【摘要】 背景:磁场能在体内或体外影响肿瘤细胞的生长与分裂,但目前尚无定论磁场能否在体外诱导人肝癌细胞的凋亡。目的:观察极低频磁场诱导人肝癌细胞SK-HEP-1凋亡的效果。设计:开放性实验。单位:上海交通大学生物技术研究所。材料:实验于2004-09/2005-01在上海交通大学生物技术研究所完成。人肝癌细胞SK-HEP-1购于上海中科院细胞库。方法:SK-HEP-1细胞以2.0×104L-1接种,生长于含有体积分数为0.1的热灭活新生牛血清和2mmol/LL-谷氨酰胺的DMEM培养基中,50Hz,20mT磁场对人肝癌细胞SK-HEP-1连续作用8d;对照组细胞则在另一个培养箱中培养,除无磁场干预外,与磁场处理组细胞的生长条件完全一致。在细胞培养的第8天以DNA梯度电泳、Hoechst33258染色和AO/EB双染色检测凋亡情况。主要观察指标:①有无DNA断裂后形成的梯度。②有无异常的细胞核。③凋亡细胞的比例。结果:①DNA梯度电泳检测细胞核间DNA裂解情况:SK-HEP-1细胞在极低频磁场处理8d后产生DNA片段条带,对照组中(无磁场处理)则未观察到此特征性DNA条带。②Hoechst33258荧光染色细胞凋亡分析:Hoechst33258染色被用来研究细胞中核的变化,在极低频磁场处理过的细胞中,存在许多包含有细胞核片段的凋亡小体,但在对照组中则几乎没有。同时,磁场处理的细胞观察到有细胞质皱缩现象,在某些细胞中,甚至细胞膜都不完整。③AO/EB双染细胞凋亡分析:极低频磁场处理后活细胞的比例(9.2%)与对照组(91.8%)相比极为低下,且伴有很高的细胞凋亡率(72.3%),对照组细胞凋亡率为4.2%,凋亡细胞中的大多数属于凋亡早期。磁场处理后坏死细胞的比例(18.5%)也比对照组(4%)要高。AO/EB双染结果显示AO/EB双染后细胞的不同形态,其中对照组的细胞呈亮绿色的完整圆球状,而磁场处理后的细胞则呈现出不规则的细胞形态且有凝集的细胞核。结论:50Hz,20mT极低频磁场能在体外诱导人肝癌细胞SK-HEP-1发生凋亡。更多还原

【Abstract】 BACKGROUND: Magnetic field can affect the growth and division of cancer cells both in vivo and in vitro, however, the effects on apoptosis of human liver cancer cells induced by magnetic field is still unclear. OBJECTIVE: To explore the inducing effect of extremely low frequency (ELF) magnetic field on apoptosis of human liver cancer cell SK-HEP-1. DESIGN: An open experiment with cells as the observational subjects. SETTING: Institute of Biotechnology, Shanghai Jiaotong University. MATERIALS: The experiment was carried out at the Institute of Biotechnology, Shanghai Jiaotong University from September 2004 to January 2005. The subject was human liver cancer cell line SK-HEP-1, purchased from cell bank of Chinese Academy of Science, Shanghai. METHODS: SK-HEP-1 cells were inoculated to T-flasks at the density of 2.0×107 cells L-1, and cultivated in the DMEM containing 0.1 volume fraction of heat-inactivated fetal bovine serum and 2 mmol/L L-glutamine. Exposure groups were exposed to 50 Hz, 20 mT magnetic field and the control groups were run concurrently under the same conditions with the exposed cultures but in a separate incubator which was free of magnetic field during 8-day culture process. The apoptosis of SK-HEP-1 cells were defined by DNA ladder assay, Hoechst 33258 staining and AO/EB staining respectively on day 8. MAIN OUTCOME MEASURES: ① DNA fragmentation pattern formation. ② The abnormal nucleus formation. ③ The percentage of apoptotic cells. RESULTS: ① Detection of internucleosomal DNA fragmentation by DNA ladder assay: After 8-day ELF magnetic field exposure, DNA fragmentation pattern was detected by DNA ladder assay, which was not observed in control groups (free of exposure). ② Fluorescence microscopy analysis of apoptosis by Hoechst 33258 staining: Hoechst 33258 staining was used to investigate the changes in the nucleus of cells, and many apoptotic bodies containing nuclear fragments were found in ELF magnetic field exposed cells, but just about none in untreated cells. At the same time, cytoplasmic shrinkage was observed in cells cultured under the exposure. And in some cells, even the cell membrane was unable to keep intact. ③ Fluorescence microscopy analysis of cell apoptosis by acridine orange/ethidium bromide (AO/EB) double staining: After ELF magnetic field exposure, the percentage of viable cells in exposure groups (9.2%) was rather low compared with the control groups (91.8%), and was accompanied with a high apoptotic rate (72.3%), while only 4.2% in control group. A large number of apoptotic cells were at the early stage of apoptosis. The rate of apoptotic cells (18.5%)after treated by magnetic field was higher than that of control group (4%). With the AO/EB double staining, control cells appeared to be round, intact and bright green while some of the exposed cells exhibited irregular cell morphology and condensed nucleus. CONCLUSION: Apoptosis of human liver cancer cell SK-HEP-1 could be induced by 50 Hz, 20 mT magnetic field in vitro.更多还原