口蹄疫病毒致乳鼠心肌炎动物模型的建立及其数字基因表达谱分析

发布时间：2018-08-06 14:49

【摘要】：口蹄疫病毒(FMDV)除引起偶蹄动物蹄部和唇部的水泡外还能引起幼龄动物急性心肌炎导致其死亡,目前对FMDV致心肌炎的动物模型及其分子机制缺乏研究。本研究通过FMDV感染乳鼠后心脏冰冻切片及石蜡切片的H.E染色、心肌透射电镜观察、FMDV和纤维连接蛋白(Fn)免疫组化分析、心脏组织FMDV特异性目的基因RT-PCR扩增、心脏研磨上清炎性相关因子定量检测等技术手段证实FMDV感染乳鼠36h可引起急性心肌炎。乳鼠表现为死亡,病理变化为心脏染色可见心肌大量炎性细胞浸润,透射电镜可见FMDV致乳鼠心肌细胞线粒体肿大、肌节走向紊乱、心肌纤维溶解,免疫组化及其灰度分析证实心脏中有FMDV复制,心肌中FN蛋白过量表达。乳鼠心肌细胞体外培养实验和FMDV感染实验表明,FMDV在体外培养的乳鼠心肌细胞中无法复制。本研究建立的FMDV感染乳鼠心肌炎动物模型及其心肌细胞的体外培养为FMDV致心肌炎分子机制的研究奠定了基础。数字基因表达谱(DGE)是一种可快速、全面地测定某一物种特定组织在实验状态下基因表达情况的高通量测序技术,目前DGE技术已经在药物研发、医学研究和基础科学研究等领域得到广泛应用。迄今为止未见DGE在FMD病毒性心肌炎研究中的报道,根据第一章研究结果证实FMDV可以引起乳鼠心肌炎,那么FMDV感染导致乳鼠心脏组织的哪些基因发生了差异表达?这些差异表达的基因在病毒性心肌炎中扮演的可能角色是什么?为此本研究在建立了FMDV致乳鼠心肌炎动物模型的基础上,利用DGE技术对感染组乳鼠和对照组乳鼠心脏进行了数字基因差异表达分析。结果发现感染组和对照组相比总共有3141个基因发生了差异表达,其中有2735个基因上调表达、406个基因发生了下调表达,在所有差异表达的基因中和病毒性心肌炎pathway相关的有47个基因,对这条通路中的部分基因进行了qRT-PCR验证和western-blot验证,本研究结果为进一步研究FMDV致乳鼠心肌炎的分子机制提供了依据,也为FMDV致其他偶蹄幼龄动物心肌炎研究提供了思路。
[Abstract]:Foot-and-mouth disease virus (FMDV) (FMDV) not only causes blisters in hoof and lip of cloven-hoofed animals, but also causes acute myocarditis in young animals. At present, there is little research on the animal model and its molecular mechanism of myocarditis induced by FMDV. The aim of this study was to observe the immunohistochemical analysis of FMDV and fibronectin (Fn) by means of H. E staining in frozen and paraffin sections of heart after FMDV infection. The specific target gene of FMDV was amplified by RT-PCR in heart tissue. Quantitative detection of inflammatory factors in the supernatant of heart grinding confirmed that FMDV infection could cause acute myocarditis in neonatal rats for 36 h. The pathological changes were myocardial inflammatory cell infiltration. Transmission electron microscope showed that the myocardial mitochondria were enlarged, the sarcomere was disordered, and the myocardial fiber was dissolved. Immunohistochemistry and gray analysis confirmed FMDV replication in the heart and overexpression of FN protein in the myocardium. In vitro culture of neonatal rat cardiomyocytes and FMDV infection test showed that FMDV could not be duplicated in cultured neonatal rat cardiomyocytes. The animal model of myocarditis induced by FMDV and the culture of cardiomyocytes in vitro laid a foundation for the study of the molecular mechanism of myocarditis induced by FMDV. Digital gene expression profile (DGE) is a high-throughput sequencing technique that can rapidly and comprehensively determine the expression of genes in a particular tissue of a species under experimental conditions. At present, DGE technology has been developed in drug research and development. Medical research and basic scientific research and other fields have been widely used. DGE has not been reported in the study of FMD viral myocarditis so far. According to the results of the first chapter, it has been confirmed that FMDV can cause myocarditis in neonatal rats. So which genes have been differentially expressed in the heart tissue of neonatal rats caused by FMDV infection? What is the possible role of these differentially expressed genes in viral myocarditis? On the basis of establishing the animal model of neonatal myocarditis induced by FMDV, the differential expression of digital genes in the hearts of infected and control groups was analyzed by DGE technique. The results showed that a total of 3141 genes were differentially expressed in the infected group and the control group, among which 2735 genes were up-regulated and 406 genes were down-regulated. There are 47 genes associated with viral myocarditis in all differentially expressed genes and pathway. Some of the genes in this pathway were verified by qRT-PCR and western-blot. The results of this study provide a basis for further study on the molecular mechanism of FMDV induced myocarditis in neonatal rats, as well as ideas for the study of FMDV induced myocarditis in other cloven-hoofed young animals.
【学位授予单位】：中国农业科学院
【学位级别】：博士后
【学位授予年份】：2016
【分类号】：S852.65

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