下调热休克蛋白B8在视神经损伤中对小鼠视网膜神经节细胞的保护作用  

Downregulation of heat shock protein B8 protects retinal ganglion cell after optic nerve injury in mice

作　　者：谢斐葭 贺涛[1] 杨宁[1] 杨家翼 花蒂豪 罗晋媛 李宗媛 邢怡桥[1] Xie Feijia;He Tao;Yang Ning;Yang Jiayi;Hua Dihao;Luo Jinyuan;Li Zongyuan;Xing Yiqiao(Eye Center,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4期298-306,共9页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金(面上项目)(81271025);湖北省自然科学基金面上类项目(青年)(2020CFB240);中央高校基本科研业务费专项资金资助(青年教师资助项目)(2042020kf0065)。

摘　　要：目的观察敲低热休克蛋白B8(HspB8)对鼠视神经钳夹(ONC)后视网膜神经节细胞(RGC)及视网膜功能的影响。方法构建敲低HspB8的重组腺相关病毒(AAV)2-小发夹HspB8(shHspB8)-绿色荧光蛋白(GFP)。雄性C57BL/6J小鼠66只随机分为正常对照组、ONC组、AAV2-shHspB8组、ONC+AAV2-shHspB8组、ONC+AAV2组,分别为10、20、16、10、10只,双眼均作为实验眼。建模后3、7 d,蛋白质免疫印迹法(Western blot)检测小鼠视网膜HspB8蛋白相对表达变化;GFP免疫荧光染色确定AAV2-shHspB8-GFP转染情况。建模后5 d,视动反应(OMR)、暗适应全视野闪光视网膜电图(ff-ERG)检测各组小鼠视敏度、振荡电位(OPs)、明视负向反应(PhNR)振幅和潜伏期;视网膜铺片计数各组小鼠RGC。采用Mann-Whitney U检验、独立样本t检验、Welch法校正t检验、单因素方差分析、Bonferroni t检验对数据进行统计分析。结果与正常对照组比较,ONC3组小鼠视网膜中HspB8蛋白相对表达量明显增加,差异有统计学意义(F=43.63,P<0.01)。与正常对照组比较,ONC5组小鼠视敏度明显降低,差异有统计学意义(P<0.01);a波、b波、OPs、PhNR振幅下降,b波潜伏期延长,差异均有统计学意义(P<0.05);ONC3组、ONC5组、ONC7组小鼠RGC存活率呈时间依赖性降低,差异有统计学意义(F=384.90,P<0.01)。病毒转染后4周,转染率达峰值。与正常对照组相比,AAV2-shHspB8组视敏度,a波和b波、OPs、PhNR振幅、RGC计数差异均无统计学意义(P>0.05);ONC5+AAV2-shHspB8组RGC计数较ONC5组明显升高,差异有统计学意义(F=10.62,P<0.01)。结论ONC诱导HspB8在视网膜中表达,导致RGC死亡并损害小鼠视功能;下调HspB8对ONC所致的RGC死亡具有保护作用。Objective To investigate the effect of heat shock protein B8(HspB8)downregulation on retinal ganglion cell(RGC)and retinal function in the mice model of optic nerve injury(ONC).Methods Adeno-Associated Virus(AAV)2 AAV2-shHspB8-GFP was constructed to knockdown HspB8.66 adult male C57/BL6 mice were randomly divided into the control group,the ONC group,the AAV2-shHspB8 group,the ONC+AAV2-shHspB8 group,and the ONC+AAV2-GFP group.There were 10,20,16,10 and 10 mice respectively,and both eyes were used as experimental eyes.Western blot was used to evaluate the expression of HspB8 on day 3 and 7 after ONC.By GFP immunofluorescence staining,the efficacy of AAV2-shHspB8-GFP transfer was accessed.Moreover,it was possible to identify functional and RGC survival differences between groups by optomotor response(OMR),dark adapted full-field flash electroretinogram(ff-ERG),oscillatory potentials(OPs),photopic negative response(PhNR)and retinal flat-mount RGC counting 5 days after ONC.Comparisons between two groups were made using Mann-Whitney U test,unpaired t-test,unpaired t-test with Welch’s correction,one-way ANOVA,and Bonferroni t test.Results Compared with the control group,the expression of HSPB8 protein in the retina of mice in ONC3 group was significantly increased,and the difference was statistically significant(F=43.63,P<0.01).Compared with the control group,the ONC group showed obviously lower visual acuity(P<0.01),lower a-wave,b-wave,OPs,PhNR amplitude,longer b-wave latency(P<0.05),and the survival rates of RGC in ONC3 group,ONC5 group and ONC7 group decreased in a time-dependent manner(F=384.90,P<0.01).Transfection of AAV2 efficiency was highest on 4 weeks after IVT.Besides,there was no significant differences between the control group and the AAV2-shHspB8 group on visual acuity,ff-ERG,OPs,PhNR and RGC survival(P>0.05).In comparison of the control group,we found that RGC survival of the ONC5+AAV2-shHspB8 group was significantly elevated(F=10.62,P<0.01).Conclusions Expression of HspB8 on the retina can be induced b

关 键 词：热休克蛋白质类 视神经损伤 视网膜神经节细胞 眼电描记术 动物实验 

分 类 号：R774.6[医药卫生—眼科]