目的 观察银杏叶提取物(EGb761)对培养幼年大鼠大视网膜神经节细胞(RGC)的保护作用;建立用荧光金逆行上丘标记鉴定体外培养大鼠RGC的方法.方法 12只鼠龄20 d Sprague-Dawley大鼠用荧光金行双侧上丘注射,标记RGC.6 d后摘除眼球,其中一只眼球行视网膜铺片,荧光显微镜下观察标记情况.另一只眼球摘除后分离视网膜,制成细胞悬液,倒置荧光显微镜下观察荧光着染的RGC形态.将细胞悬液分为对照组及浓度分别为0.03%、0.10%、0.30%、1.00%、3.00%的EGb761组,接种后3h,1、3、5、7 d锥虫蓝染料排斥法检测RGC的存活情况,同时记录细胞存活率.结果 逆行上丘标记后观察到RGC标记良好,视网膜细胞悬液中大RGC特征明显,离体后迅速死亡,荧光消失.锥虫蓝染料排斥法观察到在视网膜细胞悬液中,这种大RGC死亡迅速.加入不同浓度的EGb761后大RGC存活率明显增加,不同时间点与对照组比较大RGC存活率均有统计学意义(P<0.01),且呈明显剂量依赖关系(P<0.01).结论 EGb761对体外培养的RGC具有明显的保护作用;荧光金逆行上丘标记鉴定体外培养的RGC方法可行.%Objective To investigate the protective effects of ginkgo biloba extract (EGb) 761 on retinal ganglion cells (RGC) in rats,and to establish a method to define the rat RGC using fluorogold as a fluorescence dye.Methods RGC of 12 20-day-old Sprague-Dawley rats were labeled by injecting fluorogold into superior colliculus.The eyeball enucleation was performed 6 days later.Retinal stretched preparation was obtained from one eye to observe the label result under fluorescence microscope,and the retina from the other eye was detached to make the cell suspension to observe the configuration of stained RGC under the contrast fluorescence microscope.The cell suspension was divided into the control group and Egb761 groups with the concentration of 0.03%,0.10%,0.30%,1.00%,and 3.00%.Trypan blue dye was used to evaluate cells viability and the survival rate of the large retinal ganglion cells was calculated.Results The sign of the RGC was clear after labeled by fluorogold.The characteristics of large RGC were obvious.After detachment,large RGC died quickly in the cell suspension and the fluorescence disappeared.The result of Trypan blue staining indicated that large RGC died rapidly in the cell suspension.Large RGC in EGb761 group showed significantly better survival rates than that in control group at different time sites (P<0.01) in a dose-dependent manner (P<0.01).Conclusions EGb761 has a significant protective effect on large RGC cultivated in vitro,and retrolable method to identify RGC is feasible.
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