喜树碱诱导的草地贪夜蛾Sf9细胞凋亡

传统植物源杀虫剂喜树碱具有优异的抑制昆虫生长发育活性， 其诱导昆虫细胞凋亡的作用方式和机制尚不明确， 极大地限制了喜树碱在植物保护领域的应用开发。本研究以1 μmol/L喜树碱诱导草地贪夜蛾Spodoptera frugiperda Sf9细胞呈现细胞皱缩、微绒毛消失和染色质边集等典型细胞凋亡早期超微结构形态特征， 中期凋亡小体逐渐出现并急剧增多， DNA电泳分析可见清晰DNA片段化凋亡特征。流式细胞术分析表明1 μmol/L喜树碱诱导Sf9细胞12 h凋亡率达到最大值39.67%， 是对照的13.13倍， 随后减小。喜树碱诱导Sf9细胞凋亡在12 h和24 h 时Sf caspase-1分别出现两个活性高峰， 表明其作为效应因子在细胞凋亡级联反应过程中具有影响作用。喜树碱显著抑制Sf9细胞拓扑异构酶Ⅰ活性， 阻断解旋负超螺旋pBR322 DNA， 导致DNA损伤进而启动细胞凋亡级联反应使Sf caspase-1活性增加， 提示其信号转导过程是细胞凋亡诱导机制之一。本研究通过分析喜树碱的诱导昆虫Sf9细胞凋亡， 对揭示喜树碱诱导昆虫细胞凋亡的作用机制具有重要启示和帮助。

Induced apoptosis of camptothecin in Spodoptera frugiperda Sf9 cells

Camptothecin possesses excellent inhibitive effects on insect growth and development. The apoptotic induction mode and mechanism of camptothecin are ambiguous, which restricts the application of camptothecin in plant protection. In order to investigate its inhibitive effect at the cellular level, the apoptotic induction effect of camptothecin on Spodoptera frugiperda Sf9 cells was studied. Induced with 1 μmol/L camptothecin, Sf9 cells showed typical morphological changes such as cell shrinkage, loss of microvilli and chromatin condensation under the transmission electron microscope. DNA Ladder assay indicated that camptothecin induced apoptosis in Sf9 cells. Apoptotic rate of Sf9 cells treated with 1 μmol/L camptothecin increased within 12 h, and reached the maximum of 39.67% at 12 h, which was 13.13 times as high as that of the control. In addition, the activity of Sf caspase-1, the homolog of mammalian effector caspase, increased after apoptosis induction in Sf9 cells, which reached the maximum at 12 h and 24 h, respectively. Furthermore, camptothecin inhibited Topo Ⅰof Sf9 cells to unwind supercoiled pBR322 DNA, which caused DNA damage to activate caspase. This suggests that the signal transduction pathway of caspase cascade is one of mechanisms of apoptosis induction. This study provides important clues to understanding the mechanisms of apoptosis induction.