骨髓Sca-1+亚群干细胞归巢对高眼压视网膜损伤修复机制的研究

Glaucoma is a disorder that leads to retinal ganglion cells (RGCs) apoptosis and optic nerve degeneration, as well as visual field loss, and, eventually, blindness. The cell death is irreversible and the retinal ganglion cells have very limited ability to regenerate their axons after injury. Recent efforts have allowed the possibility of the pluripotent bone marrow stem cell based regeneration of retinal cells and repair of neurodegenerative diseases. However, the biologic capacities of each bone marrow stem cell subpopulation in repairing the damage tissue are varied. The present study is designed to explore our current understanding of the cellular homing identity of the bone marrow-derived Sca-1+ specific cell subpopulation and their therapeutic potentials for cell therapy against degenerative retinal diseases. Here 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 in vitro and their homing efficiency in bone marrow reconstituted chimeras in vivo in conjunction with the model of ocular hypertensive glaucoma. We hypothesize that the bone marrow-derived Sca-1+ subset of stem cells exerts beneficial paracrine actions on the injured retina by releasing biologically active factors such as FGF2 and its signal pathway 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 glaucoma, 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 ocular diseases and identify the most efficient therapy for cell application in ophthalmological treatment.

青光眼可导致视网膜神经节细胞（Retinal ganglion cells，RGCs）的损伤和凋亡，RGCs损伤后的再生能力有限，最终导致视功能不可逆性损害直至视力丧失。骨髓干细胞具有多分化潜能，可以归巢到损伤视网膜参与组织修复；利用细胞表面标记对骨髓干细胞分选，寻找优质干细胞亚群，提高归巢效率和修复效果，为青光眼等视神经退行性疾病的治疗开启了新思路。我们拟对骨髓干细胞中自我更新和复制能力占优势的Sca-1+干细胞亚群进行分选富集，在体外和体内模型中观察Sca-1+亚群干细胞对压力损伤视网膜细胞的修复能力和对视功能的改善；分析骨髓Sca-1+亚群干细胞的旁分泌作用及FGF信号通路在骨髓Sca-1+亚群干细胞归巢促视网膜损伤修复中的机制；探讨骨髓干细胞亚群分选提高干细胞归巢效率的新策略，增强视网膜损伤后细胞治疗的效果。本课题将为青光眼等视网膜损伤及神经退行性眼病的细胞治疗提供新方法和新思路。

青光眼是一种神经退行病变，可导致视网膜神经节细胞（Retinal ganglion cells，RGCs）的损害，引起视功能严重损伤，导致视力丧失。骨髓干细胞具有定向分化的潜能，归巢到受损脏器参与修复。此项研究主要是探索骨髓重建促进年老视网膜组织损伤修复的机制。利用干细胞抗原-1（Stem cell antigen-1，Sca-1）对GFP供体小鼠骨髓进行分选，对野生型C57BL/6受体小鼠进行骨髓移植及重建；骨髓重建8周后，通过眼内灌注升高眼内压的方法对骨髓嵌合体小鼠造成视网膜急性高眼压损伤；免疫组织荧光观察比较Sca-1细胞在骨髓嵌合体小鼠损伤视网膜归巢及分化的情况；明暗试验和视动试验观察骨髓嵌合体小鼠视功能的改变；并利用qPCR和Western blot检测视网膜神经节细胞损伤有关基因及蛋白的改变。研究发现急性高眼压损伤小鼠视网膜7天后，视功能检测显示Sca-1+骨髓嵌合体小鼠的视功能优于Sca-1-组；免疫组织荧光显示与骨髓Sca-1-亚群干细胞相比，Sca-1+干细胞具有更强的归巢能力和分化能力，归巢的骨髓源性干细胞可以在损伤视网膜分化成为大胶质细胞和小胶质细胞；TUNEL法染色结果显示，在视网膜急性损伤后，骨髓Sca-1 +亚群干细胞可以减少宿主视网膜细胞的凋亡，并且增加碱性成纤维生长因子2（Fibroblast growth factor 2，FGF2）mRNA和蛋白在损伤视网膜的表达，眼内注射FGF2抗体后，FGF2蛋白表达被抑制，Akt磷酸化程度减低，骨髓Sca-1+亚群干细胞的抗凋亡作用减弱；体外细胞视网膜组织共培养模型也同样显示，骨髓Sca-1+亚群干细胞能够促进视网膜组织片细胞的存活，阻断FGF2后，Akt磷酸化水平降低，骨髓Sca-1+亚群干细胞对视网膜的保护作用减弱。因此，骨髓Sca-1+亚群干细胞在视网膜急性高眼压损伤后能够归巢到损伤组织，减少视网膜细胞的凋亡，促进视功能恢复；FGF2/Akt通路在骨髓Sca-1+亚群干细胞归巢和促视网膜损伤修复中可能发挥重要作用。