中华预防医学杂志
中華預防醫學雜誌
중화예방의학잡지
CHINESE JOURNAL OF
2011年
5期
399-403
,共5页
马俊香%段化伟%黄传峰%杨海军%戴宇飞%牛勇%宾萍%刘庆%郑玉新
馬俊香%段化偉%黃傳峰%楊海軍%戴宇飛%牛勇%賓萍%劉慶%鄭玉新
마준향%단화위%황전봉%양해군%대우비%우용%빈평%류경%정옥신
焦炉逸散物%MGMT基因%甲基化%彗星实验%DNA修复
焦爐逸散物%MGMT基因%甲基化%彗星實驗%DNA脩複
초로일산물%MGMT기인%갑기화%혜성실험%DNA수복
Polycyclic compounds%DNA-modified methylases%DNA methylation%Comet assay%DNA repair
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.
目的 通過觀察細胞遺傳損傷指標和O6-甲基鳥嘌呤DNA甲基轉移酶(MGMT)甲基化改變,探討焦爐逸散物暴露誘導的損傷效應機製.方法 採用1 μmol/L苯併[a]芘[B(a)P]誘導人支氣管上皮細胞16HBE 48 h後,分彆用1‰二甲基亞砜(DMSO)和2.5、5.0、10.0、20.0 μg/ml濃度的焦爐逸散物有機提取物對16HBE細胞連續染毒5 d,構建細胞損傷模型;採用甲基化特異性PCR(MSP-PCR)檢測細胞MGMT甲基化改變,併用RT-PCR檢測細胞MGMT mRNA改變,免疫印跡法檢測MGMT蛋白改變;採用堿性單細胞凝膠電泳技術檢測細胞DNA損傷水平.結果 與DMSO組相比,MGMT基因在各處理組均有高甲基化改變,併隨劑量的增加,其mRNA和蛋白錶達水平都呈下降趨勢.DMSO組及2.5、5.0、10.0、20.0μg/ml各劑量組MGMT mRNA及其蛋白的灰度比值分彆為1.0、0.96、0.96、0.85、0.32和1.0、1.0、1.1、0.41、0.52.焦爐逸散物有機提取物處理後,細胞齣現不同程度的DNA損傷,DMSO組及2.5、5.0、10.0、20.0μg/ml各劑量組彗星Olive尾距分彆為(2.98±1.43)、(4.76±1.79)、(10.09±1.75)、(11.38±1.77)、(11.67±1.88),損傷呈明顯的劑量-效應關繫(F=41.22,P＜0.05);進一步分析髮現,細胞的遺傳損傷指數與MGMT蛋白及mRNA錶達水平呈負相關.結論 焦爐逸散物引起的DNA損傷與MGMT高甲基化所緻的MGMT基因錶達水平降低有關.
목적 통과관찰세포유전손상지표화O6-갑기조표령DNA갑기전이매(MGMT)갑기화개변,탐토초로일산물폭로유도적손상효응궤제.방법 채용1 μmol/L분병[a]비[B(a)P]유도인지기관상피세포16HBE 48 h후,분별용1‰이갑기아풍(DMSO)화2.5、5.0、10.0、20.0 μg/ml농도적초로일산물유궤제취물대16HBE세포련속염독5 d,구건세포손상모형;채용갑기화특이성PCR(MSP-PCR)검측세포MGMT갑기화개변,병용RT-PCR검측세포MGMT mRNA개변,면역인적법검측MGMT단백개변;채용감성단세포응효전영기술검측세포DNA손상수평.결과 여DMSO조상비,MGMT기인재각처리조균유고갑기화개변,병수제량적증가,기mRNA화단백표체수평도정하강추세.DMSO조급2.5、5.0、10.0、20.0μg/ml각제량조MGMT mRNA급기단백적회도비치분별위1.0、0.96、0.96、0.85、0.32화1.0、1.0、1.1、0.41、0.52.초로일산물유궤제취물처리후,세포출현불동정도적DNA손상,DMSO조급2.5、5.0、10.0、20.0μg/ml각제량조혜성Olive미거분별위(2.98±1.43)、(4.76±1.79)、(10.09±1.75)、(11.38±1.77)、(11.67±1.88),손상정명현적제량-효응관계(F=41.22,P＜0.05);진일보분석발현,세포적유전손상지수여MGMT단백급mRNA표체수평정부상관.결론 초로일산물인기적DNA손상여MGMT고갑기화소치적MGMT기인표체수평강저유관.
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.
