纳秒脉冲脉宽参数与细胞凋亡的量效关系研究

Study of dose-effect relationship between the pulse duration of nanosecond pulsed electric field and cell apoptosis

目的研究纳秒脉冲脉宽参数与细胞凋亡之间的量效关系。方法将纳秒脉冲电场参数组合(电场强度7、10、14 kV/cm,脉宽50、100、200 ns,脉冲个数30,频率1 Hz)作用于离体SKOV3细胞,利用Annexin V FITC/PI双染法检测细胞凋亡率和坏死率;通过激光共聚焦显微镜检测细胞膜和线粒体跨膜电位变化情况;仿真计算纳秒脉冲作用时膜电位、微孔密度及电导率的实时动态变化。结果脉冲处理后细胞膜跨膜电位下降程度随脉宽增加而增加,而线粒体跨膜电位下降程度随脉宽增加而减小,且100 ns脉冲同时诱导最大的细胞凋亡率(32.14±3.76)%及较小的坏死率(5.04±1.72)%;仿真计算中膜电位变化规律与实验结果吻合,其中50 ns脉冲诱导最高的微孔密度(2.93×10^(15)/m^(2))和电导率(0.004 S/m)。结论通过合理选择纳秒脉冲脉宽参数可以在一定程度上调控细胞的生物电效应。

Objective Investigation of the dose-effect relationship between the pulse duration of nanosecond pulsed electric field and cell apoptosis.Methods Nanosecond pulsed electric field(field intensity of 14,10,7 kV/cm,pulse duration of 50,100 and 200 ns,pulse number of 30 and repetition frequency of 1 Hz)was applied to SKOV3 cells in vitro,and Annexin V/PI double staining was used to detect cell apoptosis and necrosis rate.Then changes in transmembrane potential of cell membrane and mitochondria membrane were examined by confocal laser scanning microscope,finally the real-time dynamic changes in transmembrane potential,pore density and conductivity during nanosecond pulsed electric field application were achieved by simulation study.Results The results indicated that with the increase of pulse duration,the decline degree of cell membrane transmembrane potential increased,while the decline degree of mitochondria membrane transmembrane potential reduced after the application of nanosecond pulsed electric field,and 100 ns pulse simultaneously induced the maximum apoptosis rate of(32.14±3.76)%and smaller necrotic rate of(5.04±1.72)%.The changes law of membrane transmembrane potential in simulation study was consistent with the experimental results.The 50 ns pulse induces the highest pore density of 2.93×10^(15)/m^(2) and conductivity of 0.004 S/m.Conclusion The research conclusions demonstrated that cell bioelectric effects might be regulated by rationally choosing the pulse duration parameter of nanosecond pulsed electric field.