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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 investigate the methylation status of CPG island in tumor suppressor gene p16 promoter region in coal-fired arsenism patients. Methods Totally 51 and 52 peripheral blood samples from coal-burning arsenic poisoning patients and normal controls were collected respectively. DNA was extracted by phenol-chloroform method. UV spectrophotometry was used to determine the amount of DNA, genomic DNA was denatured to single-stranded, bisulphite-modified single-stranded DNA, all unmethylated cytosines were converted to uracil, and methylated cytosines were not change. Specific primers for methylated and unmethylated alleles were designed and amplified by nested PCR. Finally, the target fragments were detected by gel electrophoresis. Results The detection rate of methylation of CPG island in promoter region of p16 gene was found to be 94.1% (48/51) and 71.1% (37/52) respectively in patients with arsenism of coal-fired arsenic poisoning. The difference was statistically significant. Conclusion CpG island methylation in p16 promoter is associated with coal-burning arsenism.