脉冲电场对肝癌细胞的凋亡诱导效应及线粒体跨膜电位的影响研究

为研究脉冲电场对肿瘤细胞的杀伤作用,采用Annexin V-FITC凋亡试剂盒和流式细胞技术检测电脉冲(1kV/cm、100μs和1 Hz)作用后人肝癌细胞HepG2和人肝细胞L02在不同作用时间点(8、15、30和60 s)下的凋亡效应。实验结果表明:与对照组相比,8 s刺激时间不足以诱导HepG2细胞凋亡,而15、30和60 s的刺激时间与可以明显的诱导HepG2凋亡,并且凋亡率随刺激时间增加而增大,说明延长刺激时间可以增大肿瘤细胞的凋亡程度;另外,在刺激60 s时,肿瘤细胞凋亡率是正常细胞的2倍,这表明肿瘤细胞比正常细胞对电脉冲的刺激更敏感。采用罗丹明123探针染色,用激光共聚焦显微镜实时观测细胞的线粒体膜电位荧光强度变化,观察到电刺激30 s前细胞线粒体膜电位无明显变化,而30 s后开始出现膜电位陡升后急剧降低的变化现象,揭示线粒体途径可能是脉冲诱导肿瘤细胞凋亡的机理之一。

Effects of electric pulses on liver cancer cells: apoptosis induction and decrease of mitochondrial transmembrane potential

In order to investigate the effects of electric pulses on cancer cells, we carried out the experiments with exposing HepG2 and L02 to electric pulses (1 kV/cm, l00 micros, 1 Hz) for different lengths of time (8 s, 15 s, 30 s, 60 s). Annexin V-FITC Kit and Flow cytometry were used to study the apoptosis of treated cells. The results showed that the electric pulses of 1 kV/cm, l00 micros, 1 Hz for 8 s could not induce tumor cells apoptosis. Apoptosis was observed when tumor cells were stimulated for 15 s and longer, and the apoptosis percentage increased with the increase of stimulation time. Furthermore, tumor cells were more sensitive than normal cells in response to electrical pulses. Rhodamine 123 and Laser Scanning Confocal Microscope (LSCM) were used to make a real-time study of mitochondrial transmembrane potential (Deltapsim) when the tumor cells were exposed to electric pulses for 60 s. No significant change of Deltapsim was observed within 30 s stimulation. After that, the Deltapsim increased sharply and declined later, suggesting that the mitochondrial pathway may be one of the apoptosis mechanism induced by electric pulses.