小鼠骨髓细胞中p16和Ralb基因启动区CpG岛的甲基化位点分析

Analysis of Methylation Locus at CpG Islands of p16 and Ralb Genes Promoter in Mouse Bone Marrow Cells

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.

p16（Multiple suppressor gene） and Ralb （v-ral simian leukemia viral oncogene homolog B, ras related; GTP binding protein） are strongly related with the development of acute leukemia,and the methylation of those two gene CpG island in promoter regions is of great significance to the expression of genes.This paper aims at detecting the information of methylation sites of CpG island locating in the promoter regions of the p16 and Ralb gene,and comparing methylation differences of the two genes between in vivo and in primarily cultivated C57BL/6J mouse＇s bone marrow cells in vitro.Here we used ＂MethPrimer＂software to predict the CpG islands of p16 and Ralb and to design methylation-specific primers.Bisulfite sequencing PCR （BSP ） was used to investigate the information of CpG islands methylation sites in p16 and Ralb promoter regions.The results showed that one CpG island was predict in p16 gene,and 21 CpG sites of p16 were all in the state of unmethylation ; two CpG islands were found in Ralb gene.BSP sequencing showed that 5 CpG sites in the first CpG island of Ralb were all methylated,while 17 CpG sites in the second island were all unmethylated.The CpG islands methylation status in p16 and Ralb promoter regions were consistent both in vivo and in vitro.From what has been discussed above,we concluded that the methylation pattern of p16 and Ralb in cultured mouse bone marrow cells were not influenced by the outside cultured environment,and the study of methylation status about these two genes in vivo could be replaced by the study in vitro.