论文部分内容阅读
目的:应用重亚硫酸氢盐测序法(bisulfite sequencing PCR,BSP)检测急性早幼粒细胞白血病细胞株(NB4)Wnt信号通路抑制基因启动子区CPG岛的甲基化状态,筛选NB4细胞中高甲基化的Wnt信号通路抑制基因,并探讨BSP法作为定量研究基因甲基化方法的优缺点。方法:以NB4细胞为研究对象,20例健康人单个核细胞为对照;常规提取DNA并用亚硫酸氢盐处理,然后用PCR扩增目标序列,应用重亚硫酸氢盐测序法(BSP)分析在NB4细胞中Wnt信号通路抑制基因基因的甲基化状态。并通过与甲基化特异性PCR(methylation specific PCR,M SP)、焦磷酸测序技术(pyrosequencing)比较、评价BSP法检测NB4细胞Wnt信号通路抑制基因甲基化状态的优缺点。结果:BSP产物克隆测序检测NB4细胞Wnt信号通路抑制基因甲基化发生率分别为:WIF-1基因95.26%,DKK3基因86%,SFRP1基因81.67%,SFRP2基因95.71%,SFRP4基因85%,SFRP5基因95%;20例健康人单个核细胞为对照组中Wnt信号通路抑制基因甲基化发生率分别为:WIF-1基因1.5%,DKK3基因4.2%,SFRP1基因0%,SFRP2基因0.9%,SFRP4基因2.5%,SFRP5基因1.75%。与正常人M NC相比,NB4细胞的Wnt信号通路相关抑制基因启动子甲基化率明显增高。结论:急性早幼粒细胞株NB4细胞中存在Wnt信号通路多个抑制基因高甲基化状态。此类基因异常甲基化有可能成为急性早幼粒细胞白血病的早期诊断指标和治疗靶点。
OBJECTIVE: To detect the methylation status of CPG island in the Wnt signaling pathway inhibitor gene promoter region by bisulfite sequencing PCR (BSP) Bax and Wnt signaling pathways, and to explore the advantages and disadvantages of BSP as a quantitative method for gene methylation. Methods: The NB4 cells were used as the research objects. Twenty healthy human mononuclear cells were used as controls. DNA was extracted routinely and treated with bisulfite. The target sequence was amplified by PCR and analyzed by bisulfite sequencing (BSP) Wnt signaling pathway inhibits the methylation status of gene in NB4 cells. The advantages and disadvantages of the BSP method in detecting the methylation status of Wnt signaling pathway in NB4 cells were evaluated by methylation-specific PCR (MSP) and pyrosequencing. Results: The methylation rates of Wnt signaling pathway suppressor genes in NB4 cells were 95.26% for WIF-1 gene, 86% for DKK3 gene, 81.67% for SFRP1 gene, 95.71% for SFRP2 gene, 85% for SFRP4 gene and 85% for SFRP4 gene Gene 95%. The methylation rates of Wnt signaling pathway suppressor genes in 20 healthy human mononuclear cells were 1.5% in WIF-1, 4.2% in DKK3, 0% in SFRP1 and 0.9% in SFRP2, SFRP4 gene 2.5%, SFRP5 gene 1.75%. Compared with normal human M NC, NB4 cells Wnt signaling pathway related gene promoter methylation rate was significantly higher. CONCLUSION: There is a hypermethylation status of multiple suppressor genes in Wnt signaling pathway in NB4 cells. Aberrant methylation of these genes may become an early target for diagnosis and treatment of acute promyelocytic leukemia.