miR-19b在病毒性心肌炎中作用机制的研究

发布时间：2018-09-05 08:37

【摘要】：背景病毒性心肌炎(viral myocarditis,VMC)是最为常见的心肌炎类型,主要是由柯萨奇病毒B(Coxsackievirus B,CVB)所引发。相关统计资料表明,造成婴儿猝死的疾病中大约有20%是由病毒性心肌炎或者由其引发的致死性室性心律失常所引起,且持续活动的病毒性心肌炎患者的预后情况通常较不理想,严重威胁儿童生命健康。当机体发生持续性慢性炎症时,往往能够造成心肌细胞生长抑制、肥大、凋亡率增高,此外还会进一步引发心肌纤维化、扩张型心肌病以及心力衰竭等。迄今为止,国内外依然缺乏治疗病毒性心肌炎的有效手段,因此,寻找诊断病毒性心肌炎的分子标志物有助于提前采取有效措施减缓或抑制该病的进展,同时还能以此作为分子靶标达到治疗的目的。mi R-19b属于mi RNA家族成员,在进化上高度保守,在多种生物的胚胎心脏中均有表达。以往研究表明mi R-19b的表达缺失时,能够导致新生小鼠室间隔缺损,提示mi R-19b可能参与了早期心脏发育过程。提示mi R-19b可能在早期即参与了心脏发育过程。也有研究表明,mi R-19b的表达水平在扩张型心肌病患者心肌重构的心肌组织中发生显著上调。尽管mi R-19b在心脏发育过程中发挥着重要作用,但目前有关mi R-19b与病毒性心肌炎发生和发展相关性的研究依然相对较少。目的本研究探讨病毒性心肌炎患儿循环血中mi R-19b的表达变化及其与心肌病变严重程度的相关性,同时分析其表达对心肌细胞增殖和凋亡的影响,并对其中的相关机制进行研究,旨在为病毒性心肌炎的临床诊断及开发新的靶向治疗药物奠定基础。方法(1)本研究采集来自于2014年6月~2015年6月在吉林大学第一医院住院收治的病毒性心肌炎患儿外周血,共50例。荧光定量PCR检测外周血样本中mi R-19b表达;检测血清中c Tn I和CK-MB的表达;测定患儿左心室射血分数(EF)和短轴缩短率(FS);分析mi R-19b表达与上述检测指标的相关性。(2)采用酶两步消化法分离培养原代SD大鼠乳鼠心肌细胞。将mi R-19b mimic和mi R-19b inhibitor转染上述细胞,荧光定量PCR检测转染后细胞中mi R-19b的表达。将CVB3感染上述细胞,CCK-8实验检测细胞增殖活性,流式细胞术检测细胞凋亡率。运用生物信息学软件预测mi R-19b的靶基因,构建含有荧光素酶报告基因的TLR23’UTR(野生型)和TLR2 3’UTR-mut(突变型)质粒,并将之分别与mi R-19b mimic共转染乳鼠心肌细胞,检测上述细胞中荧光素酶的表达。分别将mi R-19b mimic和mi R-19b inhibitor共转染乳鼠细胞,随后用CVB3感染上述细胞,检测细胞中TLR2m RNA和蛋白的表达。(3)构建mi R-19b过表达病毒性心肌炎大鼠模型,检测上述模型大鼠心肌组织中mi R-19b的表达。将CVB3分别感染上述大鼠模型以及正常SD大鼠,HE染色法观察心肌组织病理学变化,TUNEL染色法检测心肌细胞凋亡,western blot法检测TLR2蛋白的表达。结果(1)在重症VMC组中,恢复期时血浆mi R-19b的表达量明显高于急性期;在轻症VMC组中,恢复期时血浆mi R-19b的表达量明显高于急性期。病毒性心肌炎患儿血清中c Tn I水平明显高于对照组患儿。在重症VMC组中,恢复期时血清c Tn I水平明显低于急性期;在重症VMC组中,恢复期时血清c Tn I水平明显低于急性期。病毒性心肌炎患儿血清中CK-MB水平明显高于对照组患儿。在重症VMC组中,恢复期时血清CK-MB水平明显低于急性期;在轻症VMC组中,恢复期时血清CK-MB水平明显低于急性期。EF检测结果表明,重症期患儿EF明显低于对照组;重症VMC组恢复期患儿EF与对照组相比无显著差异;轻症VMC组急性期患儿EF与对照组相比无明显差异;轻症VMC组恢复期患儿与对照组相比无显著差异。重症VMC患儿急性期血浆mi R-19b表达水平与血清c Tn I含量呈负相关,重症VMC患儿恢复期血浆mi R-19b表达水平与血清c Tn I含量无明显相关性;轻症VMC患儿急性期血浆mi R-19b表达水平与血清c Tn I含量呈负相关,轻症VMC患儿恢复期血浆mi R-19b表达水平与血清c Tn I含量无明显相关性。而对照组患儿血浆mi R-19b表达水平与血清c Tn I含量无显著相关性。重症VMC患儿急性期血浆mi R-19b表达水平与血清CK-MB含量呈负相关,重症VMC患儿恢复期血浆mi R-19b表达水平与血清CK-MB含量无明显相关性;轻症VMC患儿急性期血浆mi R-19b表达水平与血清CK-MB含量呈负相关,轻症VMC患儿恢复期血浆mi R-19b表达水平与血清CK-MB含量无明显相关性。对照组患儿血浆mi R-19b表达水平与血清CK-MB含量无显著相关性。重症VMC患儿急性期血浆mi R-19b含量与EF之间存在明显正相关,重症VMC患儿恢复期血浆mi R-19b表达水平与EF之间无显著相关性;轻症VMC患儿急性期血浆mi R-19b含量与EF之间无明显相关性,轻症VMC患儿恢复期血浆mi R-19b表达水平与EF之间无显著相关性。对照组患儿血浆mi R-19b表达水平与血清EF含量无显著相关性。重症VMC患儿急性期血浆mi R-19b含量与FS之间存在明显正相关,重症VMC患儿恢复期血浆mi R-19b表达水平与FS之间无显著相关性;轻症VMC患儿急性期血浆mi R-19b含量与FS之间无明显相关性,轻症VMC患儿恢复期血浆mi R-19b表达水平与FS之间无显著相关性。对照组患儿血浆mi R-19b表达水平与血清FS含量无显著相关性。(2)转染mi R-19b mimic后乳鼠心肌细胞中mi R-19b的表达明显高于对照组;转染mi R-19b inhibitor后乳鼠心肌细胞中mi R-19b的表达明显低于对照组。与对照组相比,mi R-19b mimic组在48 h、72 h、96 h时的存活率均明显增高,而mi R-19b inhibitor组相应时间点时的存活率均明显降低。与对照组相比,mi R-19b mimic组细胞的凋亡率明显降低,mi R-19b inhibitor组细胞的凋亡率明显增高。TLR2为mi R-19b的靶基因。构建的携带有荧光素酶报告基因的野生型TLR2 3’UTR载体与mi R-19b mimic共转染乳鼠心肌细胞后细胞中荧光素酶表达水平明显降低,而突变型TLR2 3’UTR载体与mi R-19b mimic共转染后细胞中荧光素酶的表达量则无明显变化。TLR m RNA检测结果表明,与对照组相比,mi R-19b mimic组细胞中TLR m RNA的表达水平明显降低,而mi R-19b inhibitor组细胞中TLR m RNA的表达水平明显增高。TLR2蛋白检测结果表明,与对照组相比,mi R-19b mimic组细胞中TLR蛋白的表达水平明显降低,而mi R-19b inhibitor组细胞中TLR蛋白的表达水平明显升高。(3)转基因大鼠模型心肌组织中mi R-19b的表达水平明显高于对照组。正常SD大鼠在感染CVB3后大量心肌细胞发生坏死崩解,细胞核以及细胞轮廓消失,在心肌细胞间隙内可观察到大量炎症细胞浸润,且浸润部位心肌纤维排列紊乱,心肌组织被破坏。心肌炎转基因大鼠感染CVB3后心肌细胞崩解数量显著减少,细胞核和细胞轮廓较为清晰,心肌细胞间隙可见少量炎性细胞浸润。感染CVB3后心肌炎转基因大鼠心肌细胞的凋亡率明显低于正常SD大鼠。感染CVB3后心肌炎转基因大鼠心肌组织中TLR2蛋白的表达明显低于正常SD大鼠。结论(1)mi R-19b在病毒性心肌炎中表达降低;(2)mi R-19b与病毒性心肌炎疾病严重程度及病程相关;(3)mi R-19b通过靶向抑制TLR2的表达促进心肌细胞增殖,抑制其凋亡,从而抑制病毒性心肌炎的发展。(4)mi R-19b对由CVB3感染所引起的心肌细胞损伤具有保护作用,其机制与mi R-19b抑制TLR2蛋白的表达,从而减轻心肌细胞炎症反应,抑制心肌细胞凋亡有关。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R542.21

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