT1 receptor antagonist effects provide on the basis of experiment.
Establishment and identification of a stable cell line expressing HERG protein in the first part
Objective: to establish a HEK293 cell line that is stable to express HERG channel protein.
Methods: HERG gene was transfected into HEK293 cells by Lipofectin 2000 transfection reagent. After G418 screening, the stable expression of HERG gene in HEK293 cells was detected by fluorescence microscopy, Western Blot and whole cell patch clamp technique.
Results: G418 screening, positive clones were cultured for 4 weeks. The fluorescence microscope, detection of Western Blot and whole cell patch clamp technique, stable expression of HERG was confirmed and recorded the HERG current after transfection, the IC50 antagonist E-4031 on its specificity by HERG channel, further showed normal expression of HERG channel protein in HEK cells.
Conclusion: the HEK293 cell line that expresses the HERG gene is established. The expression of HERG protein can form a functional channel and produce the HERG current.
The second part Ang II acts on the rapid delayed rectifier potassium (Ikr) current of the AT1 receptor inhibiting ventricular myocytes
Objective: To observe the effect of Ang II on the Ikr of ventricular myocytes of guinea pigs.
Methods: in acute isolated guinea pig ventricular myocytes and HEK293 cells co expressing HERG and human Ang II AT1 receptor genes, Ikr/HERG current was recorded by whole cell patch clamp technique, and the effect of Ang II on current was observed.
Results: Ang II in a concentration dependent manner inhibited Ikr, IC50 is 8.9 nM.Ang II (100 nM) prolonged ventricular cell action potentials of APD50 and APD90, respectively, to extend the 20% and 16%.AT1 receptor antagonist losartan (1 M) completely abolished the inhibitory effect of AngII on Ikr current. The co expression of HERG and AT1 receptor gene HEK293 cells, Ang II also inhibited HERG activation current, slow down the channel, to live and to restore the.PKC inhibitor stausporine and Bis-1 significantly reduced the inhibitory effect of Ang II on Ikr current.
Conclusion: Ang II acts on the AT1 receptor and inhibits the Ikr current in ventricular myocytes through PKC. This provides a possible mechanism for explaining the occurrence of arrhythmia in the pathological condition, such as myocardial hypertrophy, heart failure, and the increase of Ang II.
Third part Ang II suppression of Ikr/HERG current through PKC epsilon
Objective: to analyze the PKC subtype and its downstream molecular mechanism of the intermediate Ang II to inhibit the Ikr/HERG current.
Methods: in isolated guinea pig ventricular myocytes and co expression of HERG and Ang II AT1 receptor gene on HEK293 cells, observe the selective inhibitor of PKC agonist, and PKC subtype translocation inhibitory effects of peptides on Ang II, analysis of the role of intermediary PKC subtype; observe the HERG channel phosphorylation site mutation influence of Ang II effect, analysis of molecular mechanism of PKC downstream.
Results: in HEK293 cells, using PKC inhibitors stausporine and Bis-1 significantly reduced the inhibitory effect of Ang II on HERG current; long-term incubation PMA down-regulation of PKC significantly reduced the inhibitory effects of Ang II on HERG current. The intracellular calcium chelator concentration does not affect Ang II inhibition of HERG subtype specific PKC current. -6976 and G inhibitor G?? -6983 does not affect the inhibitory effect of Ang II on HERG current; translocation inhibitor PKC epsilon isoform significantly against Ang II inhibition of HERG currents. In guinea pig ventricular myocytes, translocation inhibitor PKC epsilon isoform significantly reduced the phosphorylation site of.HERG inhibition of Ang II on Ikr channel the mutation, almost abolished the inhibitory effect of Ang II on HERG current.
Conclusion: (1) Ang II mainly inhibits Ikr/HERG. (2) Ang II through PKC epsilon phosphorylation of HERG channel protein through the activation of PKC epsilon, regulating the function of HERG channel.
conclusion
1. this study has established a stable HEK293 cell line expressing the HERG gene. The expressed protein can form a functional channel to produce a HERG current.
2. Ang II acts on the AT1 receptor and inhibits the Ikr current of ventricular myocytes through PKC. This provides a possible mechanism for explaining the occurrence of arrhythmia in the pathological condition, such as myocardial hypertrophy, heart failure, and the increase of Ang II.
3. Ang II mainly inhibits Ikr/HERG. through the activation of PKC epsilon
4. Ang II may be used to phosphorylate the HERG channel through PKC epsilon, regulating the function of the HERG channel.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R33

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