基于双荧光素酶报告基因的DNA损伤与修复检测系统的建立与应用  

Development and application of a screening system for DNA damage and repair based on dual luciferase assay in human HepG2 cells

作　　者：张荣[1] 牛玉杰[2] 樊龙刚[1] 石磊[1] 王茜[1] 王秀荣[3] 

机构地区：[1]河北医科大学公共卫生学院卫生毒理学教研室,河北石家庄050017 [2]河北医科大学公共卫生学院劳动卫生与环境卫生学教研室,河北石家庄050017 [3]河北医科大学基础医学院免疫教研室,河北石家庄050017

出　　处：《癌变．畸变．突变》2013年第6期461-464,469,共5页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金(81072269;81102151);河北省自然科学基金(C2011206048)

摘　　要：目的:建立一种能够同时对环境致癌物所致HepG2细胞DNA损伤和细胞对损伤DNA的修复能力进行快速检测的系统。方法:5μmol/L氯化镉体外损伤质粒pTK-RL,质粒pgadd153-luc和体外受损的质粒pTK-RL共转染到HepG2细胞中,以16种已知致癌物或3种无遗传毒性的非致癌物刺激细胞,利用双荧光素酶检测方法同时检测DNA损伤和细胞的修复能力;双荧光素酶检测系统检测居民区、垃圾焚烧厂和农田3个不同污染区土壤中非挥发性有机提取物对DNA的损伤作用和细胞对该损伤的修复能力;彗星实验检测DNA的损伤。结果:双荧光素酶报告基因检测系统均可检出已知环境致癌物对DNA损伤与修复能力的影响,非致癌物均未检测到DNA损伤作用和对细胞DNA修复能力的影响;3个不同污染区土壤中非挥发性有机提取物均可诱导DNA损伤并抑制细胞对DNA损伤的修复能力,强度为居民区>垃圾焚烧厂>农田土壤。结论:在本研究条件下建立了可同时检测环境致癌物DNA损伤与细胞修复能力的双荧光素酶报告基因检测系统并可用于环境污染区检测。OBJECTIVE： Development of a screening system for DNA damage and repair based on dual luciferase assay in human HepG2 cells. METHODS：5μmol/L CdCl2 was used to treat the plasmid pTK-RL in vitro. Plasmid pgadd153-luc and damaged pTK-RL were co-transfected into HepG2 cells and then the reconstituted HepG2 cells were treated with sixteen DNA-damaging agents and three non-genotoxic agents. The DNA damage and DNA repair abilities of HepG2 cells were measured by dual luciferase assay. Three organic extracts from different sites of soil （residential area,garbage dump and farmland） were evaluated by dual luciferase assay. DNA damage was detected by Comet assay. RESULTS：The dual luciferase assay could identify genotoxic and non-genotoxic agents. The three different soil samples had various levels of inducing DNA damage and decreasing DNA repair capacities in HepG2 cells following the order：residential area〉garbage dump〉farmland. CONCLUSION：In this study,the dual luciferase assay was developed to measure DNA damage and repair.

关 键 词：DNA损伤 DNA修复 宿主细胞恢复活性试验 双荧光素酶报告基因 致癌物质 

分 类 号：R394.3[医药卫生—医学遗传学]