纳秒级陡脉冲电场诱导人黑色素瘤细胞裸鼠皮下移植瘤凋亡作用及机制

Experimental studies on apoptosis of A375 cells of xenografted tumors induced by nanosecond pulsed electric field and its mechanism

目的:观察纳秒级陡脉冲对人黑色素瘤细胞裸鼠皮下移植瘤的诱导凋亡作用和Caspase-3表达的影响.方法:取雌性4～6wk龄BALB/c裸鼠20只,建立人黑色素瘤皮下移植瘤模型,设对照组和纳秒级陡脉冲处理组,采用参数为300ns,20kV/cm,f=1Hz的脉冲电场处理5min,每2d处理1次,共3次,分别观察纳秒级陡脉冲处理组和对照组各时间点瘤体体积大小变化,绘制抑瘤曲线;电镜观察肿瘤超微结构改变;采用免疫荧光检测Caspase-3蛋白表达;RT-PCR方法检测Caspase-3mRNA表达;荧光分光光度计测定Ca2+浓度.结果:纳秒级陡脉冲处理组裸鼠瘤体体积明显小于对照组(P<0.01);电镜下可见现典型的细胞凋亡改变;纳秒级陡脉冲处理组Caspase-3蛋白表达和Caspase-3 mRNA表达均较对照组明显增多;Ca2+浓度显著增加.结论:纳秒级陡脉冲电场能够诱导在体人黑色素异体移植瘤细胞产生凋亡.

AIM： To investigate the effect of nanosecond steep pulse on the growth and apoptosis in A375 ceils of xenografted tumors. METHODS： After establishment of 4 to 6 week-old female nude mouse models carrying human melanoma by subcuta- neous inoculationg, 20 mice were randomly divided into 2 groups ： Treatment group and control group. The mice were exposed to nanosecond steep pulse（20 kV/cm, 300 ns, 1 Hz in 5 min, three times every 2 d ） , the changes of the tumor body volume were observed and the inhibition rate was calculated. Caspase-3 was determinated by imnmnofluorescence and RT-PCR and the apoptosis of A375 cells was detected by transmission electron microscope. The changes of [ Ca^2＋ ] i in A375 cells were observed by fluorescence spectrophotometer using Fluo-3/AM as the calcium fluorescent indicator. RESULTS： The tumor body volume in treatment group was smaller than that in control group （ P 〈 0.01 ） and typical morphological features of apoptotic cells were observed under transmission electron microscope. Immunofluorescence and RT-PCR showed that caspase-3 in treatment group increased compared with that in control group. [ Ca^2＋]i markedly increased by treatment with nanosecond steep pulse （P〈0.01）. CONCLUSION： Nanosecond steep pulse induces apoptosis of transplanted tumors.