化痰散结法对Graves病小鼠免疫耐受开关机制及其修饰的影响

Graves'disease(GD) is an organ-specific autoimmune disease, characterized by the reduction of the immune tolerance and the generation of TRAb by B cell after being activated by Th2 Cell, which leads to goiter and hypersecretion. GATA-3 is the cell-specific transcription factor of Th2 cell. Treg is critical to immune stability. Foxp3, which can regulate the normal differentiation and function of Treg, is an important switch of Treg cell development. DNA methylation and histone modification are the factors to control expression of Foxp3. GD belongs to thyromegaly (Ying Qi) and its basic treatment method is resolving phlegm and dissipating mass therapy. The previous studies proved that this treatment can inhibit thyroid cell proliferation, promote apoptosis, restore cell dynamics balance, inhibit thyroid hormone synthesis and upregulate Treg expression. This study takes the GD mouse models as the carrier, non-marine phlegm reduction drug as the object and the switching mechanism of immune tolerance and regulation as the key technical indicators. We can study the influence of the resolving phlegm and dissipating mass therapy on GD immune tolerance through determination of Th2 cell-specific transcription factor mRNA, protein expression and cytokine.We can explain how the method of resolving phlegm and dissipating mass therapy works to regulate the immune and switching function of Treg through the study on the function of Treg, Foxp3 and DNA methylation and histone modification.

弥漫性甲状腺肿伴甲亢（GD）是器官特异性自身免疫性疾病，关键是机体免疫耐受降低，Th2细胞活化B细胞产生TRAb，导致甲状腺肿大和分泌亢进。GATA-3是Th2细胞特异性转录因子，Treg是机体免疫稳定的基石，Foxp3能调控Treg正常分化和功能，是Treg细胞发育的重要开关，DNA甲基化和组蛋白修饰是调控Foxp3表达的关键。GD属“瘿气”，化痰散结法为基本治法，以往研究证实其可抑制甲状腺细胞增殖、促进凋亡，恢复细胞动力学平衡，抑制甲状腺激素合成并上调Treg表达。本研究以GD小鼠模型为载体，非海生化痰散结药物为对象，免疫耐受及调控的开关机制为关键技术指标；通过对Th2细胞特异性转录因子mRNA和蛋白表达及细胞因子测定，研究化痰散结法对GD免疫耐受的影响；通过对Treg功能、Foxp3及其DNA甲基化和组蛋白修饰的研究，阐释化痰散结法对Treg免疫及开关的调控作用。

免疫失常是GD发病的关键因素，产生过多的TRAb，刺激甲状腺滤泡细胞增生，造成甲状腺肿大和功能亢进。GD属中医“瘿气”病，化痰散结法为其基本治法。课题组前期通过3次国自然基金项目，成功构建了以Ad-TSHR289免疫制备的GD小鼠模型，证明化痰散结法可以调控甲状腺细胞增殖凋亡、激素合成和免疫失衡。本课题以化痰散结药物为对象，以GD小鼠模型为载体，围绕Th1/Th2平衡、Treg免疫抑制功能、foxp3甲基化修饰及hippo信号通路，研究化痰散结法调控GD免疫耐受开关及修饰的作用机理，并进一步探索中药对甲状腺氧化应激和血管新生的抑制作用。.研究表明：（1）优化后的GD造模方法由3次免疫减至1次，免疫后第4周即可成模，第13周成模率100%，并可维持至第18周。（2）化痰散结药物改善GD小鼠甲状腺肿大与功能亢进。给药6周后，与模型组比较，血清T4下降33.87%，TRAb下降56.52%；甲状腺肿大、充血减轻，滤泡增生及内凸减轻；细胞表面微绒毛减少、缩短，表明胞吞作用减弱。（3）化痰散结药物提高Treg细胞功能，纠正Th1/Th2失衡，从而改善GD小鼠免疫失常。与模型组比较，脾脏Th1/Th2比值降低；Th1/Th2特异性转录因子T-bet降低，GATA-3上升；血清细胞因子IFN-γ、IL-6等减少；Treg比例及Foxp3表达增加，Teff增殖抑制率升高；脾脏Treg细胞因子IL-10、TGF-β含量增加。（4）化痰散结药物可能通过调控上游hippo信号通路调节Foxp3表达。与模型组比较，在CpG岛7个位点的foxp3 DNA甲基化程度无明显变化，但Hippo信号通路的核心因子Mst1表达上调，YAP、TAZ表达下调。（5）化痰散结药物修复甲状腺抗氧化能力。与模型组比较，甲状腺氧化应激因子SOD、GSH-px、T-AOC等不同程度升高，可能是改善甲状腺形态的重要途径。（6）化痰散结药物可通过下调血管生成因子VEGFA和VEGFR2的表达降低GD小鼠甲状腺中微血管密度。.本研究建立了更优化的GD造模方法，为GD发病机制及药物研发提供技术支撑。阐释了化痰散结法治疗GD免疫失常的机制，及其调控甲状腺组织氧化应激和抑制血管新生的作用，为临床使用化痰散结法治疗GD改善免疫失常提供了现代科学依据，具有重要意义。