基于GADD153启动子的环境致癌物快速筛选系统研究

The Eukaryotic Environmental Carcinogens Screening System Based on GADD153 Promoter

[目的]构建快速检测环境中痕量致癌物DNA损伤效应的重组人肝细胞株。[方法]构建含生长抑制与DNA损伤基因153(GADD153)启动子和萤光素酶报告基因的重组稳定转染人肝肿瘤HepG2细胞;发光检测不同致癌物对稳定转染细胞的DNA损伤效应;同时RT-PCR检测内源性GADD153mRNA的表达;彗星试验(Cometassay)检测DNA的损伤效应。[结果]稳定转染GADD153-LUC细胞株可检出多环芳烃、芳香胺、重金属、农药等已知环境致癌物以及实际水样中的含量。检测结果与彗星试验结果相比,大多数检测下限均低于彗星试验的检测限,其中对苯并芘、氯化镉和2-氨基芴的检测灵敏度分别高于彗星试验1000、300和100倍;对Aems试验不敏感的重金属、四氯化碳、氟化钠等重组细胞株具有较高的检测灵敏性。10μmol/L的氯化镉可诱导内源性GADD153mRNA和萤光素酶活性表达增加,相关分析表明两者具有良好的相关性(r2=0.9539)。[结论]重组细胞株GADD153-LUC细胞可快速检测环境中痕量污染物的DNA损伤效应。

[ Objective ] Constructing a hepatic cell line that can detect DNA damage induced by trace environmental cancinogens. [ Methods ] Stable transfected hepatic HepG2 cell lines containing DNA damage and repair inducible gene 153 （ GADD153 ）promoter and luciferase reporter gene were constructed. The inducible luciferase activity was detected by bioluminescence. At the same time, endogenic GADD153mRNA level was detected by RT-PCR. Comet assay was applied to determine the cellular DNA damage. [ Results ] The stable transfected GADD153-LUC cell lines can detect different carcinogens such as polycyclic aromatic hydrocarbon, arylamine, heavy metals and pesticide etc as well as the mix sample in environment（ water sample ）. The detection limit of GADD153-LUC was lower than that of Comet assay in most carcinogens. The detection limits of benzopyrene, cadmium chloride and 2-Amino fluorine using GADD153-LUC cells were 1000, 300 and 100 times more sensitive than that by using Comet assay. There was high sensitivity in the recombinant cells to detect heavy metals, carbon tetrachloride and sodium fluoride which showed negative results in Ames test. The expression of endogenic GADD153 mRNA and the luciferase activity were increased after treated by 10 μmol/L cadmium chloride. Correlation analysis showed there was positive correlation between the expression of endogenic GADD153 mRNA and the luciferase activity （ r^2=0.9539 ）. [ Conclusion ] Recombinant GADD 153-LUC cell can detect DNA damage effects induced by environmental cancinogens.