氯化镉诱发16HBE细胞恶性转化不同阶段P16基因甲基化研究

目的对氯化镉（CdCl2）诱发16HBE细胞系恶性转化不同阶段P16基因启动子区甲基化状况进行研究，探讨镉的表遗传致癌机制。方法从各组CdCl2恶性转化不同阶段及接种裸鼠成瘤的16HBE细胞中提取全基因组DNA，采取甲基化特异性PCR法（Methylation—specific PCR，MSP）检测该基因组P16基因启动子区的甲基化状况，与非转化的16HBE对照细胞进行比较，并用去甲基化因子5-Azac（5-Aza-2＇deoxycytidine）处理有异常甲基化的细胞。结果CdCl2恶性转化及接种裸鼠成瘤的16HBE细胞P16抑癌基因启动子区CpG岛存在异常高甲基化现象，且随着细胞恶性程度的增加，甲基化现象有明显升高的趋势，同时发现有异常高甲基化的细胞经去甲基化处理后甲基化现象消失。结论P16基因启动子区的高甲基化导致抑癌基因表达关闭，无法正常发挥细胞增殖周期对细胞分裂和生长的负调控，造成细胞周期失控而导致无限制地细胞增殖，这可能是氯化镉诱导16HBE细胞恶性转化及接种裸鼠成瘤的一种表遗传致癌作用。研究结果可部分解释镉化合物的细胞转化作用及其可能的表遗传致癌机制，以及进一步对甲基化的表型逆转和药物治疗研究提供了重要依据。

Methylation in malignant transformation and tumorigenicity in inoculated athymic mouse 16HBE cells induced by cadmium chloride

Objectlve To investigate the methylation status of genome P16 gene promotor induced by cadmium chloride in athymic mouse at various malignant transforming stages and explore the sur - carcinogenic mechanisms of cadmium. Methods Genomic DNA was extracted from different stages of malignant transformation and tumorigenicity in inoculated athymic mouse 16HBE cells induced by cadmium chloride and methylating status of genome P16 gene promoter was observed with methylation - specific PCR and then compared with the controls. The abnormal methylation cells were dealt with demethylation 5 - Aza - 2＇ deoxycytidine. Results There was abnormally high methylation in CpG isle of P16 carcinoma - inhibiting gene promotor during malignant transformation and tumorigenicity in inoculated athymic mouse 16HBE cells induced by cadmium chloride. The process of methylation showed a tendency of increase along with the kintesification of malignant transformation of cells and there was a phenomenon of disappearance of methlylation of abnormal methylated cells after demethylation. Conclusion The hypermethyylation of P16 gene promoter region lead to the close of expression of carcinoma - inhibiting gene that could not normally negatively control cell division and growth, resulting in indefinite cell proliferation, this might be the sur- carcinogenic mechanism of 16HBE cell malignant transformation and tumorigenicity in inoculated athymic mouse induced by cadmium chloride.