自噬调控成纤维细胞功能保护甲状腺相关眼病效应与机制研究

The dysregulation of orbital fibroblasts are the key etiology of thyroid associated ophthalmopathy（TAO）.Autophgy is indispensible for differentiation, development and response to stress for cell and organ homeostasis. Recently autophagy is related with fibrosis of retal, lung and liver. We speculate that autophagy is also involved inoveractivation of fibroblast of TAO. We found that the key the expression of LC-3B was decreased in TAO tissue and active form of LC-3B (LC-3BII) was also decreased in primary fibroblast from TAO tissue, which indicates that autophagy is involved in TAO etiology. The proposal plan to increase the collection of TAO samples, prove the relation between autophagy and TAO, investigate the regulation of fibroblast by autophagy, identify the key autophagy checkpoint in dysregulation of fibroblast, evaluate the therapeutic function of the key autophagy checkpoint inhibitors in mice TAO model. The accomplishment of proposal will outline the new clue and target for the etiology mechanism and therapy of TAO.

眼眶成纤维细胞功能异常是甲状腺相关眼病（thyroid associated ophthalmopathy ，TAO）关键致病环节。自噬是维持细胞正常分化、发育的保护机制。新近研究发现自噬参与成纤维细胞过度活化相关的风湿性关节炎、肺纤维化和肝脏纤维化。我们推测自噬也参与调控TAO发生发展过程中成纤维细胞的活化。我们的预实验发现：自噬关键分子LC-3B及其活化形式LC-3BII在TAO患者的临床样本及分离的原代成纤维细胞中明显降低，提示TAO患者局部自噬受到抑制可能与成纤维细胞过度活化有关。本课题组拟进一步扩大收集TAO患者临床样本，统计和实验分析自噬在TAO患者局部受到抑制的现象，及其对成纤维细胞炎症功能的影响，研究自噬抑制的关键信号机制和应用自噬诱导剂对TAO小鼠模型的治疗效果。为进一步研究TAO发病机制和临床治疗TAO提供线索。

甲状腺相关眼病（Ththyroid-associated orbitopathy,TAO ）是由于眼眶组织内脂肪细胞和成纤维细胞过度活化与增生导致的炎症反应性疾病。而自噬是否参与脂肪细胞与成纤维细胞的过度活化增生没有研究报道。.本项目发现了自噬可以抑制TSHR诱导的成纤维细胞和脂肪细胞炎症性活化的效应。简述如下：.临床样本水平分析，WesternBlot实验发现在急性期TAO患者中自噬流关键蛋白p62表达水平很高，在正常眼眶组织和稳定期TAO患者中表达水平则很低，说明TAO急性期眼眶脂肪组织中自噬受到抑制；免疫组化和WesternBlot发现NF-kB关键转录因子p65的磷酸化状态在正常脂肪组织很低，而在急性期与稳定期脂肪组织中很高，说明p65的活化与TAO的发生发展紧密相关。免疫组化和免疫荧光证实，p62在TAO急性期眼眶脂肪细胞表达水平很高，进一步说明自噬可能通过抑制NF-kB关键转录因子p65活化，抑制TAO发病。.细胞水平分析，急性期TAO患者血浆（含有大量THSR活化型抗体）刺激原代脂肪细胞，ESLIA与Q-PCR检测CD40或IFNr诱导脂肪细胞产生炎症因子（TNF-a和IL-6）检测活化显著增强。WesternBlot检测p62表达明显增加，LC3剪切明显减弱，NF-kB关键转录因子p65活化增强，提示THSR抑制自噬，促进NF-kB活化。.急性期TAO患者血浆（含有大量THSR活化型抗体）刺激原代成纤维细胞，ESLIA与Q-PCR检测CD40或IFNr诱导成纤维细胞产生炎症因子（TNF-a和IL-6）检测活化显著增强。WesternBlot检测p62表达明显增加，LC3剪切明显减弱，NF-kB关键转录因子p65活化增强，提示THSR抑制自噬，促进NF-kB活化。.成纤维细胞的分化指标（α-SMA表达，MMP9产生）明显增加。