淋巴细胞和甲状腺细胞的生物钟及bmal1基因调控自身免疫甲状腺炎发病的机制研究

Autoimmune thyroiditis (AIT) represents a high prevalence and a variety of adverse effects on health. Its characteristic changes include immunocyte imbalance and thyrocyte damage, for which pathogenetic mechanisms remain unclear. Recent lines of evidence have highlighted the contribution of circadian clock and core clock gene bmal1 to the function of the immune system. Our preliminary studies also indicated that the circadian clock and bmal1 gene inside cells may become a novel explanation for pathogenesis of AIT. Therefore, we intend to apply AIT patients, AIT mouse model, Bmal1(Cd4-/-) and Bmal1(Thyroid-/-) transgenic mice to a series of studies. Aims of the present project include: 1) To demonstrate the presentation of circadian clock and bmal1 gene expression in both T lymphocyte and thyrocyte in AIT patients and mouse model. 2) To explore whether circadian clock and bmal1 gene may regulate expression levels and/or daily oscillations in transcription factors and cytokines, leading to imbalance of T cell differentiation and function, as well as immune microenvironment in thyroid. 3) To investigate whether circadian clock and bmal1 may regulate the amount and/or rhythm of autoantigens, endogenous proinflammatory ligands and apoptosis produced by thyrocytes, subsequently engendering thyrocytes more vulnerable to the immune attack. 4) To study whether outcomes caused by alterations in circadian clock and bmal1 gene be in accord with characteristic changes in AIT, and be related to the severity of AIT. Results of the project are supposed to enrich our understanding in mechanisms of development of AIT and, from the perspective of chrono-immunology, deliver a novel and promising data for potential application.

自身免疫甲状腺炎(AIT)是患病率高、危害广的内分泌系统疾病，其特征性改变包括免疫细胞失衡和甲状腺细胞损伤，但具体发生机制尚未明晰。近年的研究表明生物钟和核心钟基因bmal1在调控机体免疫反应过程中充当重要角色，我们的前期工作也提示它们是AIT机制探索的新切入点。本项目拟以临床病例、AIT动物模型、T细胞/甲状腺bmal1敲除鼠等为研究对象，阐明AIT中淋巴细胞和甲状腺的生物钟和bmal1的表达特点；探讨它们能否通过调控相关转录因子和细胞因子等的表达水平和/或节律，造成T细胞的分化、功能失衡和产生甲状腺炎症微环境；研究它们能否通过调控甲状腺细胞自身抗原、内源性促炎症配体和细胞凋亡的水平和/或节律，导致甲状腺细胞在炎症微环境中更易损伤；分析它们调控的结果是否符合AIT特征性改变并与疾病严重程度相关。此研究将利于丰富对AIT免疫机制的认识，创新性地从时间免疫学角度提出AIT的防治靶点和策略。

自身免疫甲状腺炎(AIT)是患病率高、危害面广的内分泌疾病。其致病性免疫应答异常的具体机制尚未明晰。免疫细胞和甲状腺细胞的生物钟和核心钟基因Bmal1是研究AIT的新切入点。本课题中我们重点探讨了下述内容：AIT患者和动物模型中的生物钟现象和核心钟基因Bmal1的表达特点；实验动物在体实验论证生物钟紊乱与AIT炎症反应的相互作用；转基因动物模型论证甲状腺生物钟紊乱在AIT中的作用。研究结果显示：1) AIT患者甲状腺组织中Bmal1和Per2的表达水平显著降减少，并与甲状腺自身抗体、细胞因子和甲状腺组织促炎基因表达呈负相关。2) 在pTg诱导的AIT模型小鼠中，AIT严重程度无明显节律性变化；小鼠淋巴细胞亚群、脾脏和甲状腺组织生物钟基因存在节律性表达，并且在AIT小鼠中发生改变。3）光照紊乱未诱发AIT，但可加剧AIT小鼠的炎症反应。4）甲状腺细胞特异性Bmal1敲除可导致甲状腺局部生物钟紊乱，但敲除鼠未发生AIT；无论是否敲除甲状腺Bmal1，小鼠对pTg的免疫反应均在ZT6时强于ZT18；敲除细胞甲状腺Bmal1可引起AIT小鼠TgAb水平增高，炎症性细胞因子表达增加，加剧甲状腺损伤；这一过程与Bmal1敲除影响甲状腺的局部免疫微环境、促进甲状腺细胞与淋巴细胞间的自身免疫反应及导致CD4T细胞的免疫通路改变和亚群分布异常有关。本项目的科学意义在于揭示了生物钟核心钟基因在AIT中的表达和功能。生物钟紊乱并非AIT的始动因素，但可能影响AIT的疾病进展。以Bmal1基因为主导的甲状腺局部生物钟稳态有望为AIT提供新的治疗靶点，从而具有良好的临床应用前景。