癌变·畸变·突变 ›› 2017, Vol. 29 ›› Issue (1): 42-45,50.doi: 10.3969/j.issn.1004-616x.2017.01.008

• 论著 • 上一篇    下一篇

BaP诱导细胞恶性转化过程中DNA甲基化水平的变化

刘海龙1,2, 高玮1, 古盼1, 邓燕霞1, 黄新凤1, 吴德生1, 刘建军1, 黄海燕1   

  1. 1. 深圳市疾病预防控制中心, 现代毒理学重点实验室, 广东 深圳 518055;
    2. 南昌大学公共卫生学院, 江西 南昌 330031
  • 收稿日期:2016-07-08 修回日期:2016-11-02 出版日期:2017-01-31 发布日期:2017-01-31
  • 通讯作者: 黄海燕,E-mail:hhy424@126.com E-mail:hhy424@126.com
  • 作者简介:刘海龙,E-mail:15118840643@163.com
  • 基金资助:

    国家自然科学基金(81370080,81001261);深圳市未来产业专项资金(ZDSYS20140509101335476)

Analysis of DNA methylation during BaP-induced malignant transformation in vitro

LIU Hailong1,2, GAO Wei1, GU Pan1, DENG Yanxia1, HUANG Xinfeng1, WU Desheng1, LIU Jianjun1, HUANG Haiyan1   

  1. 1. Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, Guangdong;
    2. School of Public Health, Nanchang University, Nanchang 330031, Jiangxi, China
  • Received:2016-07-08 Revised:2016-11-02 Online:2017-01-31 Published:2017-01-31

摘要:

目的:通过分析苯并芘(BaP)诱导细胞恶性转化过程中DNA甲基化水平的变化,探讨BaP致癌的作用机制。方法:以正常人支气管上皮细胞(16HBE)为研究对象,使用梯度浓度BaP(0、10、20和40 μmol/L)染毒处理,构建不同染毒周期(1周、9周和15周)的细胞株,使用5-甲基胞嘧啶(5-mC)细胞免疫荧光检测各组细胞基因组DNA整体甲基化水平的变化,并进一步利用Western blotting和实时荧光定量-PCR技术分析不同染毒周期细胞甲基化蛋白酶(DNMT1、DNMT3a、DNMT3b、MBD2)表达的变化。结果:BaP染毒后,16HBE细胞的5-mC荧光强度表达下降,且随着染毒剂量的增加和染毒时间的延长,这种下降趋势更明显,其中40 μmol/L BaP染毒处理细胞15周时,肉眼已难以观察到可见荧光。与对照组比较,BaP染毒可下调细胞DNMT1蛋白及其mRNA的表达,并呈现明显的剂量和时间反应关系(P均 < 0.05),但DNMT3a、DNMT3b、MBD2蛋白的表达变化不明显(P均 > 0.05)。结论:BaP可诱导16HBE细胞基因组DNA整体甲基化水平下调,DNMT1在其中可能发挥重要作用。

关键词: 苯并芘, DNA甲基化, 恶性转化

Abstract:

OBJECTIVE: To investigate the role of DNA methylation as a possible mechanism of Benzo[a]pyrene (BaP) induction of malignant transformation in vitro. METHODS: Human bronchial epithelial cells (16HBE cell line) were treated with different concentrations BaP (0,10,20 and 40 μmol/L) for 1,9 and 15 weeks. Then,5-methylcytosine immunofluorescent assay was used to detect genomic DNA methylation level,Western blotting and RT-PCR were used to detect protein and mRNA levels of DNMT1,DNMT3a,DNMT3b and MBD2. RESULTS: BaP treatment decreased expression of 5-mC (methylation) in 16HBE cells in a dose- and time-dependent manner. The protein and mRNA levels of DNMT1 showed in a significant dose- and time-dependent reduction (P < 0.05),but there were no signi fi-cant changes in protein levels of DNMT3a,DNMT3b and MBD2(all P > 0.05). CONCLUSION: BaP treatment decreased the global DNA methylation levels in 16HBE cells and reduction of DNMT1 expression could play an important role.

Key words: benzo[a]pyrene, DNA methylation, malignant transformation

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