心肌线粒体功能基因在克山病心肌损伤及功能障碍中的分子机制研究

The project team has studied the genes in peripheral blood mononuclear cells of Keshan disease, involved in cardiac mitochondrial function and apoptosis, ion channels and protein transport and other related genetic information by application of oligonucleotide microarray technology. The myocardial injury, metabolic disorders, and unknown pathogenesis would be the key issues for Keshan disease. At the basis of these results, we use field investigation, experiments research and crowd study with RNAi, cell culture, western blot, qRT-PCR and so on to study the genes associated with myocardial injury in Keshan disease from the human body, cells, genes, proteins at different levels. To investigate myocardial injury related protein expression and mitochondrial metabolic function changes before and after RNAi; verify the genes referred to myocardial injury, metabolic disorders and to determine its role in myocardial injury. The establishment of the network associated with low selenium, myocardial cell injury, metabolic disorders and related genes, would be useful to explore the interrelation in molecular mechanisms of myocardial injury, harmful environmental factors and the key genes. These results could promote the etiology and pathogenesis study of Keshan disease for seeking new molecular biological markers and therapeutic targets to provide scientific basis to improve prevention, diagnosis and treatment level of Keshan disease in China.

针对克山病心肌损伤和代谢障碍发病机制不明的关键科学问题，依据近来应用寡核苷酸基因芯片所获克山病外周血单核细胞涉及心肌线粒体功能、细胞凋亡、离子通道及蛋白转运相关基因的研究进展，采用现场人群与体外心肌细胞培养、RNAi、Western blot和qRT-PCR等技术，从人体、细胞、基因、蛋白不同层面，对比研究心肌线粒体功能基因在克山病与扩张性心肌病心肌损伤中的作用机制和RNA干扰前后心肌损伤相关蛋白表达和线粒体功能的改变，揭示其对心肌差异表达蛋白和线粒体代谢功能的影响，明确心肌损伤、代谢障碍的关键基因及其功能。藉此为基础，进一步确定低硒对心肌细胞损伤、代谢障碍和心肌线粒体功能基因影响的作用机制，力求从环境-基因相互作用层面，阐明克山病环境有害因素对心肌、线粒体功能和代谢途径的分子机制，明确克山病心肌损伤分子生物学标志和可能的治疗靶点，为提高我国克山病早期诊断、预防和治疗水平，提供科学依据。

针对“克山病是一种心肌线粒体病”的假说，本课题对筛选的克山病相关的线粒体功能基因进行多层面验证及鉴定，确定候选基因。应用RNAi技术干扰候选基因，比较研究RNA干扰前后线粒体功能和细胞各代谢途径的变化及变化特征。建立克山病细胞损伤、代谢途径障碍与相关基因间的相互关系。分析和探索低硒、相关基因在心肌损伤、代谢途径障碍、线粒体功能之间的作用机制。确定克山病线粒体功能基因及其在克山病发病的机制中的作用。筛选克山病与扩张型心肌病差异表达基因。推进克山病线粒体功能障碍机制研究，为提高克山病防治水平，寻找新的治疗靶点。本项目的主要内容有：（1）克山病与扩张型心肌病外周血单核细胞差异表达基因的筛选及验证；（2）硒对克山病线粒体损伤相关AMPK通路的影响；（3）Bnip3L基因在克山病心肌细胞线粒体凋亡机制中活化作用的研究；（4）硒对克山病CYP450代谢通路的影响；（5）硒对内外源性NO介导心肌损伤的保护作用及其机制研究；（6）克山病和正常对照间唾液或血清之间差异糖蛋白筛选研究；（7）克山病单核苷酸多态性研究。克山病心肌损伤与心肌线粒体相关功能基因的异常表达有密切的关系，我们通过人群实验、细胞实验和动物实验验证心肌线粒体功能性相关基因的异常表达能够引起心肌细胞的损伤，但是硒对心肌细胞的损伤有一定的保护作用。 本项目资助1名博士生、5名硕士生。发表学术论文20篇，其中SCI收录11篇。