视网膜干细胞在兔慢性青光眼模型中向神经节细胞定向分化研究

青光眼是全世界主要的致盲眼病之一，最终会引起视网膜神经节细胞（RGCs）凋亡，从而导致视功能不可逆的损害。目前治疗方法主要是降眼压，但这并不能治疗RGCs凋亡引起的视功能损害。为了寻求有效的方法来保护RGCs，本研究拟将体外培养的兔视网膜干细胞（RSCs）在不同液压下与RGCs共培养，观察RSCs分化为神经节样细胞的比例；并检测各视网膜神经元特异标记物的表达，从而明确RSCs的分化情况；建立兔慢性青光眼模型，构建慢病毒将绿色荧光蛋白（GFP）整合至兔RSCs，采用直接玻璃体腔注射法和以超薄聚酰亚胺膜为细胞载体进行RSCs移植，观察其在视网膜上迁移、分化、定位及宿主视网膜功能的变化，并观察神经节样细胞的分化比例。比较两种方法的优劣。检测可能参与RGCs定向分化的基因表达水平变化，明确RSCs向RGCs定向分化可能的机制。从而为青光眼及其他视神经变性疾病的神经元再生提供新的来源及治疗思路。

One of the main causes of blindness in the world is glaucoma, which is actually a complex of ocular diseases that include progressive loss of retinal ganglion cells (RGCs), damage to the optic nerve, and loss of vision. A common treatment is to reduce intraocular pressure (IOP), but this approach does not halt visual loss caused by the death of retinal ganglion cells (RGCs). Therefore, in order to find an effectively way to protect RGCs from apoptosis, retinal stem cells are isolated from rabbit retina in this study. Next, the stem cells are cocultured with rabbit retinal ganglion cells (RGCs) at different pressures in vitro, the neurons differentiation from stem cells are detected by immunofluorescence staining, and then calculate the proportion of RGCs differentiated from stem cells. The expression of nestin, Thy1.1, Rhodopsin, PKC-α, HPC-1, calbindin and GS are measured by qPCR and western blot. Next, we will build a rabbit model of chronic glaucoma, and the green fluorescent protein (GFP) will integrated into rabbit RSCS by lentiviral. Retinal stem cells are transplantated into the vitreous cavity of rabbit chronic glaucoma model by Intravitreal transplantation directly or transplantated by a cellular scaffolds of Ultrathin Polyimide Membrane into vitrectomized eyes. Then observe the migration, localization, differentiation of retinal stem cells, and detect the retinal function of the host. After animals are sacrificed, we observe the differentiation proportion of ganglion cells, detect the inflammation reaction of vitreous cavity and the expression of Atoh7, Brn-3b, Isl-1, Notch, which may be involved in RGCs differentiation are measured by qPCR and western blot. Thus, it provides a new source of optic nerve regeneration and opening up a new avenue for gene therapy in glaucoma and other degenerative diseases of the optic nerve.