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

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.

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。

本研究在体外成功进行体外原代分离兔视网膜干细胞及视网膜神经节细胞。在不同压力下，将两者共培养。结果发现，在20、40、60和80 mmHg的压力下，视网膜干细胞均可分化为神经节样细胞；在40mmHg的压力下，神经节样细胞占分化所得细胞的比例最高；压力过高，会影响视网膜干细胞的分化。同时，在20、40、60和80 mmHg的压力下，干细胞特异的nestin的mRNA及蛋白表达下降，40 mmHg下降程度最大，视网膜神经节细胞的标记物THY1.1mRNA及蛋白表达均上升，40 mmHg上升程度最大。说明体外培养的视网膜干细胞具有向视网膜神经节样细胞分化的能力，并且在一定压力条件下，能够促进视网膜干细胞的分化。在体内实验中，成功建立兔慢性青光眼模型，采用HE染色观察兔视网膜各层形态变化，结果发现，随着高眼压时间延长，兔内层视网膜结构逐渐紊乱，神经节细胞及内核层神经元细胞数明显减少。在慢性青光眼模型兔玻璃体腔注射兔视网膜干细胞（GFP+）1个月后，处死模型兔并制作病理切片，检测到兔视网膜干细胞（GFP+）停留在视网膜表面，仍然保持干细胞特性，未发现明显分化。但是宿主的视网膜形态较模型对照组紊乱减轻。说明视网膜干细胞对青光眼模型兔视网膜具有保护作用，其作用机制有待我们进一步研究。