贵州兴仁县燃煤型砷中毒患者p16基因甲基化研究

Research into methylation of p16 gene in the patients suffering from coal-burnt arsenism in Xingren County Guizhou Province

目的了解抑癌基因 p16启动子区 CPG 岛在燃煤型砷中毒患者中甲基化的情况和意义。方法分别采集燃煤型砷中毒患者与正常人群外周血标本各51例和52例,采用酚-氯仿法提取DNA。紫外分光光度法测定 DNA 的含量,将基因组 DNA 变性成为单链,用亚硫酸氢盐修饰单链DNA,所有未甲基化的胞嘧啶被转变为尿嘧啶,而甲基化的胞嘧啶则不变。设计针对甲基化和非甲基化等位基因的特异引物,进行巢式 PCR 扩增,最后经凝胶电泳检测目的片段。结果燃煤型砷中毒患者与正常对照 p16基因启动子区 CPG 岛甲基化检出率分别为94.1%(48/51)和71.1%(37/52),两者间差异有统计学意义。结论 p16启动子区 CPG 岛甲基化与燃煤型砷中毒有关联。

Objective To probe into the situation and significance of p16 gene CPG island methylation in patients with arseniasis caused by coal-burning pollution. Methods DNA was extracted using the Phenol-Chloroform method from leukocytes of 51 patients suffered from coal-burnt arsenism and 52 healthy volunteers. The quantity of the DNA was determined by UV spectrophotometry . Target DNA was denatured by NaOH, then the single strand DNA was modified by sodium bisulfite, converting all unmethylated （ but not the methylated） cytosines to uracil. Subsequently a nested amplification with primers specific for methylated versus unmethylated DNA was performed, and PCR products were detected by gel electrophoresis. Results Hypermethylation of the p16 CPG island was presented in 94. 1% of the patients suffering from coal-burnt arsenism and in 73. 1% of the healthy volunteers. There was statistical difference （P 〈 0. 05 ） between them. Condusions Methylation of p16 genne CPG island should have important pertinence in the metabolism of coal-burnt arsenism.