组蛋白乙酰化修饰在ITP发病中的作用及机制研究

Immune thrombocytopenia (ITP) is an organ-specific autoimmune disease characterized by decreased platelet count. The exact etiology of ITP is still unclear. Th1 polarization of the immune response, up-regulation of Th17 cells and decreased number of Treg cells have been demonstrated in ITP patients. It is known that epigenetic changes include three systems: DNA methylation, RNA-associated silencing such as microRNA (miRNA), and histone modifications. It had been reported that the abnormal DNA methylation, dysregulation of miRNA and abnormal histone methylation were involved in the development of ITP. The epigenetic mechanisms of histone acetylation in ITP still remain unknown. In preliminary experiment, the results show that the global histone H3 and H4 hypoacetylation was observed in active ITP CD4+ T cells compared with controls, indicating that the abnormal histone acetylation play a vital role in the pathogenesis of ITP. The level of global histone acetylation was controlled by the balance of histone acetylases (HATs) and histone deacetylase (HDACs). HDAC inhibitors not only can reverse the skewed expression of multiple genes involved in autoimmune diseases and but also can improve their clinical symptoms. Therefore, it is necessary to screen and identify the specific HATs and/or HDACs to explain why global histone H3 and H4 were in hypoacetylation. Regulatory T cell (Treg), characterized by expression of CD4, CD25, and transcription factor forkhead box P3, is a subpopulation of CD4+T cells specialized for immune suppression. Recently, a series of studies in ITP patient have found that the frequency of Tregs is reduced and Treg function is impaired. It is worth emphasizing that, in this study, it was found that the H3 acetylation at the Foxp3 gene promoter region (-504/-416) was lower in CD4+ T cells from ITP patients compared with normal controls. It is important to clarify the mechanism of why the Foxp3 is in hypoacetylation, and whether this abnormality is related to the dysregulation of Treg in ITP patients. Glucocorticoid and rituximab have also been shown to return the ratio of Th1/Th2 and the number of Th17 and Treg cells to the normal levels in ITP patients. It was speculated that the HDACi such as SAHA and TSA also can treat the ITP patients by correcting the Th1/Th2 and Treg/Th17 balance, which need to be verified. In conclusion, the aim of this study was to investigate the implications of histone acetylation alterations in the development of ITP, and to provide the theory basis for therapeutic mechanism of SAHA/TSA in ITP patient.

免疫性血小板减少症（ITP）是一种器官特异性的自身免疫性疾病，至今发病机制尚未阐明。前期研究发现：ITP患者外周血CD4+T细胞整体组蛋白H3/H4处于低乙酰化状态和脾脏调节性T细胞（Treg）Foxp3启动子区域组蛋白H3呈低乙酰化水平，表明组蛋白乙酰化修饰的异常参与了ITP的发病，为后期研究提供重要思路。首先，筛选和锁定导致ITP整体组蛋白处于低乙酰化状态的关键基因和乙酰化修饰酶（HATs和HDACs）；其次，研究Foxp3乙酰化修饰变化的分子机制以揭示ITP患者 Treg细胞数量降低和功能缺陷的原因；最后，从调节Th1/Th2和Th17/Treg平衡的角度探讨组蛋白去乙酰化抑制剂（SAHA/TSA）治疗ITP的作用及机制。本课题将从组蛋白乙酰化修饰的全新角度，阐明表观遗传学在ITP发病机制中的作用，为探索新的可能有效治疗ITP的药物SAHA/TSA提供重要的理论依据。

免疫性血小板减少症（ITP）是一种器官特异性的自身免疫性疾病，至今发病机制尚未阐明。本项目进一步证实ITP患者整体组蛋白H3/H4的乙酰化水平均显著降低，ITP患者脾脏Treg细胞Foxp3启动子区域组蛋白H3乙酰化水平，结果显示：与对照组比较，ITP患者Foxp3启动子区域(-504/-416)组蛋白H3乙酰化水平显著降低，表明组蛋白乙酰化修饰的异常在ITP发病中起重要的作用。ITP患者中HDAC9表达高于健康对照（p<0.05），SIRT1、p300、TIP60在ITP患者及健康对照者中表达无显著差异。HDAC的活性在ITP患者Treg细胞中高于健康对照（p<0.01），Foxp3基因在TSA处理的Treg细胞中表达高于DMSO处理组（p<0.01）；CHIP 技术检测ITP患者Treg细胞Foxp3启动子区域组蛋白H3乙酰化水平，结果显示：与DMSO组比较比较，TSA组Foxp3启动子区域(-504/-416)组蛋白H3乙酰化水平显著升高（p<0.05）。Treg抑制实验表明 Treg细胞在ITP患者中抑制功能受损（p<0.01），TSA处理组Treg细胞抑制功能增强（p<0.01）。ITP患者外周血CD4+ T细胞整体组蛋白H3K9和H3K4甲基化水平进行初步研究，结果发现：活动性ITP患者CD4+ T细胞整体组蛋白H3K9处于低甲基化状态，表明组蛋白甲基化修饰的异常参与了ITP的发病。EZH2作为重要的表观遗传修饰物，主要对组蛋白H3K27进行甲基化，从而沉默下游基因。近年来的研究表明EZH2和Th细胞分化密切相关，推测EZH2同样在ITP发病中发挥重要的作用。通过免疫印迹法检测ITP患者CD4+T细胞EZH2表达，发现：与正常对照比较，ITP患者的EZH2明显高表达，表明EZH2参与了ITP的发病。ITP患者CD4+T细胞定向造血干细胞激酶1 (Hematopoietic progenitor kinase 1，HPK1)启动子区H3K27me3水平显著高于正常对照, 并通过染色质免疫沉淀(CHIP) 结合实时定量PCR进一步证实。本课题目前主要从组蛋白乙酰化和组蛋白甲基化的表观遗传学角度，对ITP发病机制从新的角度进行阐述，其结果可以作为下一个课题的前期工作基础，开展对ITP发病机制的深入研究。