系统性硬化病患者外周血CD4＋T细胞CD40LDNA甲基化研究  

DNA methylation status of CD40L in peripheral blood CD4＋ cells from patients with systemic sclerosis

作　　者：肖嵘[1] 廉晓日[1] 胡新红[1] 金藏拓郎[3] 姜红岩[1] 杨艳[1] 王瑶瑶[1] 李亚萍[1] 张桂英[1] 赵明[2] 陆前进[2] 

机构地区：[1]中南大学湘雅二医院皮肤科,长沙410011 [2]中南大学湘雅二医院医学表观基因组学湖南省重点实验室,长沙410011 [3]日本鹿儿岛大学

出　　处：《中华皮肤科杂志》2012年第7期517-519,共3页Chinese Journal of Dermatology

基　　金：国家自然科学基金（30972662）;科技部国际科技合作重点项目（2008DFA31980）

摘　　要：目的探讨系统性硬化病（SSc）患者外周血CD4＋T细胞中CD40L基因调控序列的甲基化状态。方法密度梯度离心法分离SSc患者（女16例，男10例）和健康对照组（女15例，男10例）外周血单一核细胞，磁珠分选CD4＋T细胞，提取DNA，亚硫酸氢钠处理DNA，巢式PCR扩增CD40L基因调控序列片段（包括启动子和增强子），转化进入大肠杆菌，每个样本挑取8个克隆进行测序。结果亚硫酸盐基因组测序结果显示，健康女性CD40L基因调控序列一半为甲基化，一半为去甲基化，女性SSc患者CD40L基因启动子和增强子区域平均甲基化水平均显著低于女性健康对照（P值均〈0．01）；男性SSc患者和男性健康对照CD40L基因启动子和增强子区域几乎全部为去甲基化，两组平均甲基化水平差异无统计学意义（P值均〉0．05）。结论女性SSc患者CD4＋T细胞中失活的X染色体上CD40L调控序列低甲基化，可能是导致CD40L在女性SSc患者CD4＋T细胞中过度表达的原因之一。Objective To study the methylation status of CD40L gene regulatory regions in peripheral blood CD4＋ T cells from patients with systemic sclerosis （SSc）. Methods Peripheral blood mononuclear cells were isolated from the venous blood of 21 SSc patients and 20 healthy controls by density gradient centrifugation. CD4＋ T cells were separated by using magnetic beads. Genomic DNA was isolated from the CD4＋T cells and treated with sodium bisulfite. Nested PCR was performed to amplify the desired regulatory sequences （including the promotor and enhancer） of CD40L, and the amplicons were transformed into the Eschenehia coli DH5α. Subsequently, 8 independent clones were selected and sequenced for each of the amplified fragments. Results In healthy female controls, half of the cloned fragments of CD40L regulatory sequences were unmethylated, and the other half were methylated. The mean methylation levels of CD40L promoter and enhancer from female SSe patients were significantly lower than those from healthy female controls （both P 〈 0.01 ）. Almost all of the cloned fragments of CD40L promoter and enhancer were unmethylated in healthy male controls and male SSc patients, with no significant difference in the methylation level between male SSc patients and healthy controls （both P 〉 0.05, respectively）. Conclusions There is a low methylation level of CD40L regulatory elements on the inactive X chromosome in female SSc patients, which may contribute to the CD40L overexpression in CD4＋T cells.

关 键 词：外周血单一核细胞 CD4+T细胞 DNA甲基化 CD40L 系统性硬化病 患者 基因调控序列 基因启动子 

分 类 号：R593.2[医药卫生—内科学]