论文部分内容阅读
[目的]探讨苯并[α]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、5mmol/L BaP处理24h,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,1mmol/L BaP组低于溶剂对照组(40.062±7.746),2、5mmol/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 (16HBE) cells exposed to benzo[α]pyrene (BaP),and provide scientific evidence for toxicological mechanism of BaP.[Methods] Well-grown 16HBE 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 lmmol/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 groups showed higher methylation levels than the solvent control group (P ＜ 0.05).The mRNA expression levels of CYP1A1 were 0.009 ± 0.001,0.009 ± 0.001,and 0.017 ± 0.003 in the BaP groups,respectively,which were significantly decreased compared to the solvent control group (1.067 ± 0.064) (P＜0.05).The mRNA expression levels of GSTP1 were 0.179 ± 0.074,0.167 ± 0.048,and 0.143 ± 0.029 in the BaP groups,respectively,significantly decreased compared to the solvent control group (0.829 ± 0.116) (P＜0.05).The mRNA expression levels of GSTM1 were 0.547 ± 0.181,0.518 ± 0.141,and 0.297 ± 0.044 in the BaP groups,respectively,significantly decreased compared to the solvent control group (0.975 ± 0.183) (P＜0.05).[Conclusion] The toxicological mechanism of BaP may be related to its enhanced cell proliferation,decreased DNA methylation in the promoter regions of CYP1A1 and GSTP1,and reduced expression of CYP1A1,GSTP1,GSTM1 mRNA in 16HBE.