神经干细胞移植对大鼠视神经损伤后节细胞的保护作用

Protective effect of neural stem cell transplantation on retinal ganglion cells of rats with optic nerve injury

目的探讨神经干细胞(NSCs)移植对视神经受损SD大鼠视网膜节细胞(RGCs)的保护作用。方法将48只健康成年SD大鼠随机分为N组(NSCs移植组)和C组(对照组),均使用精确校准方法在右眼造成部分视神经损伤,左眼作为正常对照。从胚胎SD大鼠海马分离NSCs,利用细胞培养和体内移植技术,将培养后的NSCs注入N组大鼠右眼玻璃体内,C组大鼠右眼玻璃体内注入同等体积的PBS,处死前3d在双上丘注射3%快蓝逆行标记双眼RGCs。将大鼠分别于注射NSCs或PBS后7d、14d、21d、28d处死,各分为4组,每组6只,分离视网膜置于荧光显微镜下,摄影输入计算机图像分析仪计数RGC,计算RGC标识率。另取6只健康成年SD大鼠作NSCs移植,分别于移植后7d、28d处死,每时间段3只,通过视网膜免疫荧光切片观察NSCs在视网膜的存活、整合情况。结果N组大鼠各时间段RGCs标识率与C组同时间段比较均增高,有显著性差异(P<0.01);N组与C组RGCs标识率均随时间延长而降低,且前后段时间比较有显著性差异,但N组降低速度明显慢于C组。NSCs移植7d后部分移植细胞迁移进入视网膜的内网层和节细胞层,28d后可见移植细胞广泛整合至宿主视网膜内。结论NSCs移植入视神经损伤大鼠视网膜后可提高视网膜神经节细胞的存活率,对受损的节细胞具有一定的保护作用。

Objective To investigate the protective effect of neural stem cell （NSC） transplantation on retinal ganglion cells （RGCs） of Sprague-Dawley rats with optic nerve injury. Methods Forty-eight adult Sprague-Dawley rats were randomly divided into group N （NSC） and group C （control）. Optic nerve crush injury was induced in the right eyes by a calibrated method, and the left eyes served as the normal control. NSCs were taken fi＇om embryonic SD rat hippocampus and cultured in vitro and grafted in vivo. NSCs were injected into the vitreous body of right eyes of rats in group N. Rats in group C were injected with isovolumetric PBS. Three days before sacrifice, 3% fast blue was injected into the superior colliculi bilaterally. According to the time interval between the optic nerve crush and sacrifice, both group N and C were divided respectively into four subgroups （day 7, day 14, day 21 and day 28） with 6 rats in each one. The eyes were enucleated after the rat was sacrificed, and flat mounts of the retina fi＇om both eyes were observed under a fluorescence microscope. The labeled RGCs were counted by a computerized image analyzer. The label rate of RGCs was calculated for statistical analysis. In addition, 6 adult SD rats were treated as group N, and sacrificed on days 7 and 28 after transplantation with 3 rats each time. Survival and integration of graRed NSCs in the retina of the host animals were detected by the immunofluorescence technique. Results The label rate of RGCs in group N was significantly higher than that in group C at each time period （P〈0.01）. The labeled rates of RGCs in both groups N and C decreased as the time passed, but the speed of decreasing in group N was evidently slower than that in group C. Seven days after the NSCs transplantation, a few grafted cells could be seen migrating into inner retinal layer and ganglion cell layer of the host retina and on the 28th day, widespread migration of grafted NSCs could be seen into the host retina. Conclusion NSCs transplanting into the vitreous body can rescue and/or restore injured RGCs and increase survival rate of the RGCs in optic nerve-crush rat model.