基于微孔板的人HepG2细胞体外彗星试验方法的研究  

Research on the comet assay in vitro by plating HepG2 cells in 96-well plates

作　　者：王征[1] 张天宝[1] 朱勇飞[1] 朱江波[1] 谢宗华[1] 

机构地区：[1]第二军医大学卫生毒理学教研室,上海200433

出　　处：《卫生研究》2007年第1期20-23,共4页Journal of Hygiene Research

基　　金：国家"973"资助项目(No.2002CB512901)

摘　　要：目的运用微孔板体外培养技术，建立人HepG2细胞的体外彗星试验方法，为进一步建立体外高通量筛选方法提供依据。方法选用人HepG2细胞，运用96孔板体外细胞培养技术．建立微孔板的彗星试验方法，结合中性红检测细胞毒性的方法，选择20种化合物对所建方法进行验证。结果阳性和阴性时照物用所建方法与体内试验所获结果一致，验证结果显示，8种遗传毒物在体外彗星试验中均获得阳性结果，灵敏度为100％，且当细胞的相对活力〉50％时，拖尾率及尾长均与剂量呈正相关。12种非遗传毒物中，11种物质呈阴性反应，1种物质呈阳性反应，特异性为91％。结论运用微孔板体外细胞培养技术，可在1块96孔板上同时检测4—5种受试物，并在无S9的情况下能检测出直接与间接诱变剂，并可界定出引起遗传毒性的作用剂量，提高了筛选的速度，减少了受试物等的用量，具有发展成为体外高通量筛选方法的良好条件。Objectlve To provide the proof for the high throughput screening method in vitro by establishing the comet assay in vitro using HepG2 cells plated in 96-well plates. Methods Plating HepG2 cells in 96-well plates, and then detecting the cytotoxicity using the neutral red method before comet assay. Results 8 genotoxicial agents could be detected using the method and the sensitivity was 100%, the ratio of the comet cells and the length of the tail of the comet were increased with the increasing dose when the cytotoxicity less than 50%. 11 non-genotoxicial agents could be detected within 12 non-genotoxicial agents and the specificity was 91%. Conclusion （1）This method could detect the direct genotoxic agents and the indirect genotoxic agents at the absent of the $9. （2）4-5 agents could be detected in the same 96- well plate using the method. （3）Using the method, we could obtain an dose at which the agent could cause genotoxicity. （4）By plating cells in plates in vitro, increased the speed of the detection and reduced the consumption of the agents. （5） The method could be developed into the high throughput screening method in vitro.

关 键 词：HEPG2细胞 体外彗星 试验 

分 类 号：Q355[生物学—遗传学] Q-331