高强度皮秒脉冲电场对人宫颈癌HeLa细胞的生长抑制及凋亡诱导作用

摘要

目的 探讨高强度皮秒脉冲电场对体外培养的人宫颈癌细胞HeLa的生长抑制和凋亡诱导作用.方法 体外培养人宫颈癌细胞HeLa,将细胞分为处理组(在高强度皮秒脉冲电场处理)及正常对照组(未经高强度皮秒脉冲电场处理).固定脉宽、频率、场强,按脉冲个数100、200、500、1000、1500、2000分为6个处理组,利用MTT比色法检测脉冲电场对HeLa细胞的生长抑制作用和量效关系.检测细胞存活率发生显著变化的500、1000、2000组细胞的Caspase3活性.根据MTT及Caspase结果,只选取Caspase 3活性高的脉冲个数2000组加入罗丹明123探针后于激光共聚焦扫描显微镜下检测HeLa细胞线粒体跨膜电位.结果 MTT证明高强度皮秒脉冲电场对HeLa细胞有杀伤作用且存在剂量-效应关系,细胞存活率随脉冲个数的增加而降低,脉冲个数100、200、500、1000、1500、2000处理组及正常对照组HeLa细胞存活率分别为96.23％±0.76％,94.11％±2.42％,90.31％±1.77％,64.59％±1.59％,32.95％±0.73％,23.85％±2.38％,100％,各处理组与正常对照组比较差异有统计学意义(P＜0.01).脉冲个数500、1000、2000及正常对照组Caspase 3吸光度值(A值)分别为0.174±0.012,0.232±0.017,0.365±0.016,0.122±0.011,各处理组均高于正常对照组(P＜0.05).脉冲个数2000组线粒体跨膜电位荧光强度(76.66±13.38)明显低于正常对照组(155.81±2.33,P＜0.05).结论 高强度皮秒脉冲电场对HeLa细胞有生长抑制作用,其机制可能是通过线粒体途径诱导细胞凋亡.%Objective To investigate the growth and apoptosis of HeLa cells induced by intense picosecond pulsed electric field (PEF) in vitro. Methods HeLa cells cultured in vitro were divided into experimental group and control group (with or without intense picosecond PEF). With constant pulse width, frequency and voltage, the cells in experimental group were divided into 6 sub-groups according to the number of pulse (100, 200, 500, 1000, 1500, 2000), the growth inhibition of HeLa cells by PEF and the dose-effect relationship were analyzed by MTT. Caspase 3 protein activity was detected in the cells in 500, 1000 and 2000 sub-groups. Mitochondrial transmembrane potential was detected by rhodamine 123 staining with the cells in 2000 sub-groups. Results MTT assay demonstrated that intense picosecond PEF significantly inhibited the proliferation of HeLa cells in dose-dependent manner. The survival rates of cells declined along with the increase in pulse number, and were 96. 23%±0. 76%, 94. 11%±2. 42%, 90. 31%±1. 77%, 64. 59%±1. 59%, 32. 95% ±0. 73%, 23. 85%±2. 38% and 100%, respectively, in 100, 200, 500, 1000, 1500, 2000 sub-groups and control group (P<0. 01). The Caspase 3 protein activity was significantly enhanced by intense picosecond PEF, and the absorbancy indexes (A) were 0. 174 + 0. 012, 0.232±0.017, 0.365±0.016 and 0. 122± 0.011, respectively, in 500, 1000, 2000 sub-groups and control group (P<0.05). The mitochondrial transmembrane potential of HeLa cells was significantly inhibited by intense picosecond PEF, and the fluorescence intensity in 2000 sub-group (76. 66±13.38) was much lower than that in control group (155. 81±2.33, P<0.05). Conclusion Intense picosecond PEF may significantly inhibit the growth of HeLa cells, and induce cell apoptosis via mitochondrial pathway.