角膜营养不良发病中Wnt通路的作用及机制研究

Corneal dystrophy (CD) is a common hereditary ocular disease, which always affects binoculars resulting in visual acuity impairment but without effective treatment. The disease is mostly autosomal dominant inheritance, most of which was caused by the mutation of TGFBI gene. However the mechanism by which these mutations cause corneal abnormal depositions remains unknown. This project aims to investigate the regulating effect of Wnt/β-catenin signal pathway as well as its target gene MMPs in TGFBI-related corneal dystrophies. The study includes following sections: measure the protein expression levels of Wnt signaling components (GSK3β, β-catenin）and the mRNA/protein expression levels of target gene MMPs (MMP1, MMP2, MMP7, MMP9, MMP13, MMP14) in the normal/corneal dystrophy cornea tissues as well as human corneal epithelium (HCE)/corneal fibroblasts which were overexpression wild-type or mutant-type TGFBIp. Using immunofluorescence assay to detect β-catenin location and intracellular distribution, as well as TOPFLASH report test for identify the state of Wnt/β-catenin signaling pathway. Measuring the different state of Wnt signaling pathway between wild and mutant cells after treated by the activator (SB216763) and inhibitor (DDK1). Verifing the interaction between TGFBIp and integrin by co immunoprecipitation and pull down technology.Through ILK activator CA and inhibitor KD to prove TGFBIp regulate Wnt pathway through ILK. The research will contribute to better understanding the important roles of Wnt/β-catenin signal pathway in the pathogenesis of TGFBI related CD and explore the potential target point for the therapy of CD。

角膜营养不良(CD)是一类常见的遗传性眼病，多为双眼发病，严重影响视力，目前尚无有效治疗方法。其多与TGFBI基因突变相关。然而突变致角膜异常物质沉积的机制仍不明确。本项目旨在研究Wnt信号通路及靶基因MMPs在TGFBI相关性CD中的作用机制。拟通过比较野生型和突变型细胞/角膜组织Wnt通路关键信号分子表达差异；β-catenin入核、通路活化水平（TOPFLASH试验）的异同；以及Wnt通路激活剂及抑制剂作用下Wnt通路活化状态差异，来阐明突变蛋白抑制Wnt信号通路为TGFBI相关性CD的发病机制之一；通过免疫共沉淀和pull down技术验证TGFBIp与整合素的相互作用；并通过ILK激活剂CA和抑制剂KD来验证TGFBIp通过ILK调控Wnt通路。本研究是Wnt信号通路在TGFBI相关性CD的首次研究，为该病发病机制研究提供新线索，为早期干预及治疗提供潜在靶点。

角膜营养不良(CD)是一类常见的遗传性眼病，多与TGFBI基因突变相关，然而突变致角膜异常物质沉积的机制仍不明确。本项目通过研究野生型和突变型细胞中Wnt通路关键信号分子的表达、β-catenin入核、通路活化水平（TOPFLASH试验）的异同来阐明突变蛋白对Wnt通路的影响；并通过CO-IP技术研究TGFBIp与integrin的相互作用。本研究结果显示R124H、R555W突变使角膜上皮细胞中Wnt通路活性下降，β-catenin蛋白的核稳定性下降，靶基因MMP1、MMP14的mRNA 表达量下降，但它们的蛋白表达量并未出现明显的改变。本研究初步探索了TGFBI 相关性角膜营养不良发病中Wnt通路的作用及机制，为以后角膜营养不良发病机制的进一步研究提供理论依据。