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目的肿瘤抑制基因异常甲基化状态与白血病发生关系密切,发展白血病甲基化标志物成为研究热点。但白血病类型复杂,利用单个基因甲基化模式作为肿瘤标志物具有一定局限性。本研究通过探讨基于肿瘤抑制基因p73和死亡相关蛋白激酶(death-associated protein kinase gene,DAPK)异常甲基化模式对白血病诊断分型的价值和意义,寻找一种联合多基因异常甲基化模式作为白血病肿瘤标志物的方法。方法应用亚硫酸氢盐测序法(bisulfite sequencing PCR,BSP),分析正常人白细胞、单核细胞系白血病细胞株U937、粒细胞系HL-60及淋巴细胞系Jurkat基因组中,p73和DAPK基因启动子区CpG岛甲基化状态,将测序结果汇集到Office Excel 2010文件中,并计算各CpG位点甲基化率,绘制2个基因在4种细胞来源基因组中的CpG岛甲基化模式图,从而筛选特异甲基化位点组合。通过甲基化特异性PCR法(methylation specific PCR,MSP),在白血病细胞株、30例正常对照和104例白血病患者外周血标本中,验证基因异常甲基化模式的诊断效能。结果成功测序约107个含BSP产物转化质粒的菌株,并绘制出p73和DAPK基因CpG岛甲基化模式图。正常细胞基因组两者去甲基化程度远高于白血病细胞株。p73基因在正常细胞与白血病细胞株中,甲基化状态完全相反。DAPK基因在HL-60中与其他白血病细胞株甲基化状态存在明显差异。p73基因MSP甲基化检测可以鉴别正常细胞和白血病细胞,在临床白血病标本诊断中的灵敏度、特异度和准确度分别是21.5%、100.0%和43.1%,DAPK基因甲基化检测可以鉴别HL-60与其他白血病细胞株,其诊断急性非淋巴细胞白血病的灵敏度、特异度和准确性分别是59.1%、100.0%和77.2%。结论 p73和DAPK基因启动子区CpG岛在不同类型白血病细胞基因组中存在特异的甲基化位点,将其作为白血病初步诊断分型的潜在肿瘤标志物具有一定临床意义,也为今后发现白血病等肿瘤的甲基化标志物提供方法并奠定实验研究基础。
Objective Tumor suppressor gene abnormal methylation status is closely related to the occurrence of leukemia, the development of leukemia methylation markers become a research hot spot. However, the type of leukemia is complex, using a single gene methylation pattern as a tumor marker has some limitations. In this study, we investigated the value and significance of differential methylation patterns of tumor suppressor gene p73 and death-associated protein kinase gene (DAPK) in the diagnosis of leukemia, and sought a combination methylation pattern of multiple genes As a leukemia tumor marker method. Methods Bisulfite sequencing PCR (BSP) was used to analyze the promoter region of p73 and DAPK genes in normal human leukocyte, monocytic leukemia cell line U937, granulocyte line HL-60 and lymphocyte line Jurkat genome CpG island methylation status of the region, the sequencing results are pooled into the Office Excel 2010 file, and calculate the methylation rate of each CpG site, draw two genes in the four cell-derived CpG island methylation patterns, Thus screening for specific methylation site combinations. The methylation-specific PCR (MSP) was used to verify the diagnostic efficacy of aberrant methylation patterns in leukemia cell lines, 30 normal controls and 104 leukemia patients. RESULTS: Approximately 107 isolates containing BSP-transformed plasmids were successfully sequenced, and CpG island methylation patterns of p73 and DAPK genes were mapped. The demethylation of both normal cell genomes is much higher than that of leukemia cell lines. p73 gene in normal cells and leukemia cell lines, methylation status completely opposite. DAPK gene in HL-60 and other leukemia cell methylation status there is a significant difference. The sensitivity, specificity and accuracy of p73 gene MSP methylation assay can identify normal cells and leukemic cells in the diagnosis of clinical leukemia specimens were 21.5%, 100.0% and 43.1%, respectively. DAPK gene methylation detection could identify HL- 60 and other leukemia cell lines were 59.1%, 100.0% and 77.2%, respectively, for the diagnosis of acute non-lymphocytic leukemia. Conclusion The CpG islands of p73 and DAPK gene promoters have specific methylation sites in the genomes of different types of leukemia cells. It is of clinical significance as a potential tumor marker for the preliminary diagnosis of leukemia and also for the future discovery of leukemia Methylation markers of tumors provide methods and lay the foundation for experimental research.