反向点杂交检测肝癌细胞DNA甲基化方法的建立

摘要

Objective To establish the reverse dot blot(RDB) method for detection DNA methylation, and using the method to detect the aberrant methylation of 5'CpG islands in the MAGE-A3 gene. Methods The difference of methylation mode in Hep G2 and peripheral white blood cell was analyzed by polymerase phain reaction after bisulfite modified. Then the method of RDB for detecting ON A methylation was established. And the method was evaluated by methodology, which was used to detect hepatoma carcinoma pathology organization specimens,cirrhosis tissue sample,and the normal control group. Results The sensitivity,specificity and accuracy were 83% (25/30) ,100% (60/60) and 94% (85/90) respectively. RDB has good consistency with gold standard of clinical diagnosis of hepatoma carcinoma cell( Kappa =0.870,P <0.05). Conclusion The RDB method for detection DNA aberrant methylation in hepatoma carcinoma cell is established,which can be apply in clinical early diagnosis for hepatoma carcinoma.%目的 建立反向点杂交(RDB)检测DNA甲基化的方法,并应用于临床肝癌细胞MAGE-A3 5’端CpG位点异常甲基化的检测.方法 亚硫酸氢盐修饰后聚合酶链式测序分析细胞株Hep G2和外周血白细胞DNA的甲基化模式差异,建立RDB检测DNA甲基化方法,进行方法学评价,并运用于临床肝癌病理组织标本、肝硬化组织标本和正常对照组.结果 RDB检测肝癌细胞MAGE-A3 5’端CpG位点异常甲基化的灵敏度、特异度和准确性分别是83％(25/30)、100％(60/60)和94％ (85/90).其与临床肝癌诊断金标准的检验结果一致性较好(Kappa=0.870,P＜0.05).结论 建立了RDB检测肝癌细胞DNA异常甲基化的方法,并可用于肝癌的临床早期诊断.