过表达Krüppel样因子7对小鼠视神经钳夹后视网膜神经节细胞保护作用及机制研究

Protective effect and mechanism of Krüppel-like factor 7 overexpression on retinal ganglion cells in mice after optic nerve clamp

目的观察过表达Krüppel样因子7(KLF7)对视神经钳夹损伤(ONC)后小鼠视网膜神经节细胞(RGC)的保护作用和机制。方法10周龄C57BL/6J小鼠65只随机分为空白对照组(A组)、玻璃体腔注射(IVT)-KLF7组(B组)、IVT-磷酸盐缓冲液-ONC组(C组)、IVT-KLF7-ONC组(D组)、IVT-重组腺相关病毒2-重组绿色荧光蛋白-ONC组(E组),每组各13只小鼠。建立ONC模型后7 d,处死各组小鼠。全视网膜铺片免疫荧光染色计数RGC存活率;蛋白质免疫印迹法检测小鼠视网膜组织中KLF7、神经生长因子(NGF)、酪氨酸激酶A(TrkA)、磷酸化TrkA(pTrkA)、酪氨酸激酶B(TrkB)、磷酸化TrkB(pTrkB)、生长相关蛋白43(GAP43)、脑源性神经营养因子、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(BAX)、胱天蛋白酶3(Caspase-3)蛋白相对表达水平。组间比较采用单因素方差分析或Kruskal-Wallis检验。结果ONC后7 d,A组、B组、C组、D组、E组视网膜中RGC密度分别为(3707.4±12.8)、(3582.4±13.3)、(1396.3±16.1)、(1658.3±22.2)、(1323.6±16.9)个/mm^(2);与C组、E组比较,D组RGC密度显著升高,差异有统计学意义(P=0.028、0.007)。与A组、B组、C组、E组比较,D组小鼠视网膜中NGF、pTrkA、pTrkB、GAP43、Bcl-2蛋白相对表达量升高,BAX、Caspase-3蛋白相对表达量降低,差异均有统计学意义(P<0.01)。结论小鼠ONC模型中,过表达KLF7可相对提高RGC存活率,提高视网膜中NGF、pTrkA、pTrkB、GAP43、Bcl-2蛋白相对表达量,降低BAX、Caspase-3蛋白相对表达量。

Objective To explore the protective effect and mechanism of Krüppel-like factor 7(KLF7)overexpression on retinal ganglion cells(RGC)in mice after optic nerve crush(ONC).Methods A total of sixty five 10-week-old C57BL/6J mice were randomly divided into five groups:blank control group(group A),intravitreal injection(IVT)-KLF7 group(group B),IVT-phosphate buffer saline-ONC group(group C),IVTKLF7-ONC group(group D),and IVT-recombinant adeno-associated virus 2-enhanced green fluorescent protein-ONC group(group E),with 13 mice in each group.On the 7 days after the ONC model,the mice in each group were killed.RGC survival rate was counted by whole retina flat mount and immunofluorescence techniques.KLF7,nerve growth factor(NGF),tyrosine kinase A(TrkA),phosphorylated TrkA(pTrkA),tyrosine kinase B(TrkB)and phosphorylated TrkB(pTrk)were detected by western blot,growth associated protein 43(GAP43),brain-derived neurotrophic factor,B lymphoblastoma-2(Bcl-2),Bcl-2 associated X protein(BAX),Caspase-3 protein relative expression levels.One-way analysis of variance or Kruskal-Wallis test were used for comparison between groups.Results On the 7 days after the ONC model,the density of RGC in the retina of groups A,B,C,D and E were(3707.4±12.8),(3582.4±13.3),(1396.3±16.1),(1658.3±22.2)and(1323.6±16.9)/mm^(2),respectively.Compared with groups C and E,RGC density in group D was significantly increased,and the difference was statistically significant(P=0.028,0.007).Compared with groups A,B,C and E,the relative expression levels of NGF,pTrkA,pTrkB,GAP43 and Bcl-2 proteins in the retina of mice in group D were increased,while the relative expression levels of BAX and Caspase-3 proteins were decreased,with statistical significance(P<0.01).Conclusion In mouse ONC model,overexpression of KLF7 can improve RGC survival rate,increase the relative expression levels of NGF,pTrkA,pTrkB,GAP43 and Bcl-2 proteins in retina,and decrease the relative expression levels of BAX and Caspase-3 proteins.