PD-1及其配体在视神经炎发病机制及神经保护治疗中的作用研究

Retinal ganglion cells (RGCs) apoptosis is the ultimate pathway of optic neuritis, which contributes to irreversible visual loss in patients with optic neuritis. The mechanisms leading to this neurodegeneration, however, are poorly understood. A major hypothesis is that inflammation-induced axonal injury mediates apoptotic death of RGCs , but this does not explain the degeneration of neurons observed before the inflammatory cell infiltration or after the inflammatory phase has subsided. The applicant's previous research unveiled the expression of immunoregulator PD-1 in neurons and further revealed its unique function in physiological loss of neurons during retinal maturation.In recent study,The applicant found that PD-1 was also involved in pathological RGCs death.Study shows that PD-1 undergoes dynamic upregulation within central nervous system in animal with optic neuritis,which introduces a hypothesis that PD-1 may also play a role in optic neuritis-induced RGC death as it does within the developing mouse retina and axonal damage-induced retinopathy. In this project, PD-1 expression and activation, role of PD-1 blockade on RGC death will be investigated in EAE model, which is the most widely used model of optic neuritis and MS. Our investigation is aiming at determine the role of PD-1 and its underlying mechanisms during apoptosis of RGC in optic neuritis, in order to introduce new approaches to studies concerning pathogenesis of optic neuritis, and to create a brand new target for potential neuroprotective therapies to prevent permanent RGC loss from optic neuritis.

视网膜节细胞凋亡是造成视神经炎患者视力丧失的根本原因，其具体机制尚不清楚。传统的炎症假说并不能完全解释炎症期以外的神经退行性改变。本课题申请人前期工作揭示了PD-1在发育期视网膜节细胞凋亡中的重要作用；申请人近期还发现PD-1同样介导了病理状况下的视网膜节细胞凋亡。报道显示PD-1在多发性硬化（视神经炎）小鼠中枢神经系统中的表达明显升高，因此我们猜测，在发育期视网膜节细胞凋亡和轴突损伤所致的视网膜节细胞凋亡中均起重要作用的PD-1可能同样参与了视神经炎过程中视网膜节细胞的凋亡。为此，我们将建立广泛用于视神经炎和多发性硬化研究的动物模型EAE,探讨PD-1在视神经炎中的表达，活化，以及抑制PD-1对视网膜节细胞凋亡的影响，深入研究PD-1在视神经炎的RGC 凋亡中的重要作用，并阐明其作用机制，旨在为视神经炎发病机制的研究提供新的突破口，为视神经炎的神经保护治疗提供新的靶点。

背景：视神经炎是一种常见的与多发性硬化密切相关的视神经炎性脱髓鞘疾病，40%~60%的患者最终出现了永久性的视力丧失，主要为视网膜节细胞轴突受损所致。研究证实视神经炎小鼠的视网膜节细胞大量凋亡，数目明显减少。视网膜节细胞的进行性凋亡是许多视网膜和视神经疾病发展的必经之路，是多种眼病致盲的根本原因，但其机制尚不明确。申请人前期工作揭示了以往被大家公认只在免疫系统表达的PD-1在视网膜节细胞有表达，敲除PD-1基因可以抑制发育期视网膜节细胞的凋亡。.研究目的：探讨视神经炎症损伤致视网膜节细胞凋亡过程中免疫调节因子表达水平的变化及活化状态的改变，探讨抑制PD-1的生物活性对视神经炎症损伤所致视网膜节细胞凋亡的影响，为视神经炎的神经保护治疗找到新的靶点。.实验方法：用MOG35-55乳化液免疫法建立视神经炎症损伤动物模型。通过在小鼠玻璃体腔注射PD-1抗体来阻断PD-1信号通路。利用多剪接RNA结合蛋白（RNA binding protein with multiple splicing，RBPMS)法标记视网膜节细胞，通过检测视网膜节细胞的数量来评价其存活情况。通过冰冻切片免疫荧光染色、real-time PCR和Western Blot来检测免疫调节因子在视网膜上的表达情况。.结论：本课题建立了视神经炎症损伤动物模型，引进了国外评价视网膜节细胞凋亡的最新方法（RBPMS免疫标记法）。首次发现了免疫调节因子在小鼠视网膜中的表达情况——免疫荧光染色、real-time PCR和Western Blot结果显示小鼠出生后至视网膜发育成熟均可以在视网膜中检测到免疫调节因子的表达，为视神经炎的发病机制研究提供新的突破口。初步探索了抑制PD-1的生物活性对视神经炎症损伤所致视网膜节细胞凋亡的影响，为预防和治疗视神经炎症损伤寻找了新的靶点。