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抑癌基因p16和白血病致癌因子Ralb与白血病的发生密切相关,其启动子区CpG岛的甲基化对基因表达具有重要作用.本文旨在分析p16、Ralb基因启动子区CpG岛甲基化位点信息,并比较这两个基因在小鼠骨髓细胞和原代培养的骨髓细胞中甲基化状态的差异.运用“MethPrimer”软件预测p16、Ralb基因启动子区的CpG岛,设计甲基化特异性引物.利用重亚硫酸盐测序法(BSP)检测甲基化位点信息.结果显示,p16有1个CpG岛,岛上21个CpG位点全部未发生甲基化;Ralb有2个CpG岛,CpG岛1上的5个CpG位点全部呈甲基化状态,而CpG岛2上的17个CpG位点全部呈非甲基化状态,且小鼠骨髓细胞和体外原代培养的骨髓细胞中两基因的甲基化状态一致.表明p16、Ralb基因甲基化状态未受外界培养条件的影响而改变,提示在与两基因甲基化相关的研究中体外试验可替代体内试验.
Tumor suppressor gene p16 and leukemia oncogene Ralb are closely related to the occurrence of leukemia, and its promoter CpG island methylation plays an important role in gene expression.This paper aims to analyze the p16, Ralb gene promoter CpG island methylation position The difference between the methylation status of these two genes in mouse bone marrow cells and primary cultured bone marrow cells was compared by using “MethPrimer” software to predict the CpG islands in the promoter regions of p16 and Ralb genes and design a The specific primers were used to detect the methylation site information by bisulfite sequencing (BSP). The results showed that there was one CpG island in p16 and all 21 CpG sites on the island were not methylated. Two CpG islands, all of the five CpG sites on CpG island 1 were methylated, whereas 17 CpG sites on CpG island 2 were all unmethylated, and both mouse bone marrow cells and primary in vitro The methylation status of the two genes in the cultured bone marrow cells was consistent, indicating that the methylation status of p16 and Ralb genes was not affected by the external culture conditions, suggesting that the in vitro test may replace the in vivo test in the studies related to methylation of the two genes test.