半巢式甲基化特异性聚合酶链反应对肿瘤细胞株p15基因甲基化或缺失状态的检测

Detection of p15 Gene Methylation or Deletion Status in Different Malignant Cell Lines by Using Hemi-nested Methylation Specific Poly-merase Chain Reaction

本研究分析多种恶性肿瘤细胞株的p15基因甲基化或缺失状态,阐明p15基因甲基化或缺失在肿瘤发生发展中的作用。运用半巢式甲基化特异性聚合酶链反应(hemi-nested methylation specific polymerase chain reaction,hn-MSP)分析20种恶性肿瘤细胞株和正常人单个核细胞或细胞株的p15基因甲基化及缺失状态,并评价该方法的敏感性和特异性。结果表明:在所有的细胞中,Molt-4、Raji、KG1、CA46、SW480、NCE、SMMC-7221、NCI-H446细胞为部分甲基化,hn-MSP检测p15基因甲基化敏感性可达到1×10-5。正常人单个核细胞、HL-60、HeLa、HepG2、293、SGC7901、U266、CEM细胞则为p15非甲基化,而K562、NB4、GMC、Jurkat似乎显现p15基因缺失或突变。结论:p15基因甲基化或缺失在多种肿瘤,特别在恶性血液病中有较高的发生率,且对疾病的进展及预后有密切的关系,hn-MSP具有较高的敏感性和特异性,值得在临床推广应用。

This study was aimed to investigate the methylation or deletion status of p15 gene in different malignant cell lines, and further to clarify their roles in the development and progression of malignant tumors. Hemi-nested methylation specific polymerase chain reaction （hn-MSP） was adopted to analyze p15 gene methylation or deletion status in 20 malignant tumor cell lines and mononuclear cells or normal cell lines in healthy people, as well as to evaluate its sensitivity and specificity. The results showed that among all of the cell lines, Molt-4, KG1, NCE, Raji, SMMC-7221, CA46,SW480 and NCI-H446 were partial methylated with CDKN2B gene,and its sensitivity of detection of p15 gene methylation was up to 1.0 × 10^-5, also it had great specificity. Peripheral blood mononuclear cell（MNCs） from healthy volunteer,HL-60, HepG2,293, HeLa, SGC7901, U266 and CEM were unmethylated; and K562, NB4, GMC, Jurkat seemed to have deletion or mutation of p15 gene. It is concluded that the incidence of p15 gene methylation or deletion in many tumours, especially malignant hematopathy, is frequent, they correlate with disease progression and prognosis. Hn-MSP is highly sensitive and specific in analyzing p15 gene methylation, deserving in clinical application.