亚纳秒脉冲电场联合温度杀伤人宫颈癌细胞的实验研究  

Inhibitory Effects of Sub-Nanosecond Pulsed Electric Field on Hela Cells at Elevated Temperature

作　　者：郭飞[1] 张琳 刘欣[1] 张玉 Guo Fei;Zhang Lin;Liu Xin;Zhang Yu(Chongqing Key Laboratory of Complex Systems and Bionic Control,Chongqing University of Posts and Telecommunications,Chongqing 400065,China;Department of Cynecology and Obstetrics,Chongqing Traditional Chinese Medicine Hospital,Chongqing 400021,China)

机构地区：[1]重庆邮电大学重庆市复杂系统与仿生控制重点实验室,重庆400065 [2]重庆市中医院妇产科,重庆400021

出　　处：《中国生物医学工程学报》2020年第3期335-341,共7页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金(51507024);重庆邮电大学自然科学基金(A2015-67);重庆高校创新团队建设计划项目(CXTDX201601019)。

摘　　要：为研究亚纳秒脉冲联合温度对人宫颈癌(He La)细胞的杀伤效应,将脉冲电场参数组合(电场强度25、50、100 k V/cm,半峰值脉宽1 ns,脉冲个数分别为4000、1000、250,频率5 Hz),作用于处于不同温度(25℃、43℃、48℃)的He La细胞,利用MTT检测亚纳秒脉冲对细胞的生长抑制情况,通过AO/EB染色和透射电镜观察细胞形态学变化,采用免疫细胞化学S-P法检测caspase-3蛋白表达活性。实验发现:当细胞悬液温度到达48℃后,随着时间推移细胞死亡率不断增加;采用亚纳秒脉冲电场(电场强度50 k V/cm,半峰值脉宽1 ns,脉冲个数1000,频率5 Hz)处理不同温度下He La细胞,细胞悬液温度达到43℃后,细胞死亡率达到18.07%±1.98%,细胞悬液温度达到48℃,细胞死亡率增至25.11%±6.05%;采用相同能量脉冲电场处理处于相同温度(48℃)的细胞悬液时,细胞死亡率与脉冲场强正相关,100 k V/cm电场作用下得到最高细胞死亡率为31.09%±5.03%,进一步的分子生物学检测结果显示He La细胞发生凋亡效应。适当场强的亚纳秒脉冲联合温度通过诱导细胞凋亡,进而杀死He La细胞。In order to study the killing effects of combined exposure of sub-nanosecond pulsed electric field(sub-nsPEF)and temperature on He La cells,sub-nsPEFs(field intensity of 25,50 and 100 kV/cm,pulse duration of 1 ns,pulse number of 4000,1000 and 250,repetition frequency of 5 Hz)were applied on He La cells at different temperatures(25℃,43℃,48℃).The inhibition rate of cell proliferation was determined by MTT assay;morphological changes of the cells were examined by acridine orange and ethidium bromide(AO/EB)staining and transmission electron microscopy;protein expression of caspase-3 was detected by immunocytochemistry.Experimental results showed that the cell death rate was increased over time when the culture temperature reached 48℃.Next,sub-nsPEF(field intensity of 50 kV/cm,pulse duration of 1 ns,pulse number of 1000;repetition frequency of 5 Hz)was applied on cells at different temperatures.The cell death rate was 18.07%±1.98%when the temperature reached 43℃(P<0.05),and increased to 25.11%±6.05%when the temperature was reached 48℃(P<0.01).The cell death rate was proportionally correlated with the field intensity when the sub-nsPEF with same energy was applied on the cells at the same temperature.The largest cell death rate of 31.09%±5.03%was obtained with the field intensity was 100 k V/cm(P<0.01).Mechanistic study indicated that the cells underwent apoptosis after the combined treatment.These data demonstrate that the sub-nsPEF with lower field intensity and temperature can cause tumor cell death through inducing apoptosis.

关 键 词：亚纳秒脉冲 HELA细胞 MTT 透射电镜 细胞凋亡 

分 类 号：R318[医药卫生—生物医学工程]