纳秒脉冲电场可用于治疗裸鼠人恶性黑色素瘤

The effect of Nanosecond pulsed electric fields on human melanoma A375 cell xenograft in nude mice

目的观察纳秒脉冲电场(nsPEF)治疗裸鼠皮下人恶性黑色素瘤的效果及其机制。方法建立裸鼠皮下人黑色素瘤模型,随机分为:1)长期治疗组及对照组各10只,观察肿瘤生长、裸鼠存活率时间;2)短期治疗组(分2 h、4 d组)及对照组(分别为4、6和6只),以HE染色检查细胞形态,DNA琼脂糖凝胶电泳、TUNEL法分析细胞凋亡,Western blot和免疫组织化学法检测肿瘤组织BAX、BCL-2的表达。两组均用电场峰值40 kV/cm、脉冲宽度200 ns、频率1 Hz、脉冲次数1 000个的nsPEF进行治疗。结果长期治疗后即刻看到肿瘤区均变为灰白色,表面皮肤均没有出血现象,肿瘤质地变硬;随生存时间延长,肿瘤体积较对照组明显缩小(P<0.01);平均生存时间(60.6±6.9)d较对照组(17.7±5.4)d明显延长(P<0.01);短期治疗组,4 d时坏死区域增多;4 d时较对照组有明显"阶梯"状分布,凋亡率明显高于对照组(P<0.01),较对照组相比BAX表达明显增高(P<0.01)、而BCL-2表达明显降低(P<0.01)。结论 nsPEF对裸鼠皮下人黑色素瘤有明显的治疗效果,可能是通过调控BAX、BCL-2基因表达诱导细胞凋亡。

Objective To investigate the effect of nanosecond pulsed electric fields（nsPEF）on human melanoma A375 cell xenograft in nude mice and its possible mechanisms.Methods Nude mice were randomly divided into long-term treatment group and then short-term treatment group（includes 2 h group、4 d group and control group,4、6 and 6 mice,respectively） exposed to the 40 kV/cm,200 ns,1 Hz,1 000 pulses.Tumor morphology was observed with HE staining;The cell apoptosis was detected by DNA agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling（TUNEL） assay;The expression of BAX and BCL-2 protein was detected by Western blot and SP immunohistochemistry.Results Tumor tissue turned to grey without bleeding spots minutes after long-term treatment,the tumor volume was significantly smaller in treatment group than that in controlgroup,the mean survival time was significantly longer in treatment group（60.6±6.9）d than in that control group（17.7±5.4）d.Cell necrosis was found in 4 d group;DNA ladder was observed in 4 d group;the apoptosis rate of tumor cells was significantly higher in 4 d group than that in control group;the expression of BAX was significantly higher in 4 d group than in control group（P0.01）;the expression of BCL-2 was significantly lower in 4 d group than that in control group after short-term treatment（P0.01）.Conclusions nsPEF may cause melanomas complete remission without recurrence by inducing cell apoptosis under the BAX and BCL-2 gene regulation mechanism.