细胞内活性氧及其DNA损伤产物在启动细胞癌变中的作用  

Initiated Cancerazation Effect of Reactive Oxygen Species and Its Produ cts of Oxidative DNA Damage in B EP２D Cells

作　　者：朱茂祥[1] 杨陟华[1] 龚诒芬[1] 陆颖[1] 曹珍山[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850

出　　处：《癌症》2001年第10期1024-1028,共5页Chinese Journal of Cancer

基　　金：国家自然科学基金资助项目(批准号:39700030)

摘　　要：目的:研究辐射(60Coγ射线照射)和化学致癌物4-甲基亚硝胺-1-(3-吡啶基)-1-丁酮4methylnitrosamino13pyridyl1butanone,NNK诱发细胞(BEP2D)产生的活性氧(reactiveoxygenspecies,ROS)对DNA氧化损伤及其在启动细胞癌变中的作用。方法:细胞内H2O2和O2分别用2′7′-二氯荧光黄双乙酸盐DCFHDA和氢化乙锭HE标记,用流式细胞法测定荧光产物2′7′-二氯荧光黄DCF和溴乙锭EB荧光强度;8-羟基脱氧鸟嘌呤(8hydroxydeoxygunosine,OH8dG)含量用高压液相色谱结合电化学方法HPLCECD测定;用脂质体包埋法将目标DNA(来自NNK处理和60Coγ射线照射的BEP2D细胞)转染到受体细胞(C3H10T1/2)中,并用G418筛选阳性克隆细胞;用半固体琼脂试验检测细胞转化能力;用裸鼠成瘤试验鉴定细胞恶性转化。结果:NNK和60Coγ射线可诱发BEP2D细胞内ROS水平及OH8dG含量显著增高,并显示出良好的剂量效应关系;NNK处理和60Coγ射线照射的BEP2D细胞DNA分别转染到C3H10T1/2受体细胞后,细胞出现转化特性,裸鼠成瘤试验为阳性。结论:辐射和化学致癌物诱发细胞产生的ROS增高及其对DNA的氧化损伤可能启动了细胞癌变。Objective：To investigate oxidative DNA damage through reactive o xygen species（ROS）produced by ６０ Co gamma ray irradiation or ４－（methylnitrosamino）－１－（３－pyridyl） －１－butanone（NNK）treatment in HPV－１６immortalized hu man bronchial epithel ial cell（BEP２D），and their effect in initiating cancerazation．Methods ：Ethi dium bromide and ２'，７'－dichlorofluorescein，fluorescent products of the mem brane－permeable dyes hydroethine and ２' ，７' －dichloroflurescin diacetate，w ere used to monitor the i ntracellular production of hydrogen peroxides（H ２ O ２ ）and superoxide anions（O ２ ）respectively，by flow cytometry．８－hydroxydeoxygunosine（OH８dG）was e xamined with HPLC－ECD from extr acted DNA．DNA from BEP２D irradiated by ６０ Co gamma ray or treated with NNK were t ransferred into receptor C３H１０T１ ／２cells together with coli－neo plasmid thro ugh liposome，and positive clone of t ransfection was selected with Geneticin（G４１８）．Cell transformation fre quency was a ssayed by counting clone formation i n semisolid agar，and carcinog enesis was evaluated by tumor formation in nude mice．Results：The ROS products and DNA adduct OH８dG in ６０ Co gamma ray irradiated or NNK－treated BEP２D cells increased remarkably，a nd showed better dose－response correlation．After ６０ Co gamma rays irradiated or NNK－treate d DNA transfection，the receptor C ３H１０T１／２cells displayed the characte ristics of transformation，and tumor formatio n was observed in nude mice．Conclusion：Intracellular production of RO S and oxidative DNA damage induced by radi ation and ／or chemical carcinogen ma y initiate ce ll cancerazation．

关 键 词：化学致癌物 辐射致癌 DNA损伤 启动细胞 细胞内活性氧 

分 类 号：R730.2[医药卫生—肿瘤]