甲基丙烯酸环氧丙酯致人支气管上皮细胞恶性转化过程中P16基因的甲基化状态

Methylation status of P16 gene during malignant transformation of human bronchial epithelial cells induced by glycidyl methacrylate

目的分析甲基丙烯酸环氧丙酯（glycidylmethacrylate，GMA）致人支气管上皮16HBE细胞恶性转化过程中不同时点P16基因甲基化状态的差异，探讨GMA诱导16HBE细胞发生恶性转化相关DNA甲基化机制。方法收获GMA转化早期（染毒结束）、转化前期（第10代）、转化后期（第30代）的16HBE细胞，采用甲基化特异性聚合酶链反应（Methylation—specificPCR，MSP）检测该基因组P16基因启动子区的甲基化状态，并与未转化的16HBE细胞及同期培养的DMSO溶剂对照细胞进行比较。结果转化早期及转化前期，正常对照组及DMSO溶剂对照细胞中该基因启动子区均表现为非甲基化，而GMA组细胞中表现为不同程度的甲基化，阳性对照表现为甲基化；转化后期，正常对照组细胞中该基因启动子区表现为非甲基化，而DMSO溶剂对照组及GMA组细胞中均表现为部分甲基化，阳性对照表现为甲基化。结论在GMA诱导16HBE细胞恶性转化早期及前期P16基因的甲基化状态具有特异性，故可将其作为GMA诱导16HBE细胞发生恶性转化的一个早期敏感指标。

Objective To analyze the methylation status of P16 gene at the different stages of malignant transformation of human bronchial epithelial ceils （16HBE） induced by glycidyl methacrylate （GMA） and to explore the DNA methylation mechanisms. Methods The cells exposed to GMA were harvested at the end of exposure （early stage）, the 10th generation （protophase） and the 30th generation （anaphase）, respectively. The methylation status of P16 promotor was detected by Methylation-specific PCR （MSP）. The transformed 16HBE cells were compared with the normal 16HBE cells and the cells exposed to DMSO for methylation status. Results At the early stage and protophase stage, the non-methylation status in P16 gene promotor of the normal 16HBE cells and the cells exposed to DMSO appeared, the methylation status in P16 gene promotor of the 16HBE cells exposed to GMA was detected to some extension. At the anaphase stage, the methylation status in P16 gene promotor of the 16HBE cells exposed to GMA or DMSO was detected to some extension. Conclusion Methylation status of P16 gene promoter was specific at the early stage and protophase stage of malignant transforming in 16HBE cells induced by GMA, which can serve as an early sensitive biological indicator for malignant transforming in 16HBE cells induced by GMA.