DJ-1蛋白对视神经钳夹伤后小鼠视网膜神经节细胞及视功能的影响  

Effects of DJ-1 protein on retinal ganglion cells and visual function in mice after optic nerve crush injury

作　　者：欧阳灵艺 贺涛[1] 邢怡桥[1] Ouyang Lingyi;He Tao;Xing Yiqiao(Eye Center of Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院眼科中心,430060

出　　处：《中华眼底病杂志》2021年第5期377-384,共8页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金(面上项目)(81271025)。

摘　　要：目的观察Park7基因编码的DJ-1蛋白对小鼠视神经钳夹损伤(ONC)后视网膜神经节细胞(RGC)及视功能的影响。方法健康雄性C57BL/6J小鼠37只和116只分别随机分为正常组、ONC 2 d组、ONC 5 d组、ONC 7 d组和对照组、Park7组、Park7-ONC组、ONC组、绿色荧光蛋白(GFP)-ONC组。ONC 2 d组、ONC 5 d组、ONC 7 d组小鼠分别于建立ONC模型后2、5、7 d处死取材,进行后续实验。Park7组、Park7-ONC组小鼠玻璃体腔注射敲低Park7基因的重组腺相关病毒(rAAV)1μl,GFP-ONC组小鼠玻璃体腔注射仅带有GFP的rAAV 1μl;注射后4周观察病毒转染情况。ONC组、Park7-ONC组、GFP-ONC组玻璃体腔注射后23 d,建立ONC模型,建模后5 d取材进行后续实验。视网膜铺片免疫荧光染色观察RGC平均密度变化;全视野闪光视网膜电图检测各组小鼠a波、b波和明视负向反应(phNR)潜伏期及振幅变化和振荡电位(OPs)振幅变化;视动反应(OMR)检测小鼠视敏度;蛋白质免疫印迹法(Western blot)检测各组小鼠视网膜中DJ-1、Bax、B淋巴细胞瘤/白血病-2(Bcl-2)蛋白相对表达水平。多组间数据比较采用单因素方差分析;组间两两比较采用最小显著差法t检验。结果与正常组比较,ONC 2 d组、ONC 5 d组小鼠视网膜DJ-1蛋白相对表达量均显著上升,差异有统计学意义(t=16.610、5.628,P<0.01、<0.05)。病毒转染后4周,Park7组小鼠视网膜RGC层、内丛状层可见强GFP表达。与对照组比较,ONC组小鼠视网膜RGC密度明显下降,差异有统计学意义(t=16.520,P<0.000);与ONC组比较,Park7-ONC组小鼠视网膜RGC密度明显下降,差异有统计学意义(t=6.074,P<0.01)。随刺激光亮度增加,对照组小鼠暗适应a波、b波潜伏期逐渐缩短,振幅逐渐增大。刺激光亮度3 cd·s/m^(2),对照组、Park7组、Park7-ONC组、ONC组、GFP-ONC组小鼠暗适应a波、b波潜伏期及振幅比较,差异均无统计学意义(潜伏期:F=0.503、2.592,P=0.734、0.068;振幅:F=0.439、1.451,P=0.779、0Objective To investigate the effect of DJ-1 encoded by Park7 gene on retinal ganglion cells(RGC)and visual function after optic nerve crush injury(ONC)in mice.Methods Thirty-seven and 116 healthy male C57BL/6J mice were randomly divided into group normal,group ONC 2d,group ONC 5d,group ONC 7d and group control,group Park7,group Park7-ONC,group ONC and group green fluorescent protein(GFP)-ONC.Group ONC 2d,group ONC 5d and group ONC 7d were sacrificed on the 2nd,5th and 7th day after the establishment of ONC model,and the follow-up experiments were carried out.The mice in group Park7 and group Park7-ONC were injected 1μrecombinant adeno-associated virus(rAAV)with knocking down Park7 gene into vitreous cavity,and 1μl rAAV with only GFP was injected into vitreous cavity of mice in group GFP-ONC,and virus transfection was observed 4 weeks after injection.The injury of ONC was perfomed at 23 days after vitreous injection in group ONC,group Park7-ONC and group GFP-ONC,and the samples were taken for follow-up experiment 5 days after modeling.The average density of RGC was observed by immunofluorescence staining,the latencies and amplitudes of a-wave,b-wave and photopic negative response(phNR)and the amplitude of oscillatory potential(OPs)were detected by full-field flash electroretinogram,and the visual acuity of mice was measured by optomotor response(OMR).The relative expression levels of DJ-1,Bax and B lymphoblastoma/leukemia-2(Bcl-2)protein in the retina of mice in each group were detected by Western blot.One-way ANOVA was used to compare the data between groups,and t-test was used for pairwise comparison between groups.Results Compared with the normal group,the relative expression of DJ-1 protein in the retina of the ONC 2 d group and ONC 5 d group increased significantly,and the difference was statistically significant(t=16.610,5.628,P<0.01,<0.05).Four weeks after virus transfection,strong GFP expression was seen in the RGC layer and inner plexiform layer of the retina of mice in the Park7 group.Compared with the

关 键 词：视神经损伤 视网膜神经节细胞 视网膜电描记术 动物实验 DJ-1蛋白 

分 类 号：R774.1[医药卫生—眼科]