三氯乙烯对人正常肝细胞组蛋白质甲基化修饰影响的研究  

An investigation of trichloroethylene-induced effects on histone methylation in L-02 hepatic cells

作　　者：邓荣霞[1,2] 任晓虎[2] 阮嘉雯 郑剑[2] 钟佳成 卢维雪 邹晓青[1] 刘建军[2] Deng Rongxia Ren Xiaohu Ruan Jiawen Zheng Jian Zhong Jiacheng Lu Weixue Zou Xiaoqing Liu Jianjun(The Department of Chemistry, Xiangtan University, Xiangtan 411105, China)

机构地区：[1]湘潭大学化学学院,411105 [2]深圳市疾病预防控制中心现代毒理学重点实验室

出　　处：《中华预防医学杂志》2017年第4期347-352,共6页Chinese Journal of Preventive Medicine

基　　金：国家自然科学基金（81273126）;广东省自然科学基金（2016A030310033）;深圳市基础研究计划（JCYJ20150402102135491）

摘　　要：目的 筛选三氯乙烯（TCE）诱导的人正常肝细胞（L-02细胞）组蛋白异常甲基化修饰,并探讨其在三氯乙烯致肝细胞毒性中的可能作用.方法 以0、8.0 mmol/L的TCE处理L-02细胞24 h,采用酸法提取组蛋白,通过液相色谱-电喷雾串联质谱（LC-ESI-MS/MS）鉴定和定量分析组蛋白差异甲基化修饰水平,以Western blot验证组蛋白H3K79二甲基化（H3K79 me2）及H3K79三甲基化（H3K79 me3）修饰水平,检测相对表达量.使用单细胞凝胶电泳检测细胞的DNA损伤水平.采用Western blot检测p53与H2AX的磷酸化修饰（？H2AX）的相对表达量.结果 TCE处理L-02细胞24 h后,质谱鉴定出28个肽段的36个表达有差异的组蛋白甲基化位点;L-02细胞经0、8.0 mmol/L TCE处理后,组蛋白H3K79 me3的相对表达量分别为1.00±0.06、0.70±0.09（t=15.01,P=0.015）;组蛋白H3K79 me2的相对表达量分别为1.00±0.05、0.74±0.07（t=16.69,P=0.018）;DNA损伤的Olive尾距值分别为1.46±0.28、3.12±0.68（t=15.22,P=0.018）;蛋白p53的相对表达量分别为1.00±0.04、1.24±0.04（t=18.71,P=0.012）;？H2AX的相对表达量分别为1.00±0.03、1.56±0.11（t=8.32,P=0.045）.结论 TCE引起L-02细胞组蛋白中H3K79 me2与H3K79 me3修饰水平发生变化,并且诱导DNA损伤,提示TCE可能通过DNA损伤导致H3K79 me2和H3K79 me3甲基化修饰的变化.Objective To further explore TCE-induced hepatotoxicity and its mechanisms by identification of trichloroethylene （TCE） induced abnormal histone methylation in human liver cells. Methods L-02 cells were treated with 0 and 8 mmol/L TCE for 24 h. Histones were extracted by acid. Liquid chromatography electrospray ionization tandem mass spectrometry （ESI-LC-MS/MS） were used to identify and quantify TCE related histone methylations. TCE induced abnormal methylation of H3K79 me2 and H3K79 me3 were validated by Western blot analysis. The further analysis of the function of histone abnormal methylation modifications were done by single cell gel electrophoresis （SCGE） and Western blot analysis of p53 and？H2AX. Results After treatment with TCE for 24 h in L-02 cells, the 36 TCE related histone methylation sites in 28 peptide segments were identified by MS. After treatment with TCE in concentrations of 0 and 8.0 mmol/L in L-02 cells for 24 h, the relative expression level of histone H3K79 me3 were 1.00±0.06, 0.70±0.09 （t=15.01, P=0.015）;the relative expression level of histone H3K79 me2 were 1.00±0.05, 0.74±0.07 （t=16.69, P=0.018）;the Olive Tail Moment about DNA damage were 1.46±0.28, 3.12± 0.68 （t=15.22, P=0.018）; the relative expression levels of p53 were 1.00 ± 0.04, 1.24 ± 0.04 （t=18.71, P=0.012）; and the relative expression levels of ？H2AX were 1.00 ± 0.03, 1.56 ± 0.11 （t=8.32, P=0045）. Conclusion TCE can induce changes in the relative expression level of H3K79 me2 and H3K79 me3 in L-02 cell, and induce DNA damage, suggesting that TCE may induce changes in the relative expression level of H3K79 me2 and H3K79 me3 by DNA damage.

关 键 词：三氯乙烯 组蛋白类 正常人肝细胞 DNA损伤修复 

分 类 号：R114[医药卫生—卫生毒理学]