目的： 建立病毒性心肌病动物模型，应用基因芯片技术进行全基因组测定及分析，研究病毒性心肌病的心肌线粒体功能变化在心肌病发病机制中的作用。 方法：1、通过CVB〓反复增量病毒感染Balb/c小鼠，建立较理想的病毒性心肌病模型。2、用反复增量病毒感染法建立病毒性心肌病模型，通过心功能测定和病理学检测，筛选阳性标本进行重复小鼠心肌全基因微阵列芯片(共包括25000条基因)检测，采用GenePix Pro4.0图像分析软件进行分析，数据用Lowess方法进行归一化，并用qRT-PCR(quantitative real-time PCR)进行验证，确定差异表达基因，结合基因库进行综合分析。3、根据筛选的3倍差异表达线粒体基因变化，进行电镜扫描、原位细胞凋亡标记(TUNEL)检测心肌细胞凋亡、流式细胞仪检测线粒体膜电位变化(ΔΨm)，用张均田法分别检测心肌细胞、线粒体内细胞色素C浓度变化，荧光分光光度计比色法检测细胞色素C氧化酶活力等多角度观察病毒性心肌病鼠线粒体功能变化特征。 结果：1、反复增量病毒感染后104d，存活小鼠心功能较对照组降低(P<0.05)，心肌纤维化检出率为38.2%，与对照组有显著性统计学差异(P<0.05)，符合扩张型心肌病病理改变。2、基因芯片共筛选出2313条差异表达基因，其主要特点符合扩张型心肌病特点，如线粒体氧化磷酸化的基因下调为主、心肌结构基因下调、细胞外基质基因和免疫相关基因明显差异表达，同时涉及心肌细胞凋亡基因及相关凋亡调节信号转导途径基因明显差异表达。3、原位细胞凋亡标记(TUNEL)、电镜和基因芯片检测结果表明病毒性心肌病存在心肌细胞凋亡， AMID、CIDEA、Tnfrsfl2a、Grim19、Hipk2、Bad等重要的凋亡基因上凋。抗凋亡基因Birc1a，Birc5基闪出现代偿性升高，促凋亡和抗凋亡基因均有明显的差异表达。4、基因芯片检测结果表明线粒体凋亡信号途径参与心肌细胞凋亡， Amid、Bad、Grim19凋亡基因明显上调，调节凋亡信号途径基因Mapk8ip3表达增强。5、流式细胞仪检测早期线粒体膜电位下降(ΔΨm),张均田法检测线粒体内细胞色素C浓度下降。6、通过基因芯片检测结果证实Atp5g1、VDAC-1、Cox4i1， UCP2等许多线粒体能量代谢基因明显差异表达，荧光分光光度计比色法证实线粒体氧化磷酸化标志酶细胞色素C氧化酶活力下降。 结论：1、用反复增量病毒感染法成功复制了小鼠病毒性心肌病动物模型。 2、获得了病毒性心肌病发病过程中差异表达的宿主基因，证实了心肌细胞凋亡、线粒体氧化磷酸化、横纹肌收缩和抗原加工和递呈基因通路的变化在病毒性心肌病发病中起重要作用。3、心肌细胞凋亡是病毒性心肌病发病机制之一，机体促凋亡和抗凋亡基因表达失衡是产生细胞凋亡的重要原因。4、心肌线粒体凋亡是病毒性心肌病心肌细胞凋亡的重要机制，线粒体凋亡与非caspase依赖的细胞凋亡基因Amid基因及caspase依赖的细胞凋亡基因Cidea，BAD，Mapk8ip3调控相关；线粒体膜电位(ΔΨm)下降，细胞色素C释放是线粒体凋亡的重要机制。5、病毒性心肌病线粒体氧化磷酸化关键酶细胞色素C氧化酶活力下降，存在线粒体能量代谢障碍，是病毒性心肌病的重要病理生理基础，也很可能是细胞凋亡的启动因素之一。 关键词：病毒性心肌病；柯萨奇病毒B₃；微矩阵芯片；细胞凋亡；线粒体；细胞色素C；线粒体膜电位；细胞色素C氧化酶

 Object: To study the role of mitochondria genes expression on pathogenesis of the viral cardiomyopthy by establishing the viral cardiomyopathy model in mouse, determining and analyzing the entire host genome by gene chip technique. Method: 1.BALB/Cmice were infected with coxsackie virus B3m (CVB3m) to establish experimental model of dilated cardiomyopathy by repetitive and incremental infection. 2.We established the model of experimental viral cardiomyopathy by repetitive and incremental infection, measured the cardiac function and perform pathologic study. Specific mRNA expression pattern in positive model was analyzed with cDNA microarray containing 25000 genes. The gene expression profile was analyzed with GenePix Pro 4.0 image analysis software; the data was normalized with locally weighted linear regression analysis (LOWESS), and verified with quantitative real-time PCR. The results of the array were analyzed and classified with computer aided cluster analysis and the gene bank of internet. 3.Based on the three times change of mitochondria genes expression, we conduct further study on mitochondria function by using following techniques, such as: The techniques of the terminal deoxynucleotidyi transferase mediated dUTP nick-end labeling, the electron microscope scanning were applied to obtain the evidence of apoptosis, the flow cytometry to analyze mitochondrial transmembrane potential, Zhangjuntian method was to measured the density of cytochrome C in Cytoplasm and mitochondria . Fura-2/AM flourescein loading techniquee was used to evaluate the activity of cytochrome C oxidase. Results 1. 104 days later after the repetitive and increased infection, the cardiac function of survived mice were depressed compared with that of control group (P＜0.05). The positive rate of myocardial fibrosis was 38.2%, which was significantly different from control group (P＜0.05) according to the pathological change of dilated cardiomyopathy. 2.There were 2313 different expression genes analyzed by software and proved by quantitative real-time PCR. The main characteristic of differential expressed genes was consistant with that of dilated cardiomyopathy, such as down regulation of energy metabolism genes and myocardial structure genes, significantly differential expression of extracellular matrix genes and genes related to immunity. 3.The results of the terminal deoxynucleotidyi transferase mediated dUTP nick-end labeling, electron microscope and gene chip confirmed that myocardial apoptosis was existent in myocardium of viral cardiomyopathy. The important apoptosis genes, such as AmidN cell death-inducing DFFA-like effector A (CIDEA)、 Tnfrsfl2a、 Griml9、 Hipk2、 Bad, et al, were up-regulated. The anti-apoptosis genes, Bird a and Birc5 were elevated for the sake of compensation; both of the pro-apoptosis and anti-apoptosis genes had obviously up-regulated differential expression. 4.The results of gene chip confirmed that mitochondria apoptosis signal transduction passway participated apoptosis, Amid、 Griml9、 Bad, et al, were up-regulated, and the regulating gene to promote apoptosis Mapk8ip3 was up-regulated. 5.The flow cytometry showed mitochondrial transmembrane potential was decreased. The consentration of cytochrome C in Cytoplasm was higher than that in mitochondria by Zhangjuntian method. 6.The results of gene chip confirmed that lots of mitochondria genes, such as Atp5g、 VDAC、 UCP2、 Cox4il had obviously differential expression. Fura-2/AM flourescein loading technique showed that the activity of cytochrome C oxidase which was a symbolic enzyme in oxidative phosphorylation was decreaced. Conclusion 1.The method of repetitive and increased viral infection could establish viral cardiomyopathy model. 2.Host genes expression in viral cardiomyopathy model was obtained.Some gene pathway, such as apoptosis and mitochondrial oxidative phosphorylation and antigen processing and presentation, strated muscle contraction, etc., may be the key mechanism of viral cardiomyopathy. 3.The apoptosis is one of the pathogenesis of the viral cardiomyopthy, the disbalance between the expression of pro-apoptosis and anti-apoptosis gene was the main reason leading to cell apoptosis. 4.The mitochondria apoptosis is one of the pathogenesis of apoptosis in the viral cardiomyopthy, which was related to the caspase-independent apoptosis gene Amid and caspase-dependent apoptosis gene Griml9、 Bad、 Mapk8ip3, the mitochondria apoptosis was also interrelated the decrease of mitochondrial transmembrane potential and release of cytochrome C. 5.The activity of cytochrome C oxidase which was.a symbolic enzyme in oxidative phosphorylation was decreaced, what showed the abnormal functions of myocardium mitochondrium and severe energy metabolism disorder were the important pathological and physiological foundamental process of viral cardiomyopathy, which may lead to apoptosis. Key words: viral cardiomyopathy; coxsackie virus B₃; cDNA microarray chip; apoptosis; mitochondrium; cytochrome C; mitochondrial transmembrane potential; cytochrome C oxidase