前期研究发现视网膜Müller细胞分化的神经节细胞轴突再生能力有限。在此研究基础上，我们拟在体外通过CRISPR-cas9系统定向敲除鼠视网膜Müller细胞源性干细胞中PTEN/SOCS3基因，诱导轴突生长相关基因的表达，促使轴突生长，通过RNA测序筛选轴突生长差异基因，并确定调控轴突生长的机制及参与的调控网络。其次体内研究将PTEN/SOCS3基因共缺失的视网膜Müller细胞源性干细胞注入青光眼模型鼠玻璃体腔内，CTb-594显示视神经3D图像分析轴突的数量、长度及走向；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等方法观察轴突的长度、形态和数目变化以及通路中基因的表达，从而揭示PTEN/SOCS3共缺失调控视网膜Müller细胞分化的神经节细胞轴突导向生长的分子机制，为青光眼临床基因治疗奠定坚实的理论基础。

Our previous studies have shown that the retinal Müller cells differentiated ganglion cell axon has limited regeneration capacity. Based on those studies, we will knock out PTEN/SOCS3 genes in murine retinal Müller cell-derived stem cells using the CRISPR-cas9 system in vitro, induce axon growth-related genes to promote axon growth, perform bioinformatics and gene cluster analysis by RNA sequencing, screen for genes related to axonal growth difference, and find out the mechanism of PTEN/SOCS3 co-deletion in regulating axonal growth and its related cell signaling network. Secondly, in vivo studies, we will inject retinal Müller cell-derived stem cells by co-deletion of PTEN/SOCS3 into the vitreous cavity of mice with glaucoma. CTb-594 will be used to show the number, length and shape of the optic nerve axons by 3D imaging. Electroretinogram, high resolution OCT and visual evoked potentials will be used to detect the ability of integration and recovery of visual function. Immunofluorescence staining, qPCR and Western blot will be used to observe the changes of axonal length, shape and number, and changes in related gene expression. Finally, TUNEL technique will be used to analyze the apoptosis of retinal cells, thus revealing the molecular mechanism of PTEN/SOCS3 co-deletion in regulating Müller cell differentiated ganglion cells axon guidance and growth, and laying a solid theoretical foundation for clinical gene therapy of glaucoma.