5-杂氮胞苷对成人T淋巴细胞穿孔素基因启动子区域甲基化水平的影响  

Effects of 5-azaC on methylation pattern of the perforin promoter of the perforin gene in normal human T cells

作　　者：肖嵘[1] 丁艳[1] 陆前进[1] 李亚萍[1] 李勇坚[1] 杨心洁[2] 苏玉文[1] 梁云生[1] 张桂英[1] 文海泉[1] 

机构地区：[1]中南大学湘雅二医院皮肤科 [2]中南大学湘雅二医院医学试验中心,长沙410011

出　　处：《中南大学学报（医学版）》2006年第6期843-847,共5页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(30371297)

摘　　要：目的:研究DNA低甲基化对穿孔素基因mRNA和蛋白表达的影响。方法:密度梯度离心分离正常成人的外周血淋巴细胞,磁珠分离CD4和CD8细胞后进行细胞培养,DNA甲基化抑制剂5-杂氮胞苷(5-azaC)处理。分别提取DNA、RNA和蛋白质。亚硫酸氢钠处理DNA,巢式PCR进行扩增、转化入大肠杆菌,挑取克隆测序。实时定量逆转录聚合酶链反应(RT-PCR)检测穿孔素mRNA的表达,Western印迹检测穿孔素蛋白量的变化。结果:5-azaC处理后的CD4细胞和CD8细胞穿孔素mRNA和蛋白表达显著高于未处理的CD4和CD8细胞组(P<0.05)。测序结果显示5-azaC处理后的CD4细胞和CD8细胞DNA穿孔素启动子区域的甲基化水平降低,与未处理的CD4和CD8细胞组比较差异有统计学意义(P<0.05)。结论:5-azaC处理后CD4和CD8细胞穿孔素mRNA的表达和蛋白的表达都有显著的增高,这种增高与CD4和CD8细胞穿孔素基因启动子区域出现的低甲基化有关。Objective To investigate the effects of DNA hypomethylation on mRNA and protein expression of perforin promotor in T cells. Methods T cells were isolated from the peripheral venous blood of healthy donors by density gradient centrifugation. CD4 ^＋and CD8 ^＋ subsets were isolated using Miltenyi beads and protocols provided by the manufacturer. Where indicated the T cells were stimulated with PHA for 24 h, then treated with 5-azaC for an additional 72 h. Genomic DNA, mRNA, and protein were isolated from untreated and 5-azaC-treated T cells. Purified DNA was treated with sodium bisulfite, the desired sequences were amplified in sequential fragments using nested PCR. The amplified fragments were cloned into bacteria DH5 α, and 5 independent clones for each of the amplified fragments were sequenced. The expression of perforin was determined using real time RT-PCR and Westem blot. Results The perforin mRNA and protein in the CD4 ^＋ and CD8 ^＋ subsets treated with 5-azaC were significantly higher than those in the untreated subsets （ P 〈 0.05 ）. The results of bisulfite genomic sequencing showed that the methylation of perforin promotor was sig nificantly reduced in the treated cells compared with the untreated cells （ P 〈 0.05 ）. Conclusion The mRNA and protein expression of perforin significantly increases in the CD4 ^＋ and CD8 ^＋ T cells treated with 5-azaC, which is associated with DNA hypomethylation of perforin promoter in T cells.

关 键 词：穿孔素 甲基化 亚硫酸氢钠测序 5-杂氮胞苷 

分 类 号：Q75[生物学—分子生物学]