巢蛋白——一种新的视网膜损伤生物标记物

Nestin:a new biomarker for retinal injury

目的探讨Mller细胞在视网膜损伤中的作用,探讨巢蛋白(nestin)是否是一个更有价值的评价视网膜损伤的生物标记物。方法33只健康成年SD大鼠,其中3只做为正常对照,于各模型制作之前取材;6只制作大鼠视网膜缺氧模型,先于体积分数9%氧浓度箱中2h,其中3只在体积分数80%氧浓度中治疗2h后,均置于正常环境24h后取材;15只右眼制作青光眼模型,左眼作假手术对照,分别于术后2h、1d、3d、1周、3周取材(每个时间点3只);9只右眼制作视神经横断模型,分别于术后1d、3d、1周取材;作眼球矢状位冰冻切片,行nestin及谷氨酰胺合成酶(glutamine synthetase,GS)或胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)及GS免疫荧光双标记,共聚焦显微镜读片分析。结果健康成年SD大鼠视网膜Mller细胞不表达nestin和GFAP,视网膜损伤后(缺氧、青光眼、视神经横断),Mller细胞均出现nestin的诱导表达,青光眼模型中nes-tin在Mller细胞上的诱导表达随病程延长持续升高;视神经横断后1dMller细胞即出现nestin的诱导表达,3d时其表达进一步增强,1周时表达轻度减弱;在视网膜缺氧模型中Mller细胞有明显nestin的诱导表达,而给予高氧治疗后其表达明显减弱。青光眼和视神经横断后Mller细胞上出现GFAP的诱导表达,但在缺氧模型中未发现GFAP表达。结论nestin是一种新的、有价值的视网膜损伤生物标记物。视网膜损伤后nestin、GFAP、GS在Mller细胞足板处的聚集对维持视网膜结构完整性和促进功能恢复可能具有重要的意义。

Objective To investigate the role of Müller cells in the injured retinas and whether nestin be a more useful biomarker for retinal injury.Methods A total of 33 healty adult SD rats were enrolled in this study.Three rats were taken as normal controls and materials were taken out before making models.Six rats were treated as retinal hypoxia models and exposed in oxygen concentration box with 9% volume fraction for 2 hours,3 rats of which were exposed in oxygen concentration with 80% volume fraction for 2 hours and then given normal environment for 24 hours.Right eyes of 15 rats were treated as glaucoma models,left eyes were treated as sham operation controls,and materials were taken out at postoperative 2 hours,1 day,3 days,1 week and 3 weeks,respectively,3 rats at each time.Right eyes of 9 rats were treated as optic nerve transection models and materials were taken out at postoperative 1 day,3 days and 1 week,respectively.Eye balls sagittal cryosections were made.Double immunofluorescence labeling was carried out between nestin and glutamine synthetase（GS）,or between glial fibrillary acidic protein（GFAP） and GS.Confocal microscopy analysis was taken.Results In healthy adult SD rats,nestin and GFAP were not found in retinal Müller cells.After retinal injury（hypoxia,glaucoma and optic nerve transection）,induced expression of nestin was found in all Müller cells,and continually increased with the length of pathogenesis.In optic nerve transection models,induced expression of nestin was found in Müller cells at 1 day,increased at 3 days and slightly decreased at 1 week.Induced expression of nestin was obvious in Müller cells of hypoxia models,while significantly descreased after treated with hyperxia.In glaucoma and after optic nerve transection,induced expression of GFAP was found in Müller cells,but not in hypoxia models.Conclusions Nestin is a new and valuable biomarker for retinal injury.The intensive expression of nestin,GFAP and GS in the pedal plate of Müller cells suggest that they may help to maintain the retinal structural integrity and to enhance functional recovery in various retinal diseases.