系统性硬化病患者外周血CD4+ T细胞CD40L DNA甲基化研究

中华皮肤科杂志 ›› 2012, Vol. 45 ›› Issue (7): 517-519.

系统性硬化病患者外周血CD4+ T细胞CD40L DNA甲基化研究

肖嵘¹,廉晓日¹,胡新红¹,金藏拓郎²,³,姜红岩¹,³,杨艳¹,³,王瑶瑶¹,李亚萍¹,张桂英⁴,赵明³,陆前进⁴

1. 中南大学湘雅二医院皮肤科
2. 日本鹿儿岛大学
3.
4. 长沙中南大学湘雅二医院皮肤科

收稿日期:2011-07-25 修回日期:2011-10-12 出版日期:2012-07-15 发布日期:2012-07-02
通讯作者: 肖嵘 E-mail:xiaorong65@yahoo.com.cn
基金资助:
国家自然科学基金;科技部国际科技合作重点项目

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

Received:2011-07-25 Revised:2011-10-12 Online:2012-07-15 Published:2012-07-02

摘要/Abstract

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。

关键词: DNA甲基化

Abstract:

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 Escherichia 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 SSc 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.

Key words: DNA methylation

肖嵘廉晓日胡新红金藏拓郎姜红岩杨艳王瑶瑶李亚萍张桂英赵明陆前进. 系统性硬化病患者外周血CD4+ T细胞CD40L DNA甲基化研究[J]. 中华皮肤科杂志, 2012, 45(7): 517-519.

参考文献

[1] 赵辨.中国临床皮肤病学.1版.南京：江苏科学技术出版社，2011:814-815. [2] Whitacre CC. Sex differences in autoimmune disease. Nat Immunol, 2001, 2 (9):777-780. [3] Valentini G, Romano MF, Naclerio C, et al. Increased expression of CD40 ligand in activated CD4+ T lymphocytes of systemic sclerosis patients. J Autoimmun, 2000, 15(1):61-66. [4] Komura K, Sato S, Hasegawa M, et al. Elevated circulating CD40L concentrations in patients with systemic sclerosis. J Rheumatol, 2004, 31(3):514-519. [5] Komura K, Fujimoto M, Yanaba K, et al. Blockade of CD40/CD40 ligand interactions attenuates skin fibrosis and autoimmunity in the tight-skin mouse. Ann Rheum Dis, 2008, 67(6):867-872. [6] Attwood J. DNA methylation and the regulation of gene transcription.Cell Mol Life Sci,2002,59(2):241-257. [7] Subcommittee for Scleroderma Criteria of the American Rheumatism Association Diagnosticand Therapeutic Criteria Committee,Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum,1980,23: 581–590. [8] Cron, R.Q. CD154 transcriptional regulation in primary human CD4 T cells. Immunol Res, 2003,27(2-3):185-202. [9] Banchereau J, Bazan F, Blanchard D, et al. The CD40 antigen and its ligand. Annu Rev Immunol, 1994,12:881-922. [10] Toubi E,Shoenfeld Y.The Role of CD40-CD154 inter-actions in autoimmuity and the benefit of disrupting this pathway. Autoimmunity,2004,37(6-7):457-467. [11] Fukasawa C, Kawaguchi Y, Harigai M,et al. Increased CD40 expression in skin fibroblasts from patients with systemic sclerosis (SSc): role of CD40-CD154 in the phenotype of SSc fibroblasts. Eur J Immunol,2003,33(10):2792-2800. [12] Furner IJ, Matzke M.Methylation and demethylation of the Arabidopsis genome. Curr Opin Plant Biol,2011,14(2):137-141. [13] Lyon MF. Gene action in the X-chromosome of the mouse (mus musculus L.).Nature,1961, 190:372-373. [14] Singer-Sam J, Riggs AD. X chromosome inactivation and DNA methylation. EXS, 1993, 64:358-384. [15] Kiedrowski LA, Raca G, Laffin JJ,et,al. DNA Methylation Assay for X-Chromosome Inactivation in Female Human iPS Cells. Stem Cell Rev, 2011, [Epub ahead of print]. [16] Lu Q, Wu A, Tesmer L, et al. Demethylation of CD40LG on the inactive X in T cells from women with lupus. J Immunol, 2007,179(9):6352-6358.