三氯乙烯对人正常肝细胞亚细胞蛋白质组影响的研究  

Investigation of trichloroethylene-induced effects on subcellular proteomes in L-02 hepatic cells

作　　者：黄爱博 许华[1] 洪文旭[2,3] 庄志雄[2] 刘建军[2] 

机构地区：[1]暨南大学药学院,广州510632 [2]深圳市疾病预防控制中心现代毒理学重点实验室 [3]深圳市人口和计划生育科学研究所医学实验室

出　　处：《中华预防医学杂志》2015年第3期212-217,共6页Chinese Journal of Preventive Medicine

基　　金：基金项目：国家自然科学基金（21102094）;广东省医学科研基金（A2014659）;深圳市新兴产业发展专项（GJHS20120628151305456）

摘　　要：目的研究三氯乙烯（trichloroethylene）对人正常肝细胞系（L-02细胞）亚细胞蛋白质组的影响，探讨其潜在肝毒性作用机制。方法采用0和8．0mmol／L的三氯乙烯处理细胞后，分别提取三氯乙烯处理前后L-02细胞的细胞膜和细胞核总蛋白质，通过差异荧光双向凝胶电泳（2D—DIGE）筛选出差异点，运用基质辅助激光解析飞行时间串联质谱（MALDI—TOF—MS／MS）对差异蛋白点进行质谱鉴定。采用生物信息学方法对差异蛋白进行跨膜结构域（transmembranedomain）和基因本体（geneontology，GO）功能聚类的分析，用Westernblot分析验证不同浓度三氯乙烯处理下膜蛋白ATP合成酶B亚基（ATP5B）、核蛋白核不均一核糖核蛋白H2（hnRNPH2）和上游识别序列结合蛋白1（FUBP1）在L-02细胞中表达情况。结果三氯乙烯作用于L-02细胞24h后，鉴定出差异表达膜蛋白14个和差异表达核蛋白18个，在使用0、2．0、4．0和8．0mmol／L的三氯乙烯处理细胞后，ATPSB蛋白相对表达量分别为1．00±0．03、1．21±0．14、1．25±0．12、1．48±0．17（F=8．51，P=0．007）；hnRNPH2蛋白相对表达量分别为1．00±0．09、1．22±0．15、1．43±0．21、1．53±0．17（F=6．57，P=0．015）；FUBP1蛋白相对表达量分别为1．00±0．11、0．91±0．07、0．73±0．04、0．67±0．03（F=15．81，P=0．001）。生物信息分析显示，差异表达蛋白参与的生物学过程中，差异蛋白主要聚集在RNA加T（差异蛋白数为10个，P=2．46×10^-6），特别是在RNA剪接上（差异蛋白数9个，P=1．77×10^-7）。结论三氯乙烯处理可以引起亚细胞蛋白质组的改变，而这些与RNA剪接相关蛋白的异常表达为进一步研究三氯乙烯的肝毒性作用机制提供了新的线索。Objective To put the insight into the trichloroethylene （TCE）-induced effect on the differential expression of subcellular proteins in human normal liver cell line （ L-02 ） . Methods The membrane proteins and nuclear proteins of TCE-treated （8.0 mmol/L） group and controls were extracted by subcellular proteome extraction kit, respectively. The TCE-induced differentially expressions were analyzed by a two-dimensional fluorescence difference gel electrophoresis （2D-DIGE） and matrix-assisted laser desorption/ionization tandem time-of-flight spectrometry （MALDI-TOF-MS）. Bioinformatics analysis was used to reveal the biological processes and predict transmembrane domains of differential expressed proteins. The expression of ATP synthase subunit beta （ATP5 B） , heterogeneous nuclear ribonucleoprotein H2 （ hnRNP H2） and far up steam element-binding protein 1 （ FUBP1 ） were measured under TCE treatment by Western blot. Results After TCE treatment for 24 h in L-02 ceils, 14 membrane proteins and 18 nuclear proteins were identified as differential expression. After treated with TCE in concentrations of 0,2. 0,4. 0 and 8.0 mmol/L for 24 h ,the relative levels of ATP5B expression were 1. 00 ± 0. 03,1.21± 0. 14,1.25 ±0. 12and 1.48 ± 0. 17 （ F = 8.51, P = 0. 007 ）, the relative levels of hnRNP H2 expression were 1.00 ± 0. 09, 1.22 ± 0. 15,1.43 ±0. 21,1.53 ± 0. 17 （ F = 6. 57, P = 0. 015 ）, respectively; the relative levels of FUBP1 expression were 1.00 ±0.11,0.91 ±0.07,0.73±0.04 and 0.67 ±0.03 （F = 15.81,P =0.001）, respectively,which were consistent with the results in proteomics. The bioinformatics analysis showed that the most dominant biological process were involved in RNA processing （ 10 proteins, P = 2.46 × 10^-6 ） , especially in RNA splicing （9 proteins ,P = 1.77 ×10 ^-7 ）. Conclusion The exposure of TCE could alter the expression of membrane proteins and nuclear proteins in L-02 cells. These abnormal expressed proteins involved in RNA splicing would pro

关 键 词：三氯乙烯 核蛋白质类 膜蛋白质类 人正常肝细胞系 RNA剪接 

分 类 号：R114[医药卫生—卫生毒理学]