Peroxidasin信号通路在先天性白内障发病中的分子机制研究

Mutations in Peroxidasin (PXDN) cause severe anterior segment dysgenesis in humans, including congenital cataract, corneal opacity, and developmental glaucoma. Within an ENU (N-ethyl-N-nitrosourea) - mutagenesis screen, we identified the first mammalian model - ENU-induced PXDN mouse mutant line. We mapped the mutation to chromosome 12 within a critical interval of 2.4 Mb and sequence analysis of cDNA revealed a T3816A mutation resulting in a premature stop codon (Cys1272X) in the peroxidase domain. This mutation causes severe anterior segment dysgenesis and microphthalmia during eye development mimicing the manifestations in patients with PXDN mutations. Peroxidasin is highly expressed in the lens epithelial cells and inner retina. Aberrant lens development was firstly found in the mutants during eye development. Further, the proliferation and differentiation of the lens is disrupted in association with aberrant expression of transcription factor genes (Pax6 and Foxe3) in mutants at different embryonic periods. Additionally, PXDN is involved in the lens epithelium adhesion and consolidation of the basement membrane. However, The action of peroxidasin during lens development remains unclear. This study aims to investigate the molecular mechanisms of PXDN during lens development using site-direct mutagenesis、RNA sliencing, microarray, real-time PCR and western blot. The proposed work holds promise to provide insight into the pathogenesis of congenital cataract and establish peroxidasin as a potential therapeutic target.

Peroxidasin(PXDN)基因突变导致人类先天性白内障、角膜混浊和发育性青光眼。申请人与合作者构建并鉴定首个PXDN基因突变小鼠疾病模型，其眼部表型与人类相似，点突变位于过氧化物酶功能域(T3816A)，并发现PXDN在晶状体上皮细胞表达，晶状体发育异常产生的先天性白内障是造成眼前段发育异常的首发因素。进一步实验显示，PXDN基因突变影响晶状体上皮细胞的增殖和分化以及晶状体囊膜的稳定，并影响眼发育重要基因Pax6和Foxe3的表达。那么，PXDN如何调控晶状体上皮细胞的增殖和分化？该过程涉及哪些信号分子及其转导通路？本研究拟进一步采用定点突变技术、子宫内胚胎电转、RNA干扰、Real-time PCR和Western blot等方法研究PXDN调控晶状体上皮细胞生物学活性的分子基础，阐明PXDN突变产生先天性白内障的分子机制，为PXDN作为先天性白内障治疗的靶点提供充分的科学依据。

Peroxidasin基因突变导致先天性白内障、角膜混浊和发育性青光眼，但其发病机制仍不清楚。本研究构建了PXDN条件性敲除小鼠、PXDN基因LRR功能域缺陷小鼠及PXDN晶状体条件性过表达转基因小鼠，其中PXDN条件性敲除小鼠后续将与晶状体组织特异性cre小鼠杂交获得PXDN晶状体条件性敲除小鼠，对过表达PXDN的转基因小鼠，我们首先克隆PXDN全长cDNA片段，将其启动子换为晶状体特异性启动子，联合 PiggyBAC转座酶系统技术，制作晶状体条件性过表达转基因首建鼠并筛选到一个具有明显表型小鼠系，发现在在胚胎期出现原始玻璃体增生综合症，继而成年眼球出现白内障、眼前段发育异常及牛眼（眼球增大）的表型；同时，我们利用CRISPR/Cas9技术，选择缺失掉第3个和第4个亮氨酸重复结构域，构建LRR功能缺失的敲除小鼠，发现LRR功能域缺陷纯合子小鼠胚胎期晶状体前囊膜及晶状体上皮细胞破裂、晶状体纤维细胞及基质进入前房，继而10周龄小鼠的眼球外观上出现眼前段发育异常（角膜混浊、前房消失、晶体变小混浊）及小眼球。应用晶状体上皮细胞系及免疫共沉淀方法发现CARM1为PXDN新的相互作用蛋白。本项目揭示了PXDN在先天性白内障和晶状体发育中的部分机理，为后续深入和系统的研究PXDN的功能作用和分子机制提供了疾病动物模型和新思路。