摘要
目的 通过观察细胞遗传损伤指标和O6-甲基鸟嘌呤DNA甲基转移酶（MGMT）甲基化改变,探讨焦炉逸散物暴露诱导的损伤效应机制.方法 采用1 μmol/L苯并[a]芘[B（a）P]诱导人支气管上皮细胞16HBE 48 h后,分别用1‰二甲基亚砜（DMSO）和2.5、5.0、10.0、20.0 μg/ml浓度的焦炉逸散物有机提取物对16HBE细胞连续染毒5 d,构建细胞损伤模型;采用甲基化特异性PCR（MSP-PCR）检测细胞MGMT甲基化改变,并用RT-PCR检测细胞MGMT mRNA改变,免疫印迹法检测MGMT蛋白改变;采用碱性单细胞凝胶电泳技术检测细胞DNA损伤水平.结果 与DMSO组相比,MGMT基因在各处理组均有高甲基化改变,并随剂量的增加,其mRNA和蛋白表达水平都呈下降趋势.DMSO组及2.5、5.0、10.0、20.0μg/ml各剂量组MGMT mRNA及其蛋白的灰度比值分别为1.0、0.96、0.96、0.85、0.32和1.0、1.0、1.1、0.41、0.52.焦炉逸散物有机提取物处理后,细胞出现不同程度的DNA损伤,DMSO组及2.5、5.0、10.0、20.0μg/ml各剂量组彗星Olive尾距分别为（2.98±1.43）、（4.76±1.79）、（10.09±1.75）、（11.38±1.77）、（11.67±1.88）,损伤呈明显的剂量-效应关系（F=41.22,P＜0.05）;进一步分析发现,细胞的遗传损伤指数与MGMT蛋白及mRNA表达水平呈负相关.结论 焦炉逸散物引起的DNA损伤与MGMT高甲基化所致的MGMT基因表达水平降低有关.
Objective To elucidate the mechanism of carcinogenesis induced by coke oven emissions by investigating the cell genetic damage index and the methylation of O6-methylguanine-DNA methyltransferase （MGMT）. Methods The human bronchial epithelial cell 16HBE was treated by 1 μmol/L B（a） P for 48 h,and then was exposed continuously to either 1% dimethyl sulfoxide （DMSO） or organic extracts of coke oven emission （ OE-COE ） for five days at the concentrations of 0,2. 5 ,5. 0,10. 0 and 20. 0 μg/ml. The methylation-specific PCR （ MSP-PCR）, RT-PCR and immunoblotting were applied to detect the methylation status, changes of mRNA and protein of MGMT, respectively. Single cell gel electrophoresis was used to detect DNA damage induced by OE-COE. Results Compared with the control group （ DMSO）,there was a significant hypermethylation in all study groups, along with the suppression of mRNA and protein in a dose-dependent manner,and the gradation ratio of them was 1. 0,0. 96,0. 96,0. 85,0. 32 and 1.0,1.0,1. 1 ,0. 41,0. 52 ,separately. There was a significant DNA damage with a dose-effect relationship in all study groups （ F = 41.22, P 〈 0. 05 ）, and the comet Olive tail moment was （ 2. 98 ± 1. 43 ）, （4. 76 ± 1.79 ）,（ 10. 09 ± 1.75）, （ 11.38 ± 1.77）, （ 1 1. 67 ± 1. 88）. The further study found that the index of DNA damage was negatively correlated to the expression of MGMT mRNA and its protein. Conclusion The DNA damage induced by COE might be associated with the suppression of MGMT caused by its hypermethylation.
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
2011年第5期399-403,共5页
Chinese Journal of Preventive Medicine
基金
基金项目：国家杰出青年科学基金（30625031）
国家自然科学基金（30700659）
卫生行业科研专项基金（200902006）
关键词
焦炉逸散物
MGMT基因
甲基化
彗星实验
DNA修复
Polycyclic compounds
DNA-modified methylases
DNA methylation
Comet assay
DNA repair
