脉冲电场对宫颈癌细胞的杀伤作用研究-第三军医大学学报.doc

基金项目：国家自然科学基金重点项目，重庆市自然科学基金重点项目（CSTC,2009BA5039） 不可逆性电穿孔致Hela细胞凋亡与坏死的作用研究 周玮1，熊正爱1，刘颖1，李成祥2，姚陈果2 （1.重庆医科大学附属第二医院，重庆，400010；2．重庆大学输配电装备及系统安全与新技术国家重点实验室，重庆，400030） 摘要 目的 探讨脉冲电场不可逆性电穿孔处理Hela细胞时，发生细胞膜电穿孔、细胞凋亡、细胞坏死与脉冲电场的关系。 方法 采用固定脉宽100μs、频率1Hz、脉冲数8个，电压由100V逐级升至1000V的脉冲电场处理Hela细胞。利用PI示踪，流式细胞术检测研究Hela细胞膜发生穿孔的阈值电场剂量；CCK-8（cell counting kit-8,CCK-8）法检测脉冲电场处理Hela细胞的量效关系；Annexin V kit 染色，流式细胞术检测各组细胞的凋亡与坏死。 结果 Hela细胞膜发生可逆性穿孔的阈值场强为750V/cm；发生凋亡的阈值电压为1250V/cm至1500V/cm之间；坏死的阈值为1750V/cm。结论 不可逆性电穿孔杀灭Hela细胞时细胞凋亡也发挥着重要作用，而且导致凋亡与坏死的电场范围相互重叠。 关键词：脉冲电场；Hela细胞；不可逆性电穿孔；凋亡 Research on apoptosis and necrosis of Hela cells induced by irreversible electroporation Zhou Wei1, Xiong Zheng-ai1, Liu Ying1, Li Cheng-xiang2, Yao Chen-guo2 (1. the 2nd affiliated hospital of Chongqing Medical University, Chongqing, 400010, 2. State Key Laboratory of Power Transmission Equipment System Security and New Technology ; Chongqing, 400010) Abstract Objective To investigate the impact of pulse electric fields (PEFs) on membrane breakdown, necrosis and apoptosis of Hela cells when irreversible electroporation (IRE) was performed on it. Methods PEFs with permanent duration (100 μs), frequency(1Hz) and pulses(8 pieces) performed on Hela cells. The voltage was from 100V to 1000V and the amplification was 100V of each group. Propidium Iodide(PI) was used to tracing the breakdown of cytomenmbrane, then detected with flow cytometry according to the manufacturer’s protocol. CCK-8 (cell counting kit-8,CCK-8） assay was used to examine survival of Hela cells after treated with IRE. Hela cells were double-stained with Annexin V kit then analyzed with flow cytometry for apoptosis and necrosis. Results The reversible electroporation threshold of Hela cells was 750V/cm. The apoptotic threshold was between 1250V/cm to 1500V/cm. The necrotic threshold was about 1750V/cm. Conclusion Besides necrosis due to IRE, apoptosis also played an important roll during IRE ablating Hela cells. The areas of PEFs parameters ind