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建立高分辨熔解曲线法(HRM)定量检测哈萨克族食管癌患者癌组织中FHIT、CDKN2A甲基化水平,并分析甲基化水平与食管癌病理参数的相关性。选取30例食管癌癌患者癌组织及30例癌旁组织,提取DNA,进行甲基化修饰。将标准品DNA(甲基化DNA与非甲基化DNA相互的掺入)稀释成0,5%,25%,50%,75%,100%甲基化DNA,并进行重复性和灵敏性评价。应用HRM定量检测癌组织及癌旁组织甲基化水平,探讨FHIT、CDKN2A基因启动子区甲基化水平与食管癌发生发展的关系。100%,80%,50%,30%,10%,0甲基化标准品的高分辨率熔解曲线从右向左依次排列,待测基因在标准曲线上的位置即表示其甲基化程度。HRM最低检测限为1%,明显高于MSP结果最低检测限10%。在30例食管癌癌患者癌组织中,检测出存在FHIT甲基化的为36.7%。其中8例甲基化程度为0—5%,3例为5%—10%。CDKN2A甲基化的为100%,其中7例甲基化程度为0—5%,11例为5%—10%,12例为10%—25%。与正常组织比较,抑癌基因的甲基化与癌症的发生无明显相关性。进一步讨论甲基化与食管癌的病理级别以及甲基化与分化程度的关系,二者均无相关性。通过HRM技术成功建立了定量检测哈萨克族食管癌组织中FHIT、CDKN2A甲基化程度的方法,FHIT、CDKN2A基因启动子甲基化与哈萨克族食管癌的发生发展无明显相关性,但多个抑癌基因的甲基化有望成为其早期发生发展的分子指标。
To establish a high resolution melting curve method (HRM) for quantitative detection of FHIT and CDKN2A methylation levels in Kazakh esophageal cancer patients and to analyze the correlation between methylation level and pathological parameters of esophageal cancer. Thirty esophageal cancer tissues and 30 adjacent tissues were selected for DNA extraction and methylation modification. The standard DNA (methylated DNA and unmethylated DNA intercalate with each other) was diluted to 0, 5%, 25%, 50%, 75%, 100% methylated DNA and subjected to repeatability and sensitivity Evaluation. HRM quantitative detection of cancer tissue and adjacent tissue methylation levels, FHIT, CDKN2A promoter methylation and esophageal cancer occurrence and development of the relationship. High-resolution melting curves of 100%, 80%, 50%, 30%, 10%, 0 methylated standards are arranged in order from right to left. The position of the gene under test on the standard curve indicates the degree of methylation . The minimum detection limit for HRM was 1%, significantly higher than the minimum detection limit of 10% for MSP results. In 30 cases of esophageal cancer, the presence of FHIT methylation was detected in 36.7% of cases. Among them, the methylation degree was 0-5% in 8 cases and 5% -10% in 3 cases. Methylation of CDKN2A was 100%, of which 7 cases had a methylation level of 0-5%, 11 cases 5% -10% and 12 cases 10% -25%. Compared with normal tissues, there was no significant correlation between the methylation of tumor suppressor gene and the occurrence of cancer. Further discussion of methylation and esophageal cancer pathological grade and the relationship between methylation and differentiation, both no correlation. The method of quantitative detection of methylation of FHIT and CDKN2A in Kazakh esophageal cancer tissue was successfully established by HRM technique. There was no significant correlation between the promoter methylation of FHIT and CDKN2A gene and the occurrence and development of Kazakh esophageal cancer. However, The methylation of oncogene is expected to become a molecular indicator of its early development.