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

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.

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。

胞外金属蛋白酶是诸多病原菌重要的毒力因子，具有很强的致病性，目前关于金属蛋白酶致病作用机制尚不明确。本项目以重要海水经济鱼类大菱鲆为试验对象，探索了鳗弧菌金属蛋白酶对大菱鲆头肾细胞的毒性损伤作用及分子机制，利用RNA干扰技术研究了内源性凋亡途径在金属蛋白酶诱导大菱鲆头肾细胞凋亡中的作用。主要结果如下：获得大菱鲆Cyt C、Caspase 9和Caspase 3基因cDNA全长序列和鳗弧菌金属蛋白酶，体内注射试验结果证实金属蛋白酶能够抑制大菱鲆血清抗氧化酶活性、增加脂质过氧化物MDA含量、诱导头肾组织细胞凋亡，同时体外细胞培养试验进一步证实金属蛋白酶能够诱导大菱鲆头肾细胞凋亡、促进LDH释放、增加ROS、降低线粒体膜电位，此外，Cyt C、Caspase 9和Caspase 3基因在金属蛋白酶诱导过程中表达量显著上调，明确了金属蛋白酶毒性作用机制并说明上述基因参与了内源性线粒体凋亡过程。为了进一步解析内源性凋亡途径在金属蛋白酶诱导大菱鲆细胞凋亡中的作用，在金属蛋白酶胁迫下建立Cyt C基因的RNA干扰，证实Cyt C敲降对下游Caspase 9和Caspase 3基因及蛋白表达产生一定抑制作用，初步揭示了内源性凋亡通路基因可能在抵御金属蛋白酶毒性胁迫中具有一定作用。本项目研究结果将有助于深入研究鳗弧菌毒力因子的毒性作用机制，为探索大菱鲆病害防治的新途径提供理论依据。