前期研究发现视网膜Müller细胞源性干细胞中存在关键神经发育基因缺陷。在此研究基础上，我们拟首先在体外对视网膜Müller细胞源性干细胞去甲基化酶FTO的定向敲除，采用生物基因组和RNA芯片数据分析，明确节细胞分化和轴突再生的机制和参与的调控网络。其次体内研究将视网膜Müller细胞源性干细胞FTO基因敲除后注入青光眼模型鼠玻璃体腔内，CTb-594和荧光金显示视神经3D图像分析轴突的数量、长度及分支；ERG、OCT及VEP检测其整合能力及视功能恢复情况；免疫荧光染色、qPCR和Western blot等方法观察节细胞的分化和轴突的长度、形态和数目以及通路中基因的表达变化，从而揭示Norrin甲基化修饰经Wnt/β-catenin途径诱导原神经基因ASCL1和Ngn2的序贯表达，调控视网膜Müller细胞向节细胞分化以及轴突再生的分子机制，为青光眼临床基因治疗奠定坚实的理论基础。

Based on previous studies that revealed defects in key neurodevelopmental genes in retinal Müller cell-derived stem cells, in vitro targeted knockout of demethylase FTO will be carried out in retinal Müller cell-derived stem cells, and bio-genome and RNA chip will be used for data analysis to clarify the mechanism and regulation network of retinal ganglion cells (RGCs) differentiation and axonal regeneration. Next, we plan on carrying out in vivo study, in which FTO gene knockout Müller cell-derived stem cells were injected into the vitreous cavity of glaucoma model mice. CTb-594 and Fluoro-Gold will be used to produce optic nerve 3D images for analyzing the number, length and branching direction of axons; ERG, OCT and VEP will be used as detection of integration ability and visual function recovery; immunofluorescence staining, qPCR and Western blot will be used to observe the differentiation of ganglion cells, the length, shape and number of axons, and the expression of genes involved in the pathway. Our project aims at revealing the molecular mechanism of Norrin methylation mediated Wnt/β-catenin pathway inducing sequential expression of the proneural gene ASCL1 and Ngn2 in regulating retinal Müller cell conversion into Retina Ganglion Cells and axonal regeneration, and hope to lay a solid theoretical foundation for clinical gene therapy of glaucoma.