去甲斑蝥素诱导鳞翅目昆虫细胞凋亡的线粒体途径研究

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 pathway of norcantharidin induced apoptosis in insect cells will be determined. The toxicological mechanism of norcantharidin killing insect cells will be explored to provide scientific basis for further development and utilization of norcantharidin as pesticide.

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。

研究发现去甲斑蝥素（NCTD）和斑蝥素（CTD）能引起昆虫细胞凋亡，但对昆虫细胞凋亡途径了解甚少。本研究就是探索NCTD诱导昆虫细胞凋亡的线粒体信号传导途径，为开发利用NCTD及类似物提供科学依据。.采用AO/EB复染法荧光显微观察NCTD和CTD处理草地贪夜蛾Sf9细胞6h，浓度12.5μg/mL时细胞凋亡比例分别是26.59%和74.0%。用MTT法测定显示NCTD处理浓度低于62.5μg/ mL12h内，细胞抑制率呈时间和剂量依赖性；高于62.5μg/mL时，细胞抑制率剂量依赖性不明显。.采用荧光探针和激光共聚焦显微镜（LSCM）检测显示NCTD引起Sf9细胞中活性氧含量和线粒体Ca2+浓度呈先上升后下降，导致线粒体膜电位降低。再使用Ca2+通道阻断剂和K+通道阻断剂，Bax或MPTP通道阻抑剂，检测发现钙通道和钾通道参与了CTD诱导的Sf9细胞膜电位去极化和离子透性改变。还证实了Bax通道而非MPTP通道在诱导细胞凋亡中起重要作用。.将Sf9细胞裂解，差速离心分离线粒体和胞浆，再用Western blot检测证明NCTD处理Sf9有大量Cyt C从线粒体释放到胞浆中。克隆、表达Sf-caspase-1和Sf9线粒体核酸内切酶G（EndoG）基因并制备多克隆抗体，再用Western blot检测发现NCTD处理Sf9细胞后未激活Sf9-caspase-1，EndoG在线粒体和细胞质中的分布变化不明显。.转录组测序表明，NCTD诱导Sf9核糖体结构蛋白和ATP合成酶a亚基基因上调，但ATP合成酶抑制剂蛋白基因下调，说明昆虫细胞凋亡与核糖体蛋白和线粒体氧化呼吸链基因有关。.建立了基于昆虫酶活的药剂筛选测定方法，检测了NCTD和CTD对20种昆虫PP2A酶的抑制作用。.本研究确认了NCTD诱导昆虫细胞线粒体Cyt C的释放通道，开启了昆虫细胞凋亡的线粒体信号途径，为深入揭示去甲斑蝥素致毒机理奠定了基础。