BaP诱导细胞恶性转化过程中DNA甲基化水平的变化

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。

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

OBJECTIVE: To investigate the role of DNA methylation as a possible mechanism of Benzoa]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.