鳗弧菌金属蛋白酶致大菱鲆头肾细胞氧化损伤及其过程中线粒体凋亡途径作用研究

项目名称： 鳗弧菌金属蛋白酶致大菱鲆头肾细胞氧化损伤及其过程中线粒体凋亡途径作用研究

项目编号： No.31502187

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 农业科学

项目作者： 任海

作者单位： 河北科技师范学院

项目金额： 16万元

中文摘要： 大菱鲆（Scophthalmus maximus）是重要的海水养殖鱼类，鳗弧菌致大菱鲆死亡报道屡见不鲜。研究证实金属蛋白酶作为鳗弧菌重要的毒力因子能够导致机体死亡，目前关于金属蛋白酶致大菱鲆死亡的作用机制尚不明确。本课题拟通过内源性线粒体凋亡途径中重要基因Cyt c、Caspase 9和Caspase 3，研究金属蛋白酶对大菱鲆头肾的毒性作用机制及上述3种基因在线粒体凋亡通路中的表达，对线粒体凋亡通路在鳗弧菌金属蛋白酶对大菱鲆毒性作用中的功能开展研究。本研究通过流式细胞技术、RT-qPCR和Western blot等进行细胞损伤及基因表达特征分析，从体内和体外试验揭示金属蛋白酶在细胞凋亡中的毒性作用机制，并利用RNA干扰技术探讨线粒体凋亡通路在金属蛋白酶致大菱鲆毒性作用下的响应，以期找到阻断病原传播的重要途径，为大菱鲆及其它水产动物的健康养殖提供科学依据。

中文关键词： 金属蛋白酶；大菱鲆；线粒体；细胞凋亡

英文摘要： Scophthalmus maximus is an important mariculture species in China, there are many reports about the death of S. maximus induced by Vibrio anguillarum. The results confirmed that metalloproteases as a major toxic factor in V. anguillarum can subject the body to death. However, the molecular mechanism about how metalloproteases cause the death of S. maximus is unclear. In this project, three major genes Cyt c, Caspase 9 and Caspase 3 in the endogenous mitochondria apoptotic pathway were used. We probed into the toxic effect mechanism of metalloproteases on the head-kidney of S. maximus and detected the expressions of the three genes in the mitochondrial apoptotic pathway. We also investigated the functions of the mitochondrial apoptotic pathway during the process when V. anguillarum metalloproteases exerted toxic effect on S. maximus. We expect to confirm that the mitochondrial apoptotic pathway is involved as a major pathway when the V. anguillarum metalloproteases exert toxic effect on S. maximus. Then cell injuries and gene expressions were characterized with flow cytometry, RT-qPCR and Western blot. Through experiments in vivo and in vitro, we aim to reveal the toxic effect of metalloproteases in apoptosis and to understand the response mechanism of the endogenous mitochondrial apoptotic pathway against bacterial virulence factors using the RNA interference technique. We expect to find out the critical pathway to prevent the spread of pathogens. This study serves as a scientific basis for healthy breeding of S. maximus and other aquatic animals.

英文关键词： metalloprotease;Scophthalmus maximus;mitochondria;apoptosis