利用原代肝细胞模型检测细胞内磷酸化组蛋白H2AX表达筛选遗传毒物的方法验证  

Method validation of phosphorylated histone H2AX level detection using primary cultured hepatocytes in genotoxic agent screening

作　　者：孟涛[1] 苗盼盼[1] 纪玉青 牛勇[1] 宾萍[1] 戴宇飞[1] 郑玉新[1] 

机构地区：[1]中国疾病预防控制中心职业卫生与中毒控制所,化学污染与健康安全重点实验室,北京100050

出　　处：《中国药理学与毒理学杂志》2016年第2期135-143,共9页Chinese Journal of Pharmacology and Toxicology

基　　金：十二五国家科技支撑计划资助项目(2014-BAI12B02);中国疾病预防控制中心职业卫生与中毒控制所青年科技基金资助项目(2014YS02);中国疾病预防控制中心职业卫生与中毒控制所青年科技基金资助项目(2015YS01)~~

摘　　要：目的验证以原代小鼠肝细胞为受试细胞,以细胞内磷酸化组蛋白H2AX（γH2AX）表达改变为检测指标的体外检测化学物遗传毒性的实验方法。方法采用改良的两步胶原酶灌注法分离获取小鼠肝细胞,并运用三明治法体外培养肝细胞。选择4种已知的遗传毒物平阳霉素、苯并芘、苯乙烯和氧化苯乙烯及2种非遗传毒物硫唑嘌呤和环孢素A作为受试物,用受试物0~40μmol·L-1处理原代培养肝细胞0~24 h,CCK-8法检测细胞毒性,并用流式细胞术检测γH2AX的表达水平。结果利用所建立的方法筛选上述4种遗传毒物均获得阳性结果,2种非遗传毒物均呈阴性反应。直接遗传毒物平阳霉素和氧化苯乙烯作用3 h,间接遗传毒物苯并芘和苯乙烯作用6 h时,γH2AX表达量最高（P〈0.01）。4种遗传毒物处理组在最适时间点（直接遗传毒物：3 h;间接遗传毒物：6 h）及最适作用浓度（10μmol·L-1）诱发细胞γH2AX的表达水平依次为25.7,18.4,12.4和14.2（平均荧光强度）,分别为DMSO溶剂对照组的18,12,8和10倍（P〈0.01）,且γH2AX的表达水平与遗传毒物浓度呈显著正相关（P〈0.01）。非遗传毒物硫唑嘌呤和环孢素A处理组与DMSO溶剂和空白对照组相比,γH2AX的水平均无明显变化。结论利用原代肝细胞模型检测γH2AX表达水平可在无S9的情况下有效区分遗传毒物与非遗传毒物和直接遗传毒性与间接遗传毒性。γH2AX表达水平可作为鉴别遗传毒物与非遗传毒物的有效参考指标。OBJECTIVE To establish an in vitro test method and to evaluate the genotoxicity of chemicals using primary cultured mouse hepatocytes and the changes in phosphorylated histone H2AX （γH2AX） expression levels to provide a more reliable marker of the identification of genotoxicity.METHODS Hepatocytes were isolated from BALB/c mice by an improved two-step collagenase diges-tion method and then cultured in sandwich configuration.The primary cultured hepatocytes were treat-ed with various concentrations of four known genotoxic agents bleomycin （BLM）,benzo （a） pyrene[B （a） p],styrene and styrene-7,8-oxide （SO） within the range of 40 μmol·L-1 and two non-genotoxic agents azathioprine （Aza） and ciclosporin A （CsA） at different time points within 24 h.The cytotoxicity induced by these toxicants was assessed by CCK-8 assay.Then,the changes in γH2AX expression levels in treated cells were determined by flow cytometry.RESULTS The four genotoxic agents could be detected and two non-genotoxic agents could not be detected by this method.The γH2AX expression level was the highest when hepatocytes were exposed to BLM and SO for 3 h,or B （a） p and styrene for 6 h （P〈0.01）.The production of γH2AX was 25.67,18.36,12.43 and 14.25 for the four types of genotoxic agents,respectively,and was approximately 19,13,9 and 11 times that of the vehicle control group （P〈0.01） at the optimum time point and concentration.There was a significant positive corre-lation between the indicated concentrations of genotoxic chemicals and γH2AX expression levels （P〈 0.01）.In addition,the production of γH2AX indicated no marked increase in two non-genotoxic agents such as Aza and CsA in comparison with the control group.CONCLUSION This test method can effec-tively distinguish genotoxic agents from non-genotoxic agents,and direct genotoxic agents from indirect genotoxic agents in the absence of S9.γH2AX might be a reliable marker for the identification of the potential genotoxicity of chemicals.

关 键 词：遗传毒物 肝细胞 原代培养 组蛋白类 流式细胞术 

分 类 号：R114[医药卫生—卫生毒理学]