以neuritin为靶点修复大鼠视神经损伤的作用及机制

Low intrinsic regeneration ability of RGC is critical for optic nerve difficult to regenerate. Recent reports suggest that enhancing intrinsic regeneration ability of RGC by targeting to special molecules induce sustained and robust regeneration in the injured optic neve of mouse. Neuritin is a crucial, commom downstream molecule through which NTF play their neurotrophic role. It is also a necessary, crucial molecule through which testosterone and electrical stimulation potentiate nerve regeneration. The expression of neuritin is abundant in the visual nervous system, which imply that neuritin may be a crucial NTF in regulating intrinsic regeneration ability of RGC. Our previous study indicated that neuritin be significantly down-regulated in the optic neve and retina during early phase after rat optic neve injury. It is so far not reported that neuritin is given to treat optic nerve injury..We will display the changes of spatial and temporal expression patterns in the visual nervous system after crush of rat optic nerve. Then, vitreal injection of neuritin or neuritin adeno-associated viral vector will immediately be performed after optic nerve to increase neuritin content in the visual nervous system. To explore effects of the targeting neuritin on repairing the injured rat optic nerve and their mechanisms, retina culture in vitro, morphology, molecular biology, functional evaluation and so on will be used to search optic neve regeneration, survival and gene expressions involved of RGC and optic neve glia, functional restoration. At the same time, the effects and mechanisms will be studied in the injured RGC5 cell line in vitro. The results will provide a new treatment and its rationale for repairing human optic neve and central nervous system injury.

RGC内在再生能力低下是视神经伤后难再生的关键。最近研究表明找准靶点增强RGC内在再生能力，受损视神经能持续、活跃地再生。Neuritin是众多NTF发挥神经营养作用的关键和共同下游因子，为介导雄激素和电刺激促进神经再生的必须和关键分子，在视觉系统高表达，提示其为影响RGC内在再生能力的关键NTF。我们预试验发现视神经伤后早期的视神经和视网膜中Neuritin表达均显著下调。未见用Neuritin治疗视神经损伤的研究报道。本课题拟首先研究大鼠视神经压榨伤后视觉系统Neuritin基因表达的时空变化；伤后即玻璃体注射Neuritin或Neuritin基因重组慢病毒，用视网膜体外培养、形态学、分子生物学和功能检测等方法，观察视神经再生、RGC存活、基因表达和功能恢复等，结合对体外受损RGC5的相应研究，研究Neuritin修复视神经伤的作用及机制，为治疗视神经及中枢神经伤提供新手段和理论依据。

受损中枢神经包括视神经难以再生，关键是中枢神经神经元内在再生能力低下。Neuritin（Nrt）是神经细胞内神经营养因子发挥作用的关键的、共同下游因子及雄激素、电刺激促进神经再生的必须分子。本项目聚焦于增强视网膜神经节细胞（RGC）内在再生能力，从视网膜Nrt过表达入手，治疗受损视神经并探讨相关作用机制。首先观察到Nrt蛋白表达于大鼠视网膜RGC及部分星形胶质细胞，视神经RGC轴突、少突胶质细胞及血管内皮细胞。大鼠视神经不完全损伤后1 d，视网膜及视神经中Nrt mRNA和蛋白即早高表达，其后下调至或低于正常水平，提示为视神经再生失败原因之一及添加Nrt予以治疗的必要性。在体外用星状孢子碱诱导RGC细胞系RGC5为成熟RGC样细胞，以丙烯酰胺损伤RGC5模拟轴突切断。RGC5细胞表达Nrt蛋白，诱导后表达上调，受损后表达下调。低剂量Nrt促进受损RGC5的存活，且强于CNTF组。Nrt慢病毒载体转染约90%RGC5细胞，Nrt mRNA表达上调约14倍。Nrt蛋白和Nrt过表达可下调受损RGC5活化型Caspase 3的表达并减轻其凋亡，可促进受损RGC5突起再生，且作用强于CNTF组；Nrt过表达上调受损RGC5中pAKT、pERK的表达，通过活化AKT、ERK通路发挥其神经营养作用。在体内，首先将Nrt慢病毒载体玻璃体注射感染大鼠视网膜，感染约30%RGC和绝大部分色素上皮层细胞。大鼠视神经损伤前1周玻璃体注射Nrt慢病毒载体，促进了RGC的存活和再生以及视觉功能恢复。后改用Nrt腺相关病毒2载体玻璃体注射感染大鼠视网膜，感染约70%RGC，视网膜Nrt mRNA和蛋白的表达分别提高19倍、2.3倍。大鼠视神经损伤前4周玻璃体注射Nrt腺相关病毒2载体，结果表明视网膜过表达的Nrt可能结合RGC胰岛素受体，上调视网膜pSTAT3/ STAT3、pAKT1/ AKT1、AKT2的表达；进而上调Bcl2、下调Bax的表达，促进RGC存活；下调Caspase3和Cleaved Caspase3的表达，减轻RGC凋亡；上调GAP43的表达，增加存留和再生的轴突；最终促进了视觉功能的恢复。总之，通过体内外研究证明了视网膜Nrt过表达对视神经损伤后RGC的保护和促进其轴突再生的作用并揭示了相关作用机制，为临床治疗视神经及中枢神经损伤提供了新的有力手段和理论依据。