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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。
BACKGROUND: Magnetic fields can affect the growth and division of tumor cells in vivo or in vitro. However, it is still not known whether magnetic fields induce the apoptosis of human hepatoma cells in vitro. Objective: To observe the effect of ultra-low frequency magnetic field on the apoptosis of human hepatocellular carcinoma cell line SK-HEP-1. Design: Open experiment. Unit: Shanghai Jiao Tong University Institute of Biotechnology. MATERIALS: Experiments were performed at Institute of Biotechnology, Shanghai Jiao Tong University from September 2004 to January 2005. Human hepatoma SK-HEP-1 cells were purchased from Shanghai Chinese Academy of Sciences. Methods: SK-HEP-1 cells were inoculated with 2.0 × 104 L-1 and grown in DMEM containing heat-inactivated neonatal calf serum and 2 mmol / L LL-glutamine at a volume fraction of 0.1. The cells of SK-HEP-1 cells were treated with SK-HEP-1 for 8 days. The cells in the control group were cultured in another incubator. Except for no magnetic field intervention, the growth conditions of cells in the field-treated group were completely consistent. On day 8 of cell culture, apoptosis was detected by DNA gradient electrophoresis, Hoechst 33258 staining and AO / EB double staining. MAIN OUTCOME MEASURES: ① Gradients formed after DNA breakage. ② with or without abnormal nuclei. ③ the proportion of apoptotic cells. Results: DNA fragmentation was detected by DNA gradient electrophoresis. SK-HEP-1 cells produced DNA fragments after 8 days of ultra-low frequency magnetic field treatment. In the control group (no magnetic field treatment), no DNA bands were observed. ②Hoechst33258 fluorescence staining of apoptosis analysis: Hoechst33258 staining was used to study changes in the nucleus of cells, in ultra-low frequency magnetic field treated cells, there are many apoptotic bodies containing nuclear fragments, but in the control group is almost no. At the same time, cells treated with magnetic field were observed cytoplasmic shrinkage phenomenon, and in some cells, even the cell membrane is not complete. (3) Apoptosis analysis of AO / EB double stained cells: The proportion of viable cells after ultra-low frequency magnetic field treatment (9.2%) was extremely low compared with that of control group (91.8%) and was associated with a high apoptosis rate of 72.3% The rate of apoptosis in the control group was 4.2%, and most of the apoptotic cells belonged to the early stage of apoptosis. The proportion of necrotic cells after magnetic field treatment (18.5%) was also higher than that of the control group (4%). AO / EB double staining showed different morphological changes of cells after AO / EB double staining, in which the cells in the control group showed a bright green globular shape, while the magnetic field treated cells showed irregular cell morphology with aggregated nuclei . Conclusion: 50 Hz, 20 mT ELF magnetic field can induce apoptosis of human hepatocellular carcinoma cell line SK-HEP-1 in vitro.