急性白血病细胞中ID4基因甲基化的PCR定量检测系统的建立及其特异性和敏感性

ID4基因启动子区甲基化发生率在急性白血病中极高，甲基化程度与急性白血病关系密切。因缺少合适的研究方法，其具体的甲基化量化水平与疾病的关系，人们一直缺乏认识。本研究旨在建立ID4甲基化定量PCR体系，同时验证该方法的特异性及敏感性，并初步尝试在初治急性白血病骨髓样本中评估ID4的甲基化水平。首先构建质粒并建立体系标准曲线，分别使用MSP和定量MSP对细胞系样本进行检测，以传统MSP的结果为对照，验证新方法的特异性；按不同比例将甲基化阴性和阳性细胞混合，用甲基化定量PCR扩增验证新方法敏感性。结果表明，本研究成功建立了符合绝对定量要求的标准曲线；通过细胞系验证，MSP结果与定量MSP结果完全一致；同时在甲基化阴性细胞背景下，定量MSP可以稳定的检测到1：10-5水平的ID4甲基化阳性细胞。在急性白血病骨髓样本检测中，定量MSP甲基化阳性检出率高于MSP。结论：本研究成功建立了完整的ID4基因甲基化定量PCR体系，该方法特异性好、敏感性高。

Establishment of Methylation-Specific Quantitative PCR System for /D4 Gene in Acute Leukemia Cells and Its Specificity and Sensitivity

DNA methylation of ID4 gene promoter occurred frequently in patients with acute leukemia and was found to be highly related to the tumor progression. Due to lack of the appropriate methylation detection methads, the relation between the quantification of ID4 methylation and the states of acute leukemia is still unclear. This study purposed to set up a methylation-specific quantitative PCR system for ID4 and investigate the specificity and sensitivity of this methylation detection. The plasmids combined with target gene as well as with internal reference were constructured, and the standard curves were set up by using above mentioned plasmids. The specificity of this detection system in cell lines was verified through techniques of MSP and quantitative MSP. The sensivity of this detection system was verified by mixing methylation-positive and negative cell lines in varying proportions and throngh amplification of guatitative MSP. The results showed that the standard curves were establish successfully. The results of quantitative MS-PCR in cell fines were consistent with those of MS-PCR, and as low as 1：10-3 of ID4 methylation positive cells could be detected by the new methylation detection assay. In newly diagnosed acute leukemia patients, the positive rate of quantitative MSP was higher. It is concluded that a complete quantitative MSP system for ID4 methylation detection has been. established and this quantitative MSP method has good specificity and high sensitivity.