Brn3b基因对视神经损伤条件下视网膜神经节细胞的保护作用

The protective effect of Brn3b gene on retinal ganglion cells in the condition of optic nerve injury

目的探讨Brn3b过表达对视神经损伤条件下视网膜神经节细胞(retinal ganglion cells,RGCs)的保护作用。方法选取雄性健康成年BALB/c小鼠60只,用微型视神经夹将小鼠右眼球后视神经夹持损伤,按视神经损伤后的天数依次分为第1、3、5、7、14天组,小鼠左侧正常眼作为空白组,明确视神经损伤天数条件。选取雄性健康成年BALB/c小鼠40只,用微量进样器以小鼠右眼玻璃体内注射的方法将腺相关病毒载体转染小鼠视网膜,建立Brn3b过表达模型并分组:Brn3b过表达组[转染Brn3b过表达腺相关病毒载体(Brn3b overexpressed adeno-associated viral vector,AAV-CMV-Brn3b)]和阴性对照组[转染空白腺相关病毒载体(blank adeno-associated viral vector,AAV-CMV-GFP)阴性对照物],每组20只;之后,取Brn3b过表达组和阴性对照组各10只,用微型视神经夹将小鼠右眼球后视神经夹持损伤,构建模拟小鼠视神经损伤(controlled optic nerve crush,CONC)模型并分组:CONC-Brn3b过表达组和CONC-阴性对照组。利用视网膜铺片和切片免疫荧光标记相关蛋白表达量,检测Brn3b过表达对视神经损伤条件下RGCs、Brn3b和Caspase3蛋白表达的影响,并对其共定位情况做出统计分析。结果与阴性对照组相比,Brn3b过表达组小鼠视网膜Brn3b的表达水平明显增加。在小鼠CONC模型制作后的第7天RGCs的总凋亡数量达到65%,第14天RGCs的凋亡数量未见进一步改变。免疫荧光标记的蛋白表达量及其共定位分析显示,在视神经损伤条件下,与CONC-阴性对照组相比,CONC-Brn3b过表达组小鼠RGCs的凋亡量以及凋亡因子Caspase3的表达量均明显减少,差异均有统计学意义(均为P<0.001)。结论 Brn3b基因对视神经损伤条件下RGC具有明确的保护作用,Brn3b基因对凋亡因子Caspase3的表达可能具有一定的抑制作用。

Objective To investigate the protective effects of Brn3 b overexpression on retinal ganglion cells（ R GC s） in the condition of optic nerve injury. Methods Sixty healthy BALB/c adult male mice were selected and the right eyeball posterior optic nerves of the mouse were clamped with a mini optic nerve clip. According to the number of days after the optic nerve injury,the mice were divided into the 1 st,3 rd,5 th,7 th and 14 th day group,and the normal left eyes of mice were as the blank group. Forty healthy BALB/c adult male mice were selected and the adeno-associated virus vector was transfected into the retina of mice by microinjector in the vitreous cavity of the right eyes to establish Brn3 b overexpression model,and then grouped into Brn3 b overexpression group,which was transfected with Brn3 b overexpressed adeno-associated viral vector（ AAV-C MV-Brn3 b） and negative control group,which was transfected with blank adeno-associated viral vector（ AAV-C MV-GFP）,with 20 mice in each group. Subsequently,10 mice were taken respectively from the Brn3 b overexpression group and the negative control group,and the right eyeball posterior optic nerves of the mice were clamped with a mini optic nerve clip to build the controlled optic nerve crush（ C O NC） model and then grouped into C O NC-Brn3 b overexpression group and C O NC-negative control group. The expression of R GC s,Brn3 b and C aspase3 under the condition of optic nerve injury was detected by using the expression of Brn3 b overexpression in the retinal plaque and section immunofluorescence labeling protein,and the results were statistically analyzed. Results C ompared with the negative control group,the expression level of retinal Brn3 b increased significantly in the Brn3 b overexpression group. The total apoptosis of R GC s was 65% on day 7 after the C O NC modeling,and the apoptosis of R GC s was not significantly changed on day 14. Immunofluorescence labeled protein and co-localization analysis show ed that,compared with the C O NC-negative control group,R GC s apoptosis and the expression of apoptosis factor C aspase3 volume in the C O NC-Brn3 b overexpression group were significantly decreased under the condition of optic nerve injury,and the differences were statistically significant（ all P 0. 001）. Conclusion Brn3 b gene has a definite protective effect on R GC under the condition of optic nerve injury. Brn3 b gene may inhibit the expression of apoptotic factor C aspase3.