甲基化PCR作为白血病微量残留病检测方法的探索

Investigation on MS-PCR as a Method for Detecting Minimal Residual Disease in Acute Leukemia

本实验研究探讨以甲基化特异性聚合酶链反应(MS-PCR)作为白血病微量残留病(MRD)检测方法的可行性。将Id4基因呈完全甲基化的HL-60细胞和Id4基因呈完全非甲基化的Hek937细胞按不同比例混合,实验分为3组:A组为10%HL-60+9%Hek937,B组为1%HL-60+99%Hek937,C组为0.1%HL-60+99.9%Hek937。用MS-PCR方法检测不同白血病细胞比例下Id4基因甲基化情况。结果表明,HL-60细胞仅有155bp的Id4基因甲基化特异性扩增,呈Id4基因完全甲基化;Hek937细胞仅有156bp的Id4基因非甲基化特异性扩增,呈Id4基因完全非甲基化。A、B、C组同时有155bp的Id4基因甲基化和156bp的Id4基因非甲基化特异性扩增,显示Id4基因甲基化表达。结论:MS-PCR方法可从0.1%比例含量的白血病细胞样本中检测到Id4基因甲基化,加之Id4基因甲基化在不同类型白血病中差异不明显,因此用MS-PCR检测Id4基因甲基化状态可以作为一种检测各种类型白血病微量残留病的方法。

The aim of this study was to investigate the feasibility of monitoring minimal residual disease （MRD） of leukemia with methylation specified-polymerase chain reaction （MS-PCR）. The HL-60 cells with Id4 gene complete methylation and Hek937 cells with Id4 gene complete unmethylation were mixed in accordance with different ratios of cells and were divided into 3 groups： group A （10% HL-60 ＋ 90% Hek937 ）, group B （1% HL-60 ＋ 99% Hek937 ） and group C （0.1% HL-60 ＋99.9% Hek937）. The MS-PCR technigue was used to detect the methylation status of Id4 gene in different ratios of leukemia cells. The results indicated that the methylation specific amplification of Id4 gene with 155 bp was observed in HL-60 cells showing complete methylation of Id4 gene; while the unmethylation specific amplication of Id4 gene with 156 bp was found in Hek937 cells, showing complete unmethylation. The methylation specific amplification of Id4 gene with 155 bp and unmethylation specific amplification of Id4 gene with 156 bp were simultaneously detected in A, B and C groups, which showeO the expression of Id4 gene methylation. In conclusion, the MS-PCR technique can detect the Id4 gene methylation in leukemia cell sample containing 0. 1% of HL-60 cells, moreover the Id4 gene methylation in various leukemia cells shows no significant difference, thereby use of MS-PCR to detect the Id4 methylation level may be a potential approach for monitoring of MRD. Id4 gene promoter methylation is a candidate of biomarker for MRD detection in acute leukemias.