Tet1调控CD40L、CD70基因启动子区域低甲基化的机制及在系统性硬皮病发病中的作用

Systemic sclerosis (SSc) is an autoimmune disease that involves multiple systems characterized by autoantibody formation, vascular obliteration, excessive extracellular matrix deposition.The pathogenic mechanisms remain incompletely understood. Abnormal activation of T cells may play a central role in the pathogenesis of SSc. In our previous studies, we demonstrated that DNA demethylation of T cell results in overexpression of CD40L and CD70 genes from patients with SSc. Overexpression of these genes results in T cell autoreactivity, B cell immunoglobulin overproduction and fibroblast's collagen synthesis. These may contribute to pathogenic mechanisms in SSc. The molecular basis of DNA demethylation in SSc T cells are still unclear. Rencently, Many studies indicate that the human Tet(Ten-eleven translocation) protein family could catalyse the conversion of 5-methylcytosine(5mC) of DNA to 5-hydroxymethylcytosine (5hmC) raising the possibility that DNA demethylation may be a Tet-mediated process. Because of its demethylase function, Tet protein family play a key role in a variety of biological processes and have attracted much attention. In our previous preliminary study, elevated Tet1 mRNA and protein expression were observed in CD4+ T cells from SSc patients.The average level of methylation of the CD40L and CD70 promoter regions was significantly reduced in CD4(+)T cells transfected with pcDNA3.1-Tet1 as compared with blank plasmid-transfected controls.These results suggest that hypomethylation of CD40L and CD70 genes regulatory regions may be related to Tet1 upregulation in SSc patients. On the basis of preliminary experiments,this study is intended to explore Tet1 regulatory function of CD40L and CD70 gene methylation and its molecular mechanism. Furthermore, to reveal the role of upregulation of Tet1 protein contributing to the development of autoimmunity by promoting DNA demethylation in SSc CD4 +T cells.It will be possible to descover the key aspects and molecular mechanisms of pathological hypomethylation T cells and autoimmune reactions of SSc patients. These studies provide new insights into the mechanisms causing DNA demethylation in SSc T cells, and suggest novel approaches for the treatment of SSc and may be a target for more effective SSc treatment.

系统性硬皮病(SSc)是一种多器官、多系统受累的自身免疫性疾病，其发病机制至今尚未完全明了。异常活化的T淋巴细胞被认为在该病的发病中起着关键作用。本课题组研究证实，CD4+T细胞DNA低甲基化导致CD40L、CD70等基因过度表达是引起其异常活化及SSc发病的重要机制之一，然而，其低甲基化的机制并不清楚。近年来，Tet蛋白家族因具有去甲基化酶的功能而倍受关注。我们的初步研究发现，Tet1在SSc患者CD4+T细胞中表达水平明显增高; 在转染Tet1质粒的正常人CD4+T细胞中，上述基因启动子区域则发生了低甲基化。提示 SSc患者该细胞DNA调控区域低甲基化可能与Tet1表达上调有关。本课题拟在上述基础上，探讨Tet1对CD40L和CD70基因的调控作用及机制, 进而探讨其在SSc发生、发展中的作用。有可能揭示该病中T细胞病理性低甲基化的分子机制，并有望为SSc的治疗提供新的思路和途径。

系统性硬皮病（SSc）是以皮肤以及内脏器官结缔组织纤维化、硬化和萎缩为主要表现的自身免疫病。其具体发病机制尚不明了。异常活化的T细胞被认为在该病的发病中起关键作用。CD4+T 细胞 DNA 低甲基化导致 CD40L、CD70 等基因过度表达是引起其异常活化 及 SSc 发病的重要机制之一，然而，其低甲基化的机制并不清楚。Tet 蛋白家族因具有去甲基化酶的功能而倍受关注。本课题以此作为研究背景，探讨 Tet1 对 CD40L 和 CD70 基因的调控作用及机制, 进而探讨其在 SSc 发生、发展中的作用。.本课题主要研究内容包括：（1）Tet1 在 CD40L、CD70 基因启动子区域的结合水平，相同区域内 5hmC 的富集水平。（2）干预 Tet1 对 CD40L 和 CD70 基因表达、其启动子区域甲基化水平的影响。（3）Tet1 对 CD40L 和 CD70 基因启动子区域低甲基化的调控机制。（4）过表达 Tet1 对正常人 CD4+T 细胞功能的影响。（5）抑制 Tet1 表达对 SSc 患者 CD4+T 细胞自身反应性的逆转作用。.通过这些研究，我们发现：（1）SSc患者CD4+T细胞中Tet1表达显著升高及整体基因组DNA羟甲基化水平显著增高,且与疾病的活动度相关。（2）抑制SSc患者CD4+T细胞中Tet1的表达,可引起整体基因组DNA羟甲基化水平降低,自身免疫密切相关的甲基化敏感基因CD40L、CD70启动子羟甲基化水平降低,下调这两个基因的表达、抑制自身免疫反应。（3）在正常人CD4+T细胞中过表达Tet1同样可以使CD40L、CD70启动子区域羟甲基化水平升高，并使它们的表达上调。上述结果表明SSc患者中Tet1在CD40L、CD70 等基因过度表达，CD4+T细胞异常活化的过程中有重要的作用。