半巢式甲基化特异性聚合酶链反应在胃癌p16基因甲基化检测中的应用  

Detection of methylation status of p16 gene promoter in gastric cancer tissues by Semi-nested methylation specific PCR

作　　者：沈佳尧[1] 侯鹏[1] 祭美菊[1] 郭庆明[2] 何农跃[1] 

机构地区：[1]东南大学生物科学与医学工程系吴健雄实验室,南京210096 [2]东南大学医学院中大医院

出　　处：《中华检验医学杂志》2004年第2期85-88,共4页Chinese Journal of Laboratory Medicine

基　　金：国家973高技术计划资助项目(G1998051204);江苏省高新技术资助项目(BG2001010);国家自然科学基金资助项目(60071001)

摘　　要：目的　改进甲基化特异性聚合酶链反应 (MSP) ,采用半巢式MSP检测胃癌组织p16基因启动子区CpG岛的甲基化状态。方法　用亚硫酸氢盐修饰被测DNA后 ,采用半巢式MSP(引物分别为MS ,M1A和MS ,M2A) ,分析了 6 0例胃癌组织及相应正常组织中p16基因的甲基化状态。结果　单独用MSP ,胃癌组织中p16基因甲基化的发生率为 80 %。采用半巢式MSP ,甲基化发生率为86 7% ,比单独采用MSP提高了几个百分点 ,并提示胃癌病例的p16基因启动子区存在甲基化发生的不同模式。结论　半巢式MSP能够有效地提高灵敏度和减少假阳性。同时 ,免疫组化的结果也说明了p16基因异常甲基化与胃癌组织P16蛋白的表达密切相关。Objective A semi-nested MSP (methylation spesific PCR) was introduced to detect methylation status of p16 gene promoter in gastric cancer tissues. Method Target DNA was modified by sodium bisulfite, converting all unmethylated , but not methylated, cytodines to uracil, and subsequent amplification with semi-nested MSP primers ( MS、M1A, and MS、M2A ). It was also detected by sequencing and the expression of p16 protein was checked with immunohistochemistry. Results Hypermethylation of the p16 gene was detected in 80% (48/60) of the gastric tumor samples from the first PCR. However, the frequency increased significantly to 86.7% (52/60) of the gastric tumor samples after the second PCR. Conclusion The results showed that this technique increased the sensibility of detecting p16 hypermethylation from tumor samples.Furthermore, the aberrant methylation of p16 was observed in all of the stages, confirming that this epigenetic alteration is an early event during gastric carcinogenesis.

关 键 词：半巢式甲基化特异性聚合酶链反应 胃癌 P16基因 甲基化 检测 

分 类 号：R735.2[医药卫生—肿瘤]