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、0.247);与对照组比较,ONC组小鼠Ops、PhNR振幅明显下降,差异有统计学意义(t=15.07、12.80,P<0.000、<0.001);与ONC组比较,Park7-ONC组小鼠Ops、PhNR振幅明显下降,差异有统计学意义(t=4.042、5.062,P<0.05、<0.01);各组小鼠PhNR潜伏期比较,差异无统计学意义(F=1.327,P=0.287)。与对照组比较,ONC组小鼠视敏度明显下降,差异有统计学意义(t=23.020,P<0.000);与ONC组比较,Park7-ONC组小鼠视敏度明显下降,差异有统计学意义(t=3.669,P<0.05)。Park7组与对照组、Park7-ONC组与ONC组比较,小鼠视网膜中DJ-1蛋白相对表达量均明显下调,差异有统计学意义(t=47.140、26.920,P<0.000、<0.000);ONC组与GFP-ONC组比较,差异无统计学意义(t=0.739,P=0.983)。与ONC组比较,Park7-ONC组小鼠视网膜中Bax蛋白相对表达量明显升高,Bcl-2蛋白相对表达量明显降低,差异均有统计学意义(t=5.960、9.710,P<0.05、<0.05);Park7-ONC组Bcl-2/Bax相对表达量比值明显低于ONC组,差异有统计学意义(t=13.620,P<0.01)。结论敲低Park7基因后其编码的蛋白质DJ-1表达下调,加重ONC模型小鼠RGC损伤以及视网膜电生理反应及视功能下降。
Objective 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 control group,the RGC density of the retina in the ONC group decreased significantly,and the difference was statistically significant(t=16.520,P<0.000);compared with the ONC group,the RGC density of the retina in the Park7-ONC group decreased significantly,and the difference was statistically significant(t=6.074,P<0.01).With the increase of stimulus light intensity,the dark adaptation a wave and b wave latency of the mice in the control group gradually shortened,and the amplitude gradually increased.The stimulus light intensity was 3 cd·s/m^(2).There was no statistically significant difference in the dark adaptation a wave and b wave latency and amplitude of the control group,Park7 group,Park7-ONC group,ONC group,and GFP-ONC group(Incubation period:F=0.503,2.592;P=0.734,0.068.Amplitude:F=0.439,1.451;P=0.779,0.247).Compared with the control group,the Ops and PhNR amplitudes of the ONC group mice were significantly decreased(t=15.07,12.80;P<0.000,<0.001).Compared with the ONC group,the Ops and PhNR amplitudes of the mice in the Park7-ONC group were significantly decreased(t=4.042,5.062;P<0.05,<0.01);there was no statistically significant difference in the PhNR latency of the mice in each group(F=1.327,P=0.287).Compared with the control group,the visual acuity of the mice in the ONC group was significantly decreased,and the difference was statistically significant(t=23.020,P<0.000);compared with the ONC group,the visual acuity of the mice in the Park7-ONC group was significantly decreased,and the difference was statistically significant(t=3.669,P<0.05).Compared with the control group,Park7-ONC group and ONC group,the relative expression of DJ-1 protein in the mouse retina was significantly down-regulated,and the difference was statistically significant(t=47.140,26.920;P<0.000,<0.000).There was no significant difference between ONC group and GFP-ONC group(t=0.739,P=0.983).Compared with the ONC group,the relative expression of Bax protein in the mouse retina of the Park7-ONC group was significantly increased,and the relative expression of Bcl-2 protein was significantly reduced.The differences were statistically significant(t=5.960,9.710;P<0.05,<0.05);the relative expression ratio of Bcl-2/Bax in the Park7-ONC group was significantly lower than that in the ONC group,and the difference was statistically significant(t=13.620,P<0.01).Conclusion The expression of DJ-1 encoded by Park7 gene is down-regulated after Park7 gene was knocked down,which aggravates the RGC damage and the decrease of retinal electrophysiological response and visual function in ONC injury mice.
作者
欧阳灵艺
贺涛
邢怡桥
Ouyang Lingyi;He Tao;Xing Yiqiao(Eye Center of Renmin Hospital of Wuhan University,Wuhan 430060,China)
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
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)。
