中文摘要：

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。

英文摘要：

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.