斑蝥素对草地贪夜蛾Sf9细胞膜完整性和膜电位的影响

为明确斑蝥素对昆虫细胞膜的作用及其机理， 本研究利用草地贪夜蛾Spodoptera frugiperda的卵巢细胞系Sf9细胞作为实验材料， 采用透射电子显微技术(transmission electron microscope , TEM)、 激光共聚焦显微镜(laser scanning confocal microscope, LSCM)结合荧光探针FDA/PI及DiBAC4(3)技术研究斑蝥素(cantharidin， CTD)对Sf9细胞膜完整性及膜电位（membrane potential， MP）的影响。结果表明： 32 μmol/L CTD处理6 h和12 h后， 电镜观察均未发现细胞膜结构破损； FDA/PI染色后， 32 μmol/L CTD处理0.5 h后细胞FDA荧光强度比对照显著降低(P<0.05)， 碘化丙啶（propidium iodide， PI）染色的细胞比例与对照无显著性差异(P≥0.05)。32 μmol/L CTD处理140 s后即引起MP发生显著性去极化(P<0.05)； 64 μmol/L CTD处理瞬时MP发生显著性去极化(P<0.05)； 32 μmol/L CTD处理3 h内及64 μmol/L CTD处理2 h 内MP仍保持显著性去极化(P<0.05)， 之后去极化程度降低； 32 μmol/L CTD处理6 h及64 μmol/L CTD处理3 h时MP去极化与对照组相比已无显著性差异(P≥0.05)。结果说明, CTD处理短时间内可引起Sf9细胞膜电位去极化并维持一段时间， 同时导致细胞活性发生不可逆下降， 但未对细胞膜结构完整性产生破坏。

Effect of cantharidin on cell membrane integrity and potential in Spodoptera frugiperda Sf9 cells

To investigate the effect of cantharidin (CTD) on insect cell membrane and its mechanism, the cell membrane integrity and potential in Spodoptera frugiperda Sf9 cells treated with CTD were tested by transmission electron microscope (TEM) and laser scanning confocal microscope (LSCM) combined with fluorescent dyes FDA/PI and DiBAC4(3). The results showed that after the Sf9 cells were treated with 32 μmol/L CTD, their membrane structure was not disrupted at 6 h and 12 h after treatment, respectively. The FDA fluorescence intensities of the treated cells evidently declined compared with the control (P<0.05) while the proportion of the PI-staining cells was not significantly different from that of the control (P≥0.05) at 0.5 h after treatment under TEM observations. The membrane potential (MP) in Sf9 cells was depolarized significantly both at 140 s after treatment with 32 μmol/L CTD and in the instantaneous treatment with 64 μmol/L CTD (P<0.05). The MP in Sf9 cells depolarized obviously within 3 h after treatment with 32 μmol/L CTD or 2 h with 64 μmol/L CTD (P<0.05), and then the degree of deplorization declined. At 6 h after treatment with 32 μmol/L CTD or at 3 h with 64 μmol/L CTD, MP deplorization in treated cells was not significantly different from that in the control (P≥0.05). The results suggest that CTD causes the depolarization of cell membrane potential in Sf9 cells, which will maintain for a period of time, and induces an irreversible decline in cell activity, but not destroys the integrity of the cell membrane structure.