纳秒级陡脉冲诱导人卵巢癌细胞凋亡及对Fas介导的细胞凋亡通路的影响

Induction of Apoptosis of Ovarian Cancer Cells and Influence on Fas-mediated Apoptosis Pathway by Nanosecond Pulsed Electric Fields

为探讨纳秒级陡脉冲(nsPEFs)对人卵巢癌SKOV3细胞凋亡作用及Fas介导的细胞凋亡通路的影响,以场强为45kV/cm,脉宽分别为50、100和200ns,脉冲个数为30个的nsPEF处理的SKOV3细胞为实验组,以未经处理的SKOV3细胞为对照组,流式细胞术检测细胞晚期凋亡率和(或)死亡率,琼脂糖凝胶电泳法检测DNA Ladder,RT-PCR和Western blot检测细胞内Fas、FasL、caspase-8和Bid的表达量变化。结果显示与对照组相比,脉宽分别为50、100和200ns组细胞晚期凋亡率和(或)死亡率分别为(18.31%±0.65%)、(45.55%±3.71%)和(47.47%±7.01%),明显高于对照组(3.03%±0.57%)(P<0.05);琼脂糖凝胶电泳结果显示脉宽为50ns和100ns组有大量DNA ladder形成,而200ns组条带模糊;RT-PCR检测结果显示50ns、100ns组Fas、FasL、caspase-8和Bid mR-NA表达量均显著升高(P<0.05),200ns组mRNA表达量均显著降低(P<0.05);Western blot检测结果显示50ns、100ns组Fas、FasL、caspase-8和Bid蛋白表达量显著升高(P<0.05),200ns组蛋白表达量均显著降低(P<0.05)。提示场强45kV/cm,脉宽为50ns、100ns的nsPEFs能诱导人卵巢癌SKOV3细胞发生凋亡,机理可能与Fas介导的细胞凋亡通路被激活有关。

This paper is to investigate the apoptosis effect of ovarian cancer SKOV3 cells induced by nanosecond plused electric fileds （nsPEFs） and to study its influence on Fasmediated apoptosis. SKOV3 cell were exposed to the 45kV/cm of field intensity, 30 pulses, and 50ns, 100ns, and 200ns of pulse width, respectively. Flow eytometry were used to assay apoptosis. Agarose gel electrophoresis was used to detect DNA ladder. Real time PCR（RT-PCR） and Western blot analysis were used to measure the expression level of Fas, FasL, caspase-8 and Bid. Flow eytometry results revealed that the late apoptosis rates and （or） necrosis were significantly higher than those in control group （3.03%士0. 57%） （P〈0. 05）,with apoptosis rates and （or） necrosis being （18. 31%士0. 65%）,（45.55%士3.71 %）, （47. 47% 士 7. 01 %） in the groups of 50ns, lOOns, 200ns, respectively. A typical DNA ladder pattern of internucleosomal fragmentation was observed in the groups of 50ns and lOOns, but not clear in the 200ns group. RTPCR results revealed that the mRNA expression of Fas, FasL, caspase-8 and Bid were significantly increased in groups of 50ns, l100ns, but significantly decreased in group of 200ns （P〈0. 05）. Meanwhile, Western blot analysis demonstrated that the Fas, FasL, Caspase-8 and Bid expression were significantly higher in groups of 50ns, lOOns, but significantly lower in group of 200ns （P〈0. 05）. It indicated that 45kV/cm, 50ns, 100ns nsPEFs could induce apoptosis in ovarian cancer SKOV3 cells and activate Fas-mediated apoptosis pathway.