CAJ | 학술논문

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2 mmol/L L-谷氨酰胺的DMEM培养基中,50 Hz,20 mT磁场对人肝癌细胞SK-HEP-1连续作用8 d;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、Hoechst 33258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8 d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.②Hoechst 33258荧光染色细胞凋亡分析:Hoechst 33258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:50 Hz,20 mT极低频磁场能在体外诱导人肝癌细胞SK-HEP-1发生凋亡. 揹景:磁場能在體內或體外影響腫瘤細胞的生長與分裂,但目前尚無定論磁場能否在體外誘導人肝癌細胞的凋亡.目的:觀察極低頻磁場誘導人肝癌細胞SK-HEP-1凋亡的效果.設計:開放性實驗.單位:上海交通大學生物技術研究所.材料:實驗于2004-09/2005-01在上海交通大學生物技術研究所完成.人肝癌細胞SK-HEP-1購于上海中科院細胞庫.方法:SK-HEP-1細胞以2.0×104L-1接種,生長于含有體積分數為0.1的熱滅活新生牛血清和2 mmol/L L-穀氨酰胺的DMEM培養基中,50 Hz,20 mT磁場對人肝癌細胞SK-HEP-1連續作用8 d;對照組細胞則在另一箇培養箱中培養,除無磁場榦預外,與磁場處理組細胞的生長條件完全一緻.在細胞培養的第8天以DNA梯度電泳、Hoechst 33258染色和AO/EB雙染色檢測凋亡情況.主要觀察指標:①有無DNA斷裂後形成的梯度.②有無異常的細胞覈.③凋亡細胞的比例.結果:①DNA梯度電泳檢測細胞覈間DNA裂解情況:SK-HEP-1細胞在極低頻磁場處理8 d後產生DNA片段條帶,對照組中(無磁場處理)則未觀察到此特徵性DNA條帶.②Hoechst 33258熒光染色細胞凋亡分析:Hoechst 33258染色被用來研究細胞中覈的變化,在極低頻磁場處理過的細胞中,存在許多包含有細胞覈片段的凋亡小體,但在對照組中則幾乎沒有.同時,磁場處理的細胞觀察到有細胞質皺縮現象,在某些細胞中,甚至細胞膜都不完整.③AO/EB雙染細胞凋亡分析:極低頻磁場處理後活細胞的比例(9.2%)與對照組(91.8%)相比極為低下,且伴有很高的細胞凋亡率(72.3%),對照組細胞凋亡率為4.2%,凋亡細胞中的大多數屬于凋亡早期.磁場處理後壞死細胞的比例(18.5%)也比對照組(4%)要高.AO/EB雙染結果顯示AO/EB雙染後細胞的不同形態,其中對照組的細胞呈亮綠色的完整圓毬狀,而磁場處理後的細胞則呈現齣不規則的細胞形態且有凝集的細胞覈.結論:50 Hz,20 mT極低頻磁場能在體外誘導人肝癌細胞SK-HEP-1髮生凋亡.
배경:자장능재체내혹체외영향종류세포적생장여분렬,단목전상무정론자장능부재체외유도인간암세포적조망.목적:관찰겁저빈자장유도인간암세포SK-HEP-1조망적효과.설계:개방성실험.단위:상해교통대학생물기술연구소.재료:실험우2004-09/2005-01재상해교통대학생물기술연구소완성.인간암세포SK-HEP-1구우상해중과원세포고.방법:SK-HEP-1세포이2.0×104L-1접충,생장우함유체적분수위0.1적열멸활신생우혈청화2 mmol/L L-곡안선알적DMEM배양기중,50 Hz,20 mT자장대인간암세포SK-HEP-1련속작용8 d;대조조세포칙재령일개배양상중배양,제무자장간예외,여자장처리조세포적생장조건완전일치.재세포배양적제8천이DNA제도전영、Hoechst 33258염색화AO/EB쌍염색검측조망정황.주요관찰지표:①유무DNA단렬후형성적제도.②유무이상적세포핵.③조망세포적비례.결과:①DNA제도전영검측세포핵간DNA렬해정황:SK-HEP-1세포재겁저빈자장처리8 d후산생DNA편단조대,대조조중(무자장처리)칙미관찰도차특정성DNA조대.②Hoechst 33258형광염색세포조망분석:Hoechst 33258염색피용래연구세포중핵적변화,재겁저빈자장처리과적세포중,존재허다포함유세포핵편단적조망소체,단재대조조중칙궤호몰유.동시,자장처리적세포관찰도유세포질추축현상,재모사세포중,심지세포막도불완정.③AO/EB쌍염세포조망분석:겁저빈자장처리후활세포적비례(9.2%)여대조조(91.8%)상비겁위저하,차반유흔고적세포조망솔(72.3%),대조조세포조망솔위4.2%,조망세포중적대다수속우조망조기.자장처리후배사세포적비례(18.5%)야비대조조(4%)요고.AO/EB쌍염결과현시AO/EB쌍염후세포적불동형태,기중대조조적세포정량록색적완정원구상,이자장처리후적세포칙정현출불규칙적세포형태차유응집적세포핵.결론:50 Hz,20 mT겁저빈자장능재체외유도인간암세포SK-HEP-1발생조망.
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 fre quency (ELF) magnetic field on apoptosis of human liver cancer cell SK-HEP- 1.JESIGN: 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.