【作者】 黄雪；

【导师】 杨培增；

【作者基本信息】 郑州大学 ， 眼科学， 2023， 硕士

【摘要】 背景自身免疫性葡萄膜炎是一种慢性非感染性眼内炎性疾病,是导致视力损害甚至失明的主要病因之一。葡萄膜炎眼内存在M1型小胶质细胞的广泛浸润,和多种炎症因子的释放。鸢尾素（Irisin）是一种由运动诱导、Fndc5基因转录翻译而成的肌细胞因子,在多种疾病中发挥免疫调节功能。目的本研究旨在探究葡萄膜炎患者房水中鸢尾素水平与炎症细胞因子的相关性,并通过实验性自身免疫性葡萄膜炎（EAU）模型和体外细胞分子实验揭示鸢尾素对葡萄膜炎的作用机制。方法用ELISA法检测白塞病和Vogt-小柳原田综合征葡萄膜炎患者房水样本中鸢尾素、IL-1β、TNF-α的水平,并进行相关性分析。分别对Fndc5^-/-小鼠、野生型小鼠、腹腔注射重组鸢尾素或无菌生理盐水组小鼠,通过皮下注射光受体维甲酸结合蛋白(IRBP_651-670)诱导实验性自身免疫性葡萄膜炎（EAU）。免疫后第14天行眼部表现和病理切片评分,RT-q PCR检测视网膜炎症基因m RNA相对表达量,流式细胞术分析脾脏来源单个核细胞中Th1、Th17、Treg细胞比例,免疫荧光染色标记视网膜小胶质细胞,Western Blot检测视网膜蛋白中IBA-1、M1极化标志蛋白和M2极化标志蛋白水平。体外实验用LPS和IFN-γ刺激人小胶质细胞系（HMC3）,实验组用重组鸢尾素处理,对照组给予等量磷酸缓冲盐溶液（PBS）。Western Blot检测两组小胶质细胞系中M1和M2极化状态标志蛋白水平。结果葡萄膜炎患者房水中鸢尾素水平减少,且在白塞病葡萄膜炎患者房水中鸢尾素与TNF-α存在负相关关系。与对照组相比,腹腔注射鸢尾素处理组EAU小鼠临床评分和组织病理学评分显著降低,视网膜IFN-γ、IL-17A、TNF-α的m RNA相对表达量显著减少,IL-10 m RNA相对表达量显著增多,脾脏来源单个核细胞中,Th1、Th17细胞比例显著降低、Treg细胞比例显著升高。视网膜中M1极化标志蛋白水平显著减少,而M2极化标志蛋白显著增多。与野生型小鼠相比,Fn dc5^-/-EAU小鼠临床评分和组织病理学评分显著升高,视网膜IFN-γ、IL-17A、T NF-α的m RNA相对表达量显著增多,IL-10 m RNA相对表达量显著减少,脾脏来源单个核细胞中Th1、Th17细胞比例显著升高、Treg细胞比例显著降低,视网膜小胶质细胞数量增多且视网膜中IBA1蛋白水平增多,M1极化标志蛋白水平显著增多,而M2极化标志蛋白水平显著减少。在人小胶质细胞系（HMC3）中,与对照组相比,鸢尾素处理组M1极化标志蛋白水平显著减少,而M2极化标志蛋白水平显著增多。结论葡萄膜炎患者房水中鸢尾素含量减少,鸢尾素通过诱导视网膜小胶质细胞M1/M2极化缓解实验性自身免疫性葡萄膜炎。更多还原

【Abstract】 BackgroundAutoimmune uveitis is a chronic non-infectious intraocular inflammatory disease that is one of the leading causes of visual impairment and even blindness.Extensive infiltration of M1-type microglia and the release of multiple inflammatory factors are present in uveitis eyes.Irisin is a myocytokine induced by movement and transcribed and translated from the Fndc5 gene,which plays an immunomodulatory function in a variety of diseases.PurposeThe aim of this study was to investigate the correlation between irisin levels in aqueous humor and inflammatory cytokines in patients with uveitis,and to reveal the mechanism of irisin action on uveitis through experimental autoimmune uveitis（EAU）model and in vitro cellular and molecular experiments.MethodsThe levels of irisin,IL-1β,and TNF-αin aqueous humor samples from patients with leukoaraiosis and Vogt-Koyanagi Harada syndrome uveitis were measured by ELISA and correlation analysis was performed.Experimental autoimmune uveitis（EAU）was induced by subcutaneous injection of Interphotoreceptor retinoid binding protein（IRBP651-670）in Fndc5^-/-mice,wild-type mice,and mice in the intraperitoneal injection of recombinant irisin or stroke-physiological saline solution groups,respectively.Ocular manifestations and pathological section scoring were performed on day 14 after immunization.RT-q PCR was performed to detect the relative expression of retinal inflammatory gene m RNA,flow cytometry was performed to analyze the proportion of Th1,Th17,and Treg cells in splenic-derived mononuclear cells,immunofluorescence staining was performed to label retinal microglia,and Western Blot was performed to detect IBA-1,M1polarization marker protein and M2 polarization marker protein levels in retinal proteins.Human microglia cell line（HMC3）was stimulated with LPS and IFN-γin vitro.The experimental group was treated with recombinant irisin and the control group was treated with phosphoric acid buffer salt solution（PBS）.The levels of M1and M2 polarization marker protein in microglia cell lines were detected by Western Blot.ResultsThe level of irisin in aqueous humor decreased in patients with uveitis,and there was a negative correlation between irisin and TNF-αin patients with Behset’s disease uveitis.Compared with the control group,the clinical scores and histopathological scores of EAU mice in the intraperitoneal injection of irisin treatment group were significantly decreased,the m RNA relative expression levels of IFN-γ,IL-17A and TNF-αin retina were significantly decreased,and the m RNA relative expression levels of IL-10 were significantly increased.The proportion of Th1 and Th17 cells decreased significantly,while the proportion of Treg cells increased significantly.The level of M1 polarization marker protein in retina decreased significantly,while M2 polarization marker protein increased significantly.Compared with wild-type mice,the clinical scores and histopathological scores of Fndc5^-/-EAU mice were significantly increased,and the m RNA relative expression levels of IFN-γ,IL-17A and TNF-αin retina were significantly increased,while the m RNA relative expression levels of IL-10 were significantly decreased.The percentage of Th1 and Th17 cells in splenic-derived mononuclear cells were significantly increased and the percentage of Treg cells was significantly decreased,the number of retinal microglia and the level of IBA1 protein in the retina were increased,the level of M1 polarization marker protein was significantly increased,and the level of M2 polarization marker protein was significantly decreased.In human microglia cell line（HMC3）,compared with the control group,the level of M1polarization marker protein in irisin treatment group was significantly decreased,while the level of M2 polarization marker protein was significantly increased.ConclusionIrisin levels in aqueous humor are reduced in patients with uveitis,and irisin alleviates experimental autoimmune uveitis by inducing M1/M2 polarization in retinal microglia.更多还原