基于OPTN（E50K）突变致NTG致病机制的干细胞治疗研究

Normal tension glaucoma (NTG), a common cause of vision loss and blindness that occurs without grossly abnormal intraocular pressure. RGCs degeneration under normal pressure remains poorly understood, which now is a dilemma in the glaucoma field. The cell death is irreversible and the retinal ganglion cells have very limited ability to regenerate after injury. Mutations in optineurin (OPTN) are linked to the pathology of NTG. Emerging evidence indicated that OPTN(E50K) mutation is involved in the damage of transportation and expression for neurotrophic factors, resulting in RGCs death of mutant. Nevertheless, the role played by the pathogenic autophagy that underlies the abnormal stemness and paracrine effect of stem cell degeneration, associated with defective capacity of restoration for damaged RGCs. According to the multiple pathophysiological mechanisms underlying the E50K mutation-mediated glaucomatous RGC degeneration and the possibility of the pluripotent bone marrow stem cell based regeneration of retinal cells and repair of neurodegenerative diseases, we tried to develop a new strategy of stem cell therapy for NTG. A novel mouse model was established based on a mutation by CRISPR-Cas9 in mouse optineurin associated with hereditary, normal tension glaucoma, which improved the design and phenotype of currently available optineurin mice. Further, we propose to isolate the bone marrow Sca-1+ subset of stem cells using magnetic activated cell sorting and identify their neuroprotective effect on organotypical retinal explant of the mutants and their homing efficiency in bone marrow reconstituted chimeras in vivo in conjunction with the model of E50K mutation-mediated normal tension glaucoma. We hypothesize that the bone marrow-derived Sca-1+ subset of stem cells exerts beneficial paracrine actions on the injured retina of E50K mutation by releasing biologically active factors, regulating pathogenic autophagy, and all these may play an important role in bone marrow Sca-1+ subset of stem cell-mediated retinal neuroprotection. Our study is to evaluate the translational potential of bone marrow derived subpopulation of stem cells for the treatment of NTG, alongside targeting possible novel therapeutical factors through stem cell-independent method. It will provide a more efficient stem cell sub-type for the treatment ocular diseases and glaucoma in future. We believe that the current proposal will provide a new strategy of stem cell therapy for NTG and identify the most efficient therapy for cell application in ophthalmological treatment.

正常眼压青光眼（NTG）发病隐匿，视功能损害严重，其治疗是青光眼领域的难题。OPTN基因是NTG相关致病基因。申请者前期研究证实OPTN（E50K）突变影响神经营养因子表达及分泌是导致RGCs死亡的机制之一。另外，正常OPTN蛋白作为自噬接合体，在细胞自噬过程起关键作用；而自噬功能异常则会引起机体干细胞干性和旁分泌作用下降，进一步阻碍其对受损RGCs的修复。本项目基于OPTN（E50K）突变致RGCs损伤多重机制和骨髓优质干细胞高效归巢修复受损组织的特性，寻找NTG治疗的新方法和新思路。我们拟在体外对骨髓干细胞中自我更新能力占优势的Sca-1+干细胞亚群进行分选富集，对CRISPR-Cas9技术构建的OPTN（E50K）突变NTG小鼠进行骨髓重建，观察Sca-1+亚群干细胞对NTG致RGCs损伤的修复和对视功能的改善，探讨其作用机制，为NTG等视神经退行性疾病的干细胞治疗提供新策略。

视神经病变诱导基因OPTN的E50K突变是引起正常眼压性青光眼（Normal tension glaucoma，NTG）发生的重要病因。它在正常眼压时造成视网膜神经节细胞（retinal ganglion cell，RGCs）变性损伤并导致严重的临床表现，但病理机制仍未可知，且临床缺乏有效治疗方法。骨髓干细胞的旁分泌和可促进损伤神经元修复再生的能力具有重要的神经保护作用。因此我们利用OPTN E50K突变构建NTG小鼠模型，通过系统性分析视网膜蛋白质表达改变研究其致病机制，并验证优质骨髓干细胞亚群移植可以成为NTG治疗的新方法。通过CRISPR/Cas9基因组编辑技术构建OPTN E50K突变小鼠作为NTG动物模型，并验证其具有青光眼性视神经病变。通过研究NTG疾病过程中视网膜蛋白质表达改变并利用生物信息学方法分析，NTG中视网膜发生的蛋白改变和其它中枢神经系统退行性疾病例如阿尔茨海默病、帕金森病等相似，可能参与疾病发生的分子机制包括蛋白质合成异常、Rap1和Ras信号通路异常、以及神经营养因子信号通路失调。分子学实验也证实神经营养因子BDNF，CNTF，FGF2和IGF-1不仅在老年OPTN E50K小鼠视网膜中表达显著降低，在骨髓干细胞中也明显减少。而Sca-1+骨髓干细胞亚群在营养因子的表达上具有显著优势，但这种旁分泌优势受衰老和突变的影响而减弱。年轻野生型小鼠Sca-1+亚群可通过增加神经营养因子表达，减少体外共培养NTG小鼠视网膜凋亡。并且体内实验证实，Sca-1+亚群经静脉注射后可归巢到OPTN E50K小鼠视网膜，并通过强效的旁分泌作用促进视网膜神经营养因子表达、提高骨髓干细胞旁分泌功能，减少RGCs凋亡，显著改善老年OPTN E50K小鼠视功能。这些结果提示优质骨髓干细胞亚群移植可能成为神经退行性疾病潜在有效的治疗方式，提供新的治疗策略。

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