【摘要】：背景病毒性心肌炎(viral myocarditis,VMC)是最為常見的心肌炎類型,主要是由柯薩奇病毒B(Coxsackievirus B,CVB)所引發(fā)。相關統(tǒng)計資料表明,造成嬰兒猝死的疾病中大約有20%是由病毒性心肌炎或者由其引發(fā)的致死性室性心律失常所引起,且持續(xù)活動的病毒性心肌炎患者的預后情況通常較不理想,嚴重威脅兒童生命健康。當機體發(fā)生持續(xù)性慢性炎癥時,往往能夠造成心肌細胞生長抑制、肥大、凋亡率增高,此外還會進一步引發(fā)心肌纖維化、擴張型心肌病以及心力衰竭等。迄今為止,國內(nèi)外依然缺乏治療病毒性心肌炎的有效手段,因此,尋找診斷病毒性心肌炎的分子標志物有助于提前采取有效措施減緩或抑制該病的進展,同時還能以此作為分子靶標達到治療的目的。mi R-19b屬于mi RNA家族成員,在進化上高度保守,在多種生物的胚胎心臟中均有表達。以往研究表明mi R-19b的表達缺失時,能夠?qū)е滦律∈笫议g隔缺損,提示mi R-19b可能參與了早期心臟發(fā)育過程。提示mi R-19b可能在早期即參與了心臟發(fā)育過程。也有研究表明,mi R-19b的表達水平在擴張型心肌病患者心肌重構的心肌組織中發(fā)生顯著上調(diào)。盡管mi R-19b在心臟發(fā)育過程中發(fā)揮著重要作用,但目前有關mi R-19b與病毒性心肌炎發(fā)生和發(fā)展相關性的研究依然相對較少。目的本研究探討病毒性心肌炎患兒循環(huán)血中mi R-19b的表達變化及其與心肌病變嚴重程度的相關性,同時分析其表達對心肌細胞增殖和凋亡的影響,并對其中的相關機制進行研究,旨在為病毒性心肌炎的臨床診斷及開發(fā)新的靶向治療藥物奠定基礎。方法(1)本研究采集來自于2014年6月~2015年6月在吉林大學第一醫(yī)院住院收治的病毒性心肌炎患兒外周血,共50例。熒光定量PCR檢測外周血樣本中mi R-19b表達;檢測血清中c Tn I和CK-MB的表達;測定患兒左心室射血分數(shù)(EF)和短軸縮短率(FS);分析mi R-19b表達與上述檢測指標的相關性。(2)采用酶兩步消化法分離培養(yǎng)原代SD大鼠乳鼠心肌細胞。將mi R-19b mimic和mi R-19b inhibitor轉(zhuǎn)染上述細胞,熒光定量PCR檢測轉(zhuǎn)染后細胞中mi R-19b的表達。將CVB3感染上述細胞,CCK-8實驗檢測細胞增殖活性,流式細胞術檢測細胞凋亡率。運用生物信息學軟件預測mi R-19b的靶基因,構建含有熒光素酶報告基因的TLR23’UTR(野生型)和TLR2 3’UTR-mut(突變型)質(zhì)粒,并將之分別與mi R-19b mimic共轉(zhuǎn)染乳鼠心肌細胞,檢測上述細胞中熒光素酶的表達。分別將mi R-19b mimic和mi R-19b inhibitor共轉(zhuǎn)染乳鼠細胞,隨后用CVB3感染上述細胞,檢測細胞中TLR2m RNA和蛋白的表達。(3)構建mi R-19b過表達病毒性心肌炎大鼠模型,檢測上述模型大鼠心肌組織中mi R-19b的表達。將CVB3分別感染上述大鼠模型以及正常SD大鼠,HE染色法觀察心肌組織病理學變化,TUNEL染色法檢測心肌細胞凋亡,western blot法檢測TLR2蛋白的表達。結果(1)在重癥VMC組中,恢復期時血漿mi R-19b的表達量明顯高于急性期;在輕癥VMC組中,恢復期時血漿mi R-19b的表達量明顯高于急性期。病毒性心肌炎患兒血清中c Tn I水平明顯高于對照組患兒。在重癥VMC組中,恢復期時血清c Tn I水平明顯低于急性期;在重癥VMC組中,恢復期時血清c Tn I水平明顯低于急性期。病毒性心肌炎患兒血清中CK-MB水平明顯高于對照組患兒。在重癥VMC組中,恢復期時血清CK-MB水平明顯低于急性期;在輕癥VMC組中,恢復期時血清CK-MB水平明顯低于急性期。EF檢測結果表明,重癥期患兒EF明顯低于對照組;重癥VMC組恢復期患兒EF與對照組相比無顯著差異;輕癥VMC組急性期患兒EF與對照組相比無明顯差異;輕癥VMC組恢復期患兒與對照組相比無顯著差異。重癥VMC患兒急性期血漿mi R-19b表達水平與血清c Tn I含量呈負相關,重癥VMC患兒恢復期血漿mi R-19b表達水平與血清c Tn I含量無明顯相關性;輕癥VMC患兒急性期血漿mi R-19b表達水平與血清c Tn I含量呈負相關,輕癥VMC患兒恢復期血漿mi R-19b表達水平與血清c Tn I含量無明顯相關性。而對照組患兒血漿mi R-19b表達水平與血清c Tn I含量無顯著相關性。重癥VMC患兒急性期血漿mi R-19b表達水平與血清CK-MB含量呈負相關,重癥VMC患兒恢復期血漿mi R-19b表達水平與血清CK-MB含量無明顯相關性;輕癥VMC患兒急性期血漿mi R-19b表達水平與血清CK-MB含量呈負相關,輕癥VMC患兒恢復期血漿mi R-19b表達水平與血清CK-MB含量無明顯相關性。對照組患兒血漿mi R-19b表達水平與血清CK-MB含量無顯著相關性。重癥VMC患兒急性期血漿mi R-19b含量與EF之間存在明顯正相關,重癥VMC患兒恢復期血漿mi R-19b表達水平與EF之間無顯著相關性;輕癥VMC患兒急性期血漿mi R-19b含量與EF之間無明顯相關性,輕癥VMC患兒恢復期血漿mi R-19b表達水平與EF之間無顯著相關性。對照組患兒血漿mi R-19b表達水平與血清EF含量無顯著相關性。重癥VMC患兒急性期血漿mi R-19b含量與FS之間存在明顯正相關,重癥VMC患兒恢復期血漿mi R-19b表達水平與FS之間無顯著相關性;輕癥VMC患兒急性期血漿mi R-19b含量與FS之間無明顯相關性,輕癥VMC患兒恢復期血漿mi R-19b表達水平與FS之間無顯著相關性。對照組患兒血漿mi R-19b表達水平與血清FS含量無顯著相關性。(2)轉(zhuǎn)染mi R-19b mimic后乳鼠心肌細胞中mi R-19b的表達明顯高于對照組;轉(zhuǎn)染mi R-19b inhibitor后乳鼠心肌細胞中mi R-19b的表達明顯低于對照組。與對照組相比,mi R-19b mimic組在48 h、72 h、96 h時的存活率均明顯增高,而mi R-19b inhibitor組相應時間點時的存活率均明顯降低。與對照組相比,mi R-19b mimic組細胞的凋亡率明顯降低,mi R-19b inhibitor組細胞的凋亡率明顯增高。TLR2為mi R-19b的靶基因。構建的攜帶有熒光素酶報告基因的野生型TLR2 3’UTR載體與mi R-19b mimic共轉(zhuǎn)染乳鼠心肌細胞后細胞中熒光素酶表達水平明顯降低,而突變型TLR2 3’UTR載體與mi R-19b mimic共轉(zhuǎn)染后細胞中熒光素酶的表達量則無明顯變化。TLR m RNA檢測結果表明,與對照組相比,mi R-19b mimic組細胞中TLR m RNA的表達水平明顯降低,而mi R-19b inhibitor組細胞中TLR m RNA的表達水平明顯增高。TLR2蛋白檢測結果表明,與對照組相比,mi R-19b mimic組細胞中TLR蛋白的表達水平明顯降低,而mi R-19b inhibitor組細胞中TLR蛋白的表達水平明顯升高。(3)轉(zhuǎn)基因大鼠模型心肌組織中mi R-19b的表達水平明顯高于對照組。正常SD大鼠在感染CVB3后大量心肌細胞發(fā)生壞死崩解,細胞核以及細胞輪廓消失,在心肌細胞間隙內(nèi)可觀察到大量炎癥細胞浸潤,且浸潤部位心肌纖維排列紊亂,心肌組織被破壞。心肌炎轉(zhuǎn)基因大鼠感染CVB3后心肌細胞崩解數(shù)量顯著減少,細胞核和細胞輪廓較為清晰,心肌細胞間隙可見少量炎性細胞浸潤。感染CVB3后心肌炎轉(zhuǎn)基因大鼠心肌細胞的凋亡率明顯低于正常SD大鼠。感染CVB3后心肌炎轉(zhuǎn)基因大鼠心肌組織中TLR2蛋白的表達明顯低于正常SD大鼠。結論(1)mi R-19b在病毒性心肌炎中表達降低;(2)mi R-19b與病毒性心肌炎疾病嚴重程度及病程相關;(3)mi R-19b通過靶向抑制TLR2的表達促進心肌細胞增殖,抑制其凋亡,從而抑制病毒性心肌炎的發(fā)展。(4)mi R-19b對由CVB3感染所引起的心肌細胞損傷具有保護作用,其機制與mi R-19b抑制TLR2蛋白的表達,從而減輕心肌細胞炎癥反應,抑制心肌細胞凋亡有關。
[Abstract]:Background Viral myocarditis (VMC) is the most common type of myocarditis, mainly caused by Coxsackievirus B (CVB). Statistics show that about 20% of the sudden infant death is caused by viral myocarditis or lethal ventricular arrhythmias caused by viral myocarditis and persists. The prognosis of active viral myocarditis is usually unsatisfactory, which seriously threatens the life and health of children. When persistent chronic inflammation occurs, it often leads to myocardial cell growth inhibition, hypertrophy, increased apoptosis rate, and further leads to myocardial fibrosis, dilated cardiomyopathy and heart failure. However, there is still no effective method to treat viral myocarditis at home and abroad. Therefore, looking for molecular markers for diagnosis of viral myocarditis is helpful to take effective measures to slow down or inhibit the progress of the disease, and can also be used as a molecular target to achieve the purpose of treatment. Previous studies have shown that the absence of MIR-19b expression can lead to ventricular septal defect in neonatal mice, suggesting that MIR-19b may be involved in early cardiac development. Although mi R-19b plays an important role in cardiac development, there are few studies on the relationship between MI R-19b and the occurrence and development of viral myocarditis. Objective To investigate the expression of MI R-19b in circulating blood of children with viral myocarditis. The aim of this study is to lay a foundation for the clinical diagnosis of viral myocarditis and the development of new targeted therapies. Methods (1) This study was collected from June 2014 to June 2015. Fifty children with viral myocarditis were hospitalized in the First Hospital of Jilin University.The expression of MI R-19b in peripheral blood samples was detected by fluorescence quantitative PCR.The expressions of C Tn I and CK-MB in serum were detected.The left ventricular ejection fraction (EF) and short axis shortening rate (FS) were measured. MiR-19b mimic and MI R-19b inhibitor were transfected into these cells. The expression of MI R-19b in the transfected cells was detected by fluorescence quantitative PCR. CVB3 was infected with these cells. Cell proliferation activity was detected by CCK-8 assay and apoptosis rate was detected by flow cytometry. The target gene of MI R-19b was predicted by software. TLR23'UTR (wild type) and TLR23'UTR-mut (mutant type) plasmids containing luciferase reporter gene were constructed and co-transfected with MI R-19b mimic to detect the expression of luciferase in neonatal rat cardiomyocytes. Then the above cells were infected with CVB3 and the expression of TLR2m RNA and protein was detected. (3) A rat model of viral myocarditis with overexpression of MIR-19b was established to detect the expression of MIR-19b in the myocardium of the above-mentioned model rats. Results (1) In severe VMC group, the expression of plasma MIR-19b was significantly higher in convalescence than in acute phase; in mild VMC group, the expression of plasma MIR-19b was significantly higher in convalescence than in acute phase. In the severe VMC group, the serum levels of C Tn I in convalescent stage were significantly lower than those in acute stage; in the severe VMC group, the serum levels of C Tn I in convalescent stage were significantly lower than those in acute stage. In mild VMC group, the serum CK-MB level was significantly lower in convalescent stage than in acute stage. EF test results showed that EF in severe VMC group was significantly lower than that in control group; EF in convalescent stage in severe VMC group was not significantly different from that in control group; EF in mild VMC group was not significantly different from that in acute stage; EF in convalescent stage in mild VMC group was significantly lower than that in control group. There was no significant difference between the two groups. There was a negative correlation between the level of plasma MIR-19b and the level of serum C Tn I in the acute stage of severe VMC. There was no significant correlation between the level of plasma MIR-19b and the level of serum C Tn I in the convalescent stage of severe VMC. There was a negative correlation between the level of plasma MIR-19b and the level of serum C Tn I in the acute stage of mild VMC. There was no significant correlation between the levels of plasma M I R-19b and serum C Tn I in the acute phase of severe VMC, but no significant correlation between the levels of plasma M I R-19b and serum C Tn I in the control group. There was no significant correlation between the levels of serum CK-MB and the levels of plasma MIR-19b in mild VMC. There was no significant correlation between the levels of serum CK-MB and the levels of plasma MIR-19b in convalescent VMC. There was no significant correlation between the level of plasma MIR-19b and EF in the convalescent stage of severe VMC, but no significant correlation between the level of plasma MIR-19b and EF in the acute stage of mild VMC, and no significant correlation between the level of plasma MIR-19b and EF in the convalescent stage of mild VMC. There was no significant correlation between the levels of plasma MIR-19b and serum EF. There was a positive correlation between the levels of plasma MIR-19b and FS in the acute stage of severe VMC. There was no significant correlation between the levels of plasma MIR-19b and FS in the convalescent stage of severe VMC. There was no significant correlation between the expression of MIR-19b and FS in the convalescent stage of mild VMC. There was no significant correlation between the expression of MIR-19b and FS in the control group. Compared with the control group, the survival rate of MIR-19b mimic group was significantly higher at 48, 72 and 96 hours, while that of MIR-19b inhibitor group was significantly lower at corresponding time points. TLR2 was the target gene of MI R-19b. The expression level of luciferase in neonatal rat cardiomyocytes co-transfected with wild-type TLR2 3'UTR vector carrying luciferase reporter gene and MI R-19b mimic was significantly decreased, but the expression level of luciferase in neonatal rat cardiomyocytes co-transfected with mutant TLR2 3'UTR vector and mir-19b mic was not found. The results of TLR-m RNA detection showed that the expression of TLR-m RNA in the cells of MIR-19b mimic group was significantly lower than that of the control group, while the expression of TLR-m RNA in the cells of MIR-19b inhibitor group was significantly higher than that of the control group. (3) The expression of MIR-19b in the myocardium of transgenic rats was significantly higher than that of the control group. After infection with CVB3, a large number of myocardial cells in normal SD rats necrosis and disintegration, nucleus and cell contour disappeared, and the expression of TLR protein in the myocardial interspace was observed. A large number of inflammatory cells were infiltrated, and the myocardial fibers were arranged disorderly in the infiltration site, and the myocardial tissue was destroyed. The number of myocardial cells disintegrated significantly after the infection of CVB3 in transgenic myocarditis rats, the nucleus and cell contour were clearer, and a small amount of inflammatory cells infiltrated into the myocardial space. The expression of TLR2 protein in myocardium of transgenic rats infected with CVB3 was significantly lower than that of normal SD rats. Conclusion (1) The expression of MIR-19b was decreased in viral myocarditis; (2) MIR-19b was associated with the severity and course of viral myocarditis; (3) MIR-19b was promoted by targeting inhibition of TLR2 expression. (4) MIR-19b has protective effect on myocardial cell injury caused by CVB3 infection, and its mechanism is related to the inhibition of TLR2 protein expression by MIR-19b, which can alleviate myocardial inflammation and inhibit cardiomyocyte apoptosis.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R542.21

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