去甲斑蝥素诱导鳞翅目昆虫细胞凋亡的线粒体途径研究

项目名称： 去甲斑蝥素诱导鳞翅目昆虫细胞凋亡的线粒体途径研究

项目编号： No.31272099

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 王进忠

作者单位： 北京农学院

项目金额： 80万元

中文摘要： 去甲斑蝥素对害虫具有触杀、胃毒和拒食作用。研究结果显示，去甲斑蝥素引起鳞翅目昆虫细胞凋亡，但对昆虫细胞凋亡信号途径了解甚少。引起动物细胞凋亡信号途径有三条，其中线粒体途径在昆虫体内一直存在着争论。本研究首先用噻唑蓝法和流式细胞仪分别检测去甲斑蝥素对昆虫细胞毒性作用及细胞凋亡比例，建立剂量和时间效应关系；再使用激光共聚焦显微技术检测细胞内活性氧含量、钙离子浓度以及线粒体膜电位的变化，分析去甲斑蝥素引起昆虫细胞凋亡途径的早期传导信号作用步骤；通过使用Bax和MPTP通道阻抑剂以及丝氨酸蛋白酶抑制剂测定细胞色素c的释放与否，探索去甲斑蝥素处理鳞翅目昆虫细胞时细胞色素c的释放机制；采用caspases试剂盒测定去甲斑蝥素对昆虫细胞caspases活性的影响，确定去甲斑蝥素导致昆虫细胞凋亡过程中线粒体信号传导通路。探索去甲斑蝥素对昆虫细胞的毒理学作用机制，为进一步开发利用去甲斑蝥素农药提供科学依据。

中文关键词： 去甲斑蝥素；鳞翅目昆虫细胞；凋亡；线粒体信号途径；

英文摘要： We have proved that norcantharidin has contact toxicity, stomach poisoning action and antifeedant effect on pests. The experiment results show that norcantharidin induces apoptosis in lepidopteran cells,yet the signaling pathway of norcantharidin induced apoptosis in insect cells is not fully understood. There are three signal pathways of inducing animal cell apoptosis, but the mitochondrial pathway in insects has been highly controversial. Firstly, in the present study，cytotoxicity and apoptosis ratio of norcantharidin on insect cells will be detected by MTT assay and flow cytometry, establishing a time and dose dependent manner. Secondly，reactive oxygen species, calcium ion concentration, mitochondrial membrane potential loss will be measured by confocal microscopy in order to analyze the early steps of signal transduction pathway of norcantharidin induced insect cell apoptosis. Thirdly, by using the Bax and MPTP channel and serine protease inhibitors we will decide whether or not the release of cytochrome c from mitochondria can be detected so that we will understand the releasing mechanism of cytochrome c in norcantharidin treated lepidopteran insect cells. Activation of caspases in lepidopteran cells induced by norcantharidin will be studied using caspases assay kit, and also the mitochondrial signaling pa

英文关键词： norcantharidin；lepidopteran cells；apoptosis；mitochondrial signaling pathway；