發(fā)布時(shí)間：2018-06-03 03:31

  本文選題：心肌梗死 + Kir2.1�。� 參考：《山東大學(xué)》2016年博士論文

【摘要】：研究背景盡管當(dāng)代社會醫(yī)療水平不斷進(jìn)步,人們對健康生活方式的認(rèn)識逐步加深,但心肌梗死(myocardial infarcion, MI)仍是臨床上最常見的心血管疾病之一,是我國人民健康面臨的重大威脅。我國每年約有54萬人死于心臟性猝死,近九成猝死原因是急性MI后發(fā)生的惡性心律失常。以往的研究發(fā)現(xiàn)室性心律失常及心源性猝死與鉀離子通道的重構(gòu)有關(guān)。尤其是內(nèi)向整流鉀通道(the inward rectifier potassium channel, Kir),它是維持正常的動作電位的主要成分,維持靜息膜電位,參與動作電位早期和終末復(fù)極化。在心肌細(xì)胞中最豐富的內(nèi)向整流鉀通道是IK1通道,心臟型為Kir2.1蛋白,被KCNJ2基因編碼。MI后心室肌細(xì)胞電重構(gòu)的主要特點(diǎn)之一是IK1電流密度的下降,導(dǎo)致室性心律失常的發(fā)生或易感性增加。盡管目前臨床上已有多種抗心律失常藥物的應(yīng)用,但這些成熟的抗心律失常藥物同時(shí)存在致心律失常作用,尤其是在QT間期延長者中應(yīng)用。而MI后患者QT間期延長是提示惡性室性心律失常甚至猝死發(fā)生的重要指標(biāo)。因此目前已知的抗心律失常藥物在MI患者中的應(yīng)用頗受局限。臨床MI患者多數(shù)伴有高血壓病等危險(xiǎn)因素,而腎素—血管緊張素—醛固酮系統(tǒng)(Renin-angiotensin-aldosterone system, RAAS)拮抗劑可以顯著降低惡性心律失常的發(fā)生率,但具體機(jī)制尚未明確。因此,如能明確RAAS拮抗劑發(fā)揮抗心律失常作用的靶點(diǎn),探明其作用機(jī)制,將對發(fā)現(xiàn)新的抗心律失常藥物、改善MI預(yù)后具有重要意義。第一部分 大鼠也肌梗死后內(nèi)向整流鉀通道重構(gòu)以及纈沙坦的干預(yù)作用目的明確急性MI后大鼠梗死灶周和非梗死左心室游離壁區(qū)心肌細(xì)胞中內(nèi)向整流鉀通道(Kir2.1蛋白)的表達(dá)水平,以及纈沙坦干預(yù)對該通道的效果。并且評價(jià)藥物干預(yù)后大鼠心率、血壓、室性心律失常易感性及血流動力學(xué)指標(biāo)。方法通過永久性結(jié)扎大鼠左冠狀動脈前降支建立急性MI模型,評價(jià)造模情況。造模成功后隨機(jī)分為假手術(shù)組(Control組)、假手術(shù)組+纈沙坦組(Control+ Valsartan組)、心肌梗死組(MI組)、心肌梗死+纈沙坦組(MI+Valsartan組)。利用小動物遙測及尾動脈測壓法記錄大鼠術(shù)后心率、血壓和心電圖。7天后提取梗死灶周和非梗死左心室游離壁區(qū)心肌組織,利用qPCR和Westen blot法檢測四組KCNJ2 mRNA水平和Kir2.1蛋白水平。結(jié)果冠脈結(jié)扎術(shù)中心電監(jiān)測和術(shù)后Masson's染色證明造模成功。MI后7天,MI組大鼠心率、血壓均較Control組和Control+Valsartan組升高,而MI+Valsartan組有所下降。MI后延長的QTc和QTcd亦在纈沙坦干預(yù)后趨于正常。此外纈沙坦有效改善MI后心臟射血分?jǐn)?shù)等血流動力學(xué)指標(biāo)。同時(shí),MI組中KCNJ2 mRNA和Kir2.1蛋白水平較Control組和Control+Valsartan組降低,而MI+Valsartan組中纈沙坦改善了該離子通道的重構(gòu)。結(jié)論大鼠MI后IK1通道表達(dá)水平顯著降低,纈沙坦干預(yù)可逆轉(zhuǎn)這一現(xiàn)象,同時(shí)伴隨著心律失常易感性降低。而在正常大鼠心肌中,纈沙坦對IKI通道的調(diào)控作用并無顯著統(tǒng)計(jì)學(xué)差異。機(jī)制一：纈沙坦通過抑制心肌梗死后激活的NF-κB-miR-16信號通路調(diào)控內(nèi)向整流鉀電流第二部分 纈沙坦改善心肌梗死后內(nèi)向整流鉀通道重構(gòu)的機(jī)制研究目的MicroRNAs是調(diào)控心律失常的重要因素之一。計(jì)算機(jī)預(yù)測microRNA-16(miR-16)可以靶向調(diào)控KCNJ2基因。NF-κB可以調(diào)控miR-16的啟動子。本實(shí)驗(yàn)主要驗(yàn)證纈沙坦是否可以通過抑制MI后激活的NF-κB來下調(diào)miR-16的表達(dá),且miR-16是否可以靶向調(diào)控KCNJ2基因的表達(dá)。方法MI大鼠給予纈沙坦或生理鹽水灌胃7天,提取梗死灶周心肌組織RNA和蛋白。體外培養(yǎng)H9c2心肌細(xì)胞和急性分離的新生大鼠心室肌細(xì)胞,轉(zhuǎn)染miR-16或給予血管緊張素II及纈沙坦干預(yù)。Western blot法檢測NF-κB p65, inhibitor KBa (IκBα)和Kir2.1蛋白水平,qPCR檢測KCNJ2 mRNA和miR-16表達(dá)水平。全細(xì)胞膜片鉗記錄IK1電流。熒光素酶檢測驗(yàn)證KCNJ2是否為miR-16靶基因。CHIP驗(yàn)證NF-κB對miR-16的DNA結(jié)合水平。結(jié)果梗死灶周miR-16表達(dá)水平升高,伴隨KCNJ2/Kir2.1水平下降。體外轉(zhuǎn)染miR-16使之過表達(dá),導(dǎo)致KCNJ2/Kir2.1表達(dá)下調(diào),伴隨著IK1電流密度減低。相反的,抑制miR-16或?qū)е缕浣Y(jié)合位點(diǎn)突變增強(qiáng)了KCNJ2/Kir2.1的表達(dá)。MI大鼠接受纈沙坦干預(yù)后,升高的NF-κB p65和miR-16水平下調(diào),而降低的IκBα和Kir2.1蛋白水平升高。體外試驗(yàn)中,血管緊張素II誘導(dǎo)的miR-16表達(dá)升高和KCNJ2/Kir2.1表達(dá)下降,同樣被纈沙坦抑制。而體外經(jīng)纈沙坦處理的細(xì)胞中過表達(dá)miR-16,可消除纈沙坦對KCNJ2/Kir2.1的保護(hù)作用。體內(nèi)和體外實(shí)驗(yàn)均證明缺氧環(huán)境下NF-κB p65表達(dá)上調(diào),而IκBα表達(dá)下降,纈沙坦干預(yù)抑制了這一現(xiàn)象。而抑制NF-κB后,纈沙坦和NF-κB抑制劑對miR-16和KCNJ2/Kir2.1的調(diào)節(jié)作用相似。CHIP進(jìn)一步驗(yàn)證纈沙坦抑制NF-κB對miR-16的DNA結(jié)合水平。結(jié)論MiR-16靶向調(diào)節(jié)KCNJ2基因的表達(dá),MI后纈沙坦改善KCNJ2/Kir2.1的重構(gòu)一定程度上依賴于NF-KB-miR-16信號通路。機(jī)制二：纈沙坦通過抑制心肌梗死后激活的酪蛋白激酶2調(diào)控內(nèi)向整流鉀電流目的MI后細(xì)胞內(nèi)PKC等蛋白激酶對IKI的調(diào)節(jié)主要依賴于PIP2,并且其下游的調(diào)控機(jī)制鮮有報(bào)道。研究發(fā)現(xiàn)酪蛋白激酶2(CK2)結(jié)合并磷酸化編碼Kir2.1蛋白的KCNJ2基因轉(zhuǎn)錄因子Spl。然而纈沙坦是否可以抑制MI后激活的CK2蛋白活性來影響KCNJ2基因表達(dá)以直接改善IK1通道重構(gòu)還尚不明確。方法MI大鼠給予纈沙坦或生理鹽水灌胃7天,提取梗死灶周和非梗死左心室游離壁心肌組織RNA和蛋白。體外培養(yǎng)H9c2心肌細(xì)胞和急性分離的新生大鼠心室肌細(xì)胞,轉(zhuǎn)染CK2或給予CoCl2、CK2抑制劑TBB及纈沙坦干預(yù)。Western blot法檢測CK2和Kir2.1蛋白水平,qPCR檢測CK2 mRNA和KCNJ2 mRNA表達(dá)水平。全細(xì)胞膜片鉗記錄IK1電流。EMSA檢測體內(nèi)及體外Sp1的DNA結(jié)合活性。結(jié)果梗死灶周和非梗死左心室游離壁區(qū)心肌組織CK2蛋白表達(dá)升高,Kir2.1蛋白表達(dá)下降,伴隨著IK1電流密度減低,同時(shí)伴隨二者mRNA水平的變化。纈沙坦干預(yù)后逆轉(zhuǎn)了這一現(xiàn)象。體外培養(yǎng)的H9c2細(xì)胞中過表達(dá)CK2蛋白抑制了KCNJ2/Kir2.1的表達(dá)。相反的,抑制CK2蛋白可以增加KCNJ2/Kir2.1的表達(dá)。同樣的,在體外誘導(dǎo)缺氧環(huán)境中,纈沙坦同樣抑制缺氧后升高的CK2蛋白水平。體外經(jīng)纈沙坦處理的細(xì)胞中過表達(dá)CK2,可消除纈沙坦對KCNJ2/Kir2.1的保護(hù)作用。EMSA檢測證明CK2抑制Sp1的DNA結(jié)合活性,TBB抑制CK2后,Sp1的DNA結(jié)合活性升高。而MI后大鼠心肌組織中Sp1的DNA結(jié)合活性下降,纈沙坦干預(yù)后其活性上升。結(jié)論AT1受體拮抗劑纈沙坦抑制CK2蛋白活性,增加Kir2.1蛋白表達(dá),從而改善MI后IK1通道重構(gòu)。機(jī)制三：纈沙坦通過抑制心肌梗死后激活的I型輔助性T細(xì)胞免疫反應(yīng)調(diào)控內(nèi)向整流鉀電流目的MI導(dǎo)致Kir2.1蛋白介導(dǎo)的IK1電流密度減低,伴隨著T細(xì)胞水平上調(diào)。細(xì)胞因子IFN-γ主要Thl細(xì)胞分泌。小膠質(zhì)細(xì)胞中IFN-γ導(dǎo)致IK1電流密度下降。本實(shí)驗(yàn)主要驗(yàn)證MI后Th1細(xì)胞是否可以通過其分泌的細(xì)胞因子介導(dǎo)IK1通道重構(gòu),以及纈沙坦是否可以改善這一現(xiàn)象。方法空白對照大鼠和MI大鼠給予纈沙坦或生理鹽水灌胃7天,提取梗死灶周心肌組織RNA和蛋白。體外培養(yǎng)急性分離的梗死灶周大鼠心室肌細(xì)胞和新生大鼠心室肌細(xì)胞,分別給予纈沙坦干預(yù)和與分離的淋巴細(xì)胞共培養(yǎng)。流式細(xì)胞術(shù)分析大鼠心肌組織中Thl細(xì)胞數(shù)目。Elisa法檢測大鼠血漿中IFN-γ、IL-2和TNF-α水平。Western blot法檢測Kir2.1蛋白水平,qPCR檢測T-bet、GATA-3、IFN-γ和IL-10的mRNA表達(dá)水平。全細(xì)胞膜片鉗記錄IK1電流。結(jié)果MI后Th1細(xì)胞數(shù)目及其分泌的細(xì)胞因子水平升高,Kir2.1蛋白水平下降。而MI大鼠纈沙坦干預(yù)后,Thl細(xì)胞數(shù)及其細(xì)胞因子水平下降,同時(shí)Kir2.1蛋白表達(dá)及IK1電流密度升高。在體外淋巴細(xì)胞與心肌細(xì)胞共培養(yǎng)后,纈沙坦干預(yù)對Kir2.1/IK1的調(diào)控同上。在體外培養(yǎng)的新生大鼠心室肌細(xì)胞中,IFN-γ干預(yù)可抑制IK1電流密度,而IL-2和TNF-α對該通道無明顯作用。結(jié)論纈沙坦可以通過抑制MI后激活的Thl免疫反應(yīng)、減低IFN-γ水平,來改善IK1通道的重構(gòu)。
[Abstract]:Background although the level of medical treatment is progressing in the contemporary society, people's understanding of the healthy lifestyle is gradually deepened, but myocardial infarcion (MI) is still one of the most common cardiovascular diseases in the clinic. It is a major threat to the health of the people in our country. About 540 thousand people die of sudden cardiac death every year in China and nearly 90% of them are sudden. The cause of death is malignant arrhythmia after acute MI. Previous studies have found that ventricular arrhythmias and sudden cardiac death are related to the reconstruction of potassium channels. Especially the the inward rectifier potassium channel (Kir), which is the main component of the normal action potential, maintains the resting membrane potential and participates in the action. The most abundant inward rectifier potassium channel in cardiac myocytes is the IK1 channel, the heart type is Kir2.1 protein. One of the main characteristics of the electrical remodeling of ventricular myocytes after the KCNJ2 gene encoding.MI is the decrease of the IK1 current density, which leads to the occurrence of ventricular arrhythmia or the increase of susceptibility. There are a variety of antiarrhythmic drugs, but these mature antiarrhythmic drugs also have arrhythmia effects, especially in QT interphase prolongation. The prolongation of QT interval in patients after MI is an important indicator of malignant ventricular arrhythmias or even sudden death. The current known antiarrhythmic drugs are in MI Most of the clinical MI patients are associated with the risk factors such as hypertension, and the renin angiotensin aldosterone system (Renin-angiotensin-aldosterone system, RAAS) antagonists can significantly reduce the incidence of malignant arrhythmia, but the specific mechanism is not clear. Therefore, it is clear that the RAAS antagonist can play the role of anti arrhythmia. The target of arrhythmia effect and the mechanism of its action will be of great significance for the discovery of new antiarrhythmic drugs and the improvement of the prognosis of MI. The expression level of inward rectifying potassium channel (Kir2.1 protein) in the cell and the effect of valsartan intervention on this channel. And evaluate the heart rate, blood pressure, ventricular arrhythmia susceptibility and hemodynamic indexes after drug intervention. Methods an acute MI model was established by permanent ligation of the left coronary artery descending branch in rats, and the model was evaluated. After the success, the model was randomly divided into sham operation group (group Control), sham operation group + valsartan group (Control+ Valsartan group), myocardial infarction group (group MI), myocardial infarction + valsartan group (group MI+Valsartan). Using small animal telemetry and tail artery pressure measurement to record the heart rate, blood pressure and.7 days after.7 extraction of infarct and non infarct left ventricular travel. The levels of KCNJ2 mRNA and Kir2.1 protein in four groups were detected by qPCR and Westen blot. Results the heart rate and blood pressure of group MI rats were higher than that of Control and Control+Valsartan groups at 7 days after coronary artery ligation and postoperative Masson's staining. QTc and QTcd also tended to be normal after valsartan intervention. In addition, valsartan effectively improved the hemodynamic index of cardiac ejection fraction after MI. Meanwhile, the levels of KCNJ2 mRNA and Kir2.1 in the MI group were lower than those in the Control and Control+Valsartan groups, while valsartan in the MI+Valsartan group improved the reconstruction of the ion channel. Valsartan intervention can be significantly reduced and valsartan intervention can reverse this phenomenon, accompanied by a decrease in cardiac arrhythmia susceptibility. In normal rat myocardium, there is no significant difference in the regulatory effect of valsartan on IKI channel. Mechanism 1: Valsartan regulates inward by inhibiting the NF- kappa B-miR-16 signaling pathway activated after myocardial infarction. The mechanism of valsartan second part of valsartan to improve the remodeling of inward rectifier potassium channel after myocardial infarction objective MicroRNAs is one of the important factors in regulating arrhythmia. The computer predicted that microRNA-16 (miR-16) can regulate the KCNJ2 gene.NF- kappa B to regulate the promoter of miR-16. This experiment mainly verifies the possibility of valsartan. To reduce the expression of miR-16 by inhibiting the activation of NF- kappa B after MI, and whether miR-16 can target the expression of KCNJ2 gene. Methods MI rats were given valsartan or physiological saline for 7 days to extract the RNA and protein of Zhou Xinji tissue from the infarct. In vitro culture of H9c2 myocytes and acute isolated neonatal rat ventricular myocytes were transfected to miR-16. The levels of NF- kappa B p65, inhibitor KBa (I kappa B alpha) and Kir2.1 protein were detected by angiotensin II and valsartan, and inhibitor KBa (I kappa B alpha) and Kir2.1 protein levels were detected. The level of miR-16 expression in the infarct area increased and the level of KCNJ2/Kir2.1 decreased. The over expression of miR-16 in vitro resulted in the down expression of KCNJ2/Kir2.1 and the decrease of the IK1 current density. On the contrary, the inhibition of miR-16 or the mutation of its binding site enhanced the prognosis of KCNJ2/Kir2.1 in the.MI rats, and the increase of NF- kappa B. The levels of p65 and miR-16 decreased, while the decreased levels of I kappa B A and Kir2.1 protein were elevated. In vitro, the elevation of miR-16 expression induced by angiotensin II and the decrease of KCNJ2/Kir2.1 expression were also inhibited by valsartan. The over expression of miR-16 in valsartan treated cells in vitro could eliminate the protective effect of valsartan on KCNJ2/Kir2.1 in vivo and in vivo. In vitro, the expression of NF- kappa B p65 was up-regulated in anoxic environment, while the expression of I kappa B alpha decreased, and the intervention of valsartan inhibited this phenomenon. After the inhibition of NF- kappa B, the regulatory effect of valsartan and NF- kappa B inhibitors on miR-16 and KCNJ2/Kir2.1 was similar. The expression of the node KCNJ2 gene, after MI, the improvement of the remodeling of KCNJ2/Kir2.1 depends partly on the NF-KB-miR-16 signaling pathway. Mechanism two: Valsartan's regulation of IKI is mainly dependent on PIP2, and is dependent on PIP2, by inhibiting the activation of the casein kinase 2 after myocardial infarction, and the regulation of IKI is mainly dependent on PIP2. There are few reports on the regulation mechanism of swimming. The study found that casein kinase 2 (CK2) binding and phosphorylation of Kir2.1 protein KCNJ2 gene transcription factor Spl., however, whether valsartan can inhibit the activity of CK2 protein activated after MI to affect the KCNJ2 gene expression to directly improve the IK1 channel remodeling is still unclear. RNA and non infarcted left ventricular free wall myocardial tissue RNA and protein were extracted by irrigated saline for 7 days. H9c2 cardiomyocytes and acute isolated neonatal rat ventricular myocytes were cultured in vitro, CK2 or CoCl2, CK2 inhibitor TBB and valsartan were used to detect CK2 and Kir2.1 protein levels by.Western blot method. RNA expression level. The whole cell patch clamp recording IK1 current.EMSA detected the DNA binding activity of Sp1 in the body and in vitro. Results the expression of CK2 protein in the myocardial tissue of the infarct peripheral and non infarcted left ventricular wall increased, the expression of Kir2.1 protein decreased, accompanied by the decrease of the current density of IK1 and the change of the mRNA level in the two cases. The overexpression of CK2 protein in H9c2 cells in vitro inhibited the expression of KCNJ2/Kir2.1. On the contrary, the inhibition of CK2 protein could increase the expression of KCNJ2/Kir2.1. Similarly, in the hypoxia environment, valsartan also inhibited the level of CK2 protein increased after hypoxia. In vitro, the expression of CK2 in valsartan cells was overexpressed in the cells treated by valsartan. The.EMSA detection of valsartan to KCNJ2/Kir2.1 showed that CK2 inhibited the DNA binding activity of Sp1, and the DNA binding activity of Sp1 increased after TBB inhibition of CK2, and Sp1 DNA binding activity in the myocardium of rats after MI decreased and the activity of valsartan increased after the intervention of valsartan. Protein expression, thus improving the IK1 channel reconstruction after MI. Mechanism three: Valsartan by inhibiting the I type auxiliary T cell immune response after myocardial infarction regulates the inward rectifier potassium current purpose MI leads to the Kir2.1 protein mediated IK1 current density, accompanied by the level of T cells. The microglia is secreted by the microglia and the microglia. IFN- gamma leads to the decrease of IK1 current density in the cells. This experiment is mainly to verify whether Th1 cells can mediate IK1 channel reconstruction through its secretory cytokines after MI, and whether valsartan can improve this phenomenon. Methods blank control rats and MI rats were given valsartan or physiological saline for 7 days to extract RNA from myocardial tissue of infarct In vitro culture of ventricular myocytes and neonatal rat ventricular myocytes from acute isolated infarcted rats, valsartan intervention and co culture with isolated lymphocytes were given respectively. Flow cytometry was used to determine the number of Thl cells in rat myocardial tissue by.Elisa method to detect IFN- gamma, IL-2 and TNF- alpha levels by.Western blot assay. The level of Kir2.1 protein and the level of mRNA expression of T-bet, GATA-3, IFN- gamma and IL-10 were measured by qPCR. IK1 current was recorded by whole cell patch clamp. The results showed that the number of Th1 cells and the level of cytokines secreted and the level of Kir2.1 protein decreased after MI. IK1 current density increased. After co culture of lymphocytes and cardiomyocytes in vitro, valsartan intervention on Kir2.1/IK1 was involved. In the cultured neonatal rat ventricular myocytes, IFN- gamma intervention could inhibit the current density of IK1, while IL-2 and TNF- alpha were not significantly used for this channel. Conclusion valsartan can inhibit the T activated by MI. HL immune response, reduce IFN- gamma level, to improve the IK1 channel remodeling.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R542.22
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