苯并[a]芘对人支气管上皮细胞CYP1A1、GSTP1和GSTM1 DNA甲基化水平和mRNA表达的影响  

Effects of benzo[a]pyrene on DNA methylation and mRNA expression of CYP1A1, GSTP1, and GSTM1 in human bronchial epithelial cells

作　　者：刘爱香 李新[2] 曹晶晶 李欢[1] 张志红[1] 梁瑞峰[1] 郝忠锁 张红梅[1] LIU Ai-xiang;LI Xin;CAO Jing-jing;LI Huan;ZHANG Zhi-hong;LIANG Rui-feng;HAO Zhong-suo;ZHANG Hong-mei(Department of Environmental Health, School of Public Health, Shanxi Medical University, Taiyuan, Shanxi 030001, China;Department of Radiological Health, Center for Disease Control and Prevention, Taiyuan Iron ＆ Steel Company, Taiyuan, Shanxi 030003, China)

机构地区：[1]山西医科大学公共卫生学院环境卫生教研室,山西太原030001 [2]太钢疾病预防控制中心放射卫生科,山西太原030003

出　　处：《环境与职业医学》2018年第4期330-335,共6页Journal of Environmental and Occupational Medicine

基　　金：太原钢铁集团卫生处资助项目(编号:2014);山西省高等学校科技创新项目和哲学社会科学研究项目(编号:2016155);山西省研究生联合培养基地人才培养项目(编号:2017JD24);山西省回国留学人员科研资助项目(编号:2017-059)

摘　　要：[目的]探讨苯并[a]芘(BaP)对人支气管上皮(16HBE)细胞中CYP1A1、GSTP1和GSTM1启动子区DNA甲基化水平和mRNA表达的影响,为BaP毒理学机制的研究提供科学基础。[方法]体外培养16HBE细胞,取对数生长期的同一批次细胞,随机分为4组,包括1个溶剂对照组和3个BaP处理组,分别用二甲基亚砜(DMSO)及1、2、5 mmol/L BaP处理24 h,于倒置显微镜下观察细胞的形态学改变。采用CCK-8试剂盒测定细胞增殖能力,甲基化特异性PCR法检测CYP1A1、GSTP1和GSTM1启动子区DNA甲基化水平,实时定量PCR技术检测CYP1A1、GSTP1和GSTM1 mRNA表达的改变。采用SPSS 22.0软件的单因素方差分析进行比较,LSD法进行多组间的两两比较。[结果]与溶剂对照组相比,1、2、5 mmol/L BaP处理组新生细胞数目明显增多,细胞增殖率增高,分别为(100.00±0.47)%、(125.22±0.72)%、(122.92±1.47)%、(128.44±5.97)%(P<0.05)。3个BaP组CYP1A1启动子区甲基化水平分别为8.451±1.234、8.532±0.728、11.064±0.423,低于溶剂对照组(13.917±1.094)(P<0.05)。3个BaP处理组GSTP1启动子区甲基化水平分别为4.276±0.839、3.691±0.748、2.121±0.336,也低于溶剂对照组(10.860±1.129)(P<0.05)。GSTM1启动子区甲基化水平分别为34.693±6.603、48.407±7.824、49.803±2.516;其中,1 mmol/L BaP组低于溶剂对照组(40.062±7.746),2、5 mmol/L BaP组高于溶剂对照组(均P<0.05)。3个BaP组CYP1A1 mRNA表达水平分别为0.009±0.001、0.009±0.001、0.017±0.003,低于溶剂对照组(1.067±0.064)(P<0.05);GSTP1 mRNA和GSTM1 mRNA的表达水平分别为(0.179±0.074、0.167±0.048、0.143±0.029)和(0.547±0.181、0.518±0.141、0.297±0.044),均低于溶剂对照组(0.829±0.116、0.975±0.183)(P<0.05)。[结论]BaP对16HBE的毒作用机制可能与其细胞增殖能力增强及CYP1A1和GSTP1启动子区DNA甲基化水平降低,CYP1A1、GSTP1、GSTM1 mRNA表达降低有关。[Objective] To investigate promoter DNA methylation and mRNA expression levels of CYP1A1, GSTP1, and GSTM1 in human bronchial epithelial（16 HBE） cells exposed to benzo[a]pyrene（BaP）, and provide scientific evidence for toxicological mechanism of BaP.[Methods] Well-grown 16 HBE cells in the exponential growth from the same batch in vitro were randomly divided into four groups treated with dimethyl sulfoxide（DMSO）（solvent control group） and 1, 2, or 5 mmol/L BaP（BaP groups）, respectively. Following BaP treatment for 24 h, we observed the morphological changes under an inverted microscope, determined cell proliferation using CCK-8 kit, detected promoter DNA methylation in CYP1A1, GSTP1, and GSTM1 using methylation-specific PCR method, and measured mRNA expression levels of CYP1A1, GSTP1, and GSTM1 using real-time quantitative PCR. Comparisons among groups were made using ANOVA in SPSS 22.0 software, and pairwise comparisons were analyzed by LSD.[Results] The numbers of newborn cells of the three BaP groups were increased compared to the solvent control group, and the cell viabilities were（125.22±0.72）% ,（122.92±1.47）% , and（128.44±5.97）% in the 1, 2, and 5 mmol/L BaP groups, respectively, which were significantly increased compared to the solvent control group [（100.00±0.47）% ]（P 〈 0.05）. The methylation levels of CYP1A1 promoter were 8.451±1.234, 8.532±0.728, and 11.064±0.423 in the three BaP groups, respectively, lower than that of the solvent control group（13.917±1.094）（P 〈 0.05）. The methylation levels of GSTP1 promoter were 4.276±0.839, 3.691±0.748, and 2.121±0.336 in the three BaP groups, respectively, also lower than that of the solvent control group（10.860±1.129）（P 〈 0.05）. The methylation levels of GSTM1 promoter were 34.693±6.603, 48.407±7.824, and 49.803±2.516 in the three BaP groups, respectively; the 1 mmol/L BaP group showed a lower methylation level than the solvent control group（40.062±7.746）, but the 2 and 5 mmol/L BaP

关 键 词：苯并[A]芘 人支气管上皮细胞 细胞增殖 DNA甲基化 MRNA表达 

分 类 号：R114[医药卫生—卫生毒理学]