shRNA沉默APE/Ref1对体外培养大鼠耳蜗螺旋神经节细胞过氧化氢损伤的影响  

Effects on survival of shRNA mediated APE/Ref1 gene silencing in rat spiral ganglion cells in oxidative stress

作　　者：姜振东[1] 钟诚[1] 李太军[1] 向召兰[1] 张学渊[1] 

机构地区：[1]第三军医大学附属西南医院耳鼻咽喉头颈外科,重庆400038

出　　处：《中华耳鼻咽喉头颈外科杂志》2014年第2期145-150,共6页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

摘　　要：目的 通过转染pGenesil-APE/Ref1-shRNA抑制体外培养大鼠耳蜗螺旋神经节细胞（spiral ganglion cell,SGC）中无嘌呤无嘧啶核酸内切酶/氧化还原因子1（apurinic/apyrimidimic endonuclase/redox factor 1,APE/Ref1）的表达,观察SGC在过氧化氢（H2O2）所致氧化应激环境中的活性及凋亡情况,探讨APE/Ref1在SGC抗氧化损伤过程中的作用.方法 体外培养大鼠SGC,加入pGenesil-APE/Ref1-shRNA转染72 h,更换培养基后加入不同浓度的H2O2 （0、10、25、50、100、300μmol/L）干预1h,更换正常培养基后继续培养24h.通过免疫印迹、分光光度检测法、原位缺口末端标记法（TUNEL）、四甲基偶氮唑蓝法（MTT）分别检测SGC APE/Refl、磷酸化组蛋白H2AX的表达、caspase3活性、细胞活性以及凋亡情况.结果 pGenesil-APE/Ref1-shRNA能明显抑制APE/Ref1的表达,H2O2损伤浓度在50 ～ 300 μmol/L时,与对照组相比,pGenesil-APE/Ref1-shRNA组磷酸化组蛋白H2AX表达增加,caspase 3活性增加,细胞凋亡率增高,细胞活性降低,差异具有统计学意义（P值均＜0.05）.结论 pGenesil-APE/Ref1-shRNA抑制APE/Refl表达后,大鼠体外培养SGC抗氧化损伤能力降低;APE/Ref1可能通过DNA修复功能,实现对SGC的保护作用.Objective To investigate the effects of reducing APE/Refl expression in the cultures of rat spiral ganglion cells with oxidative damage induced by H2O2.Methods Primary cultured rat spiral ganglion cells were infected with small interfering RNA to APE/Refl （Apel siRNA） for 72 h,followed by treating with H2O2（0,10,25,50,100 and 300 μmol/L） for 1 h,and then cultured in normal medium for 24 h.Western blot were used to detect the level of APE/Refl protein and phosphorylation of histone protein H2AX in the infected cells.The caspase3 activation was tested by spectrophotometric method.The cell viability was determined by MTT and the apoptosis of spiral ganglion cells was determined by terminal-deoxynucleotidyl transferase mediated nick and labeling （TUNEL）.Results Western blot showed that infection with Ape1siRNA resulted in APE/Ref1 reduced expression in the spiral ganglion cells.Exposing spiral ganglion cultures with reduced expression of APE/Ref1 to H2O2 （50,100,300 μmol/L） for 1 h resulted in increasing in the phosphorylation of histone protein H2AX.The reduction in APE/Refl significantly reduced cell viability in cultures 24 h after 1 h expression to 50-300 μmol/L H2O2.The apoptosis of cells and caspase 3 activity was detected significantly improved.Conclusions The induced of APE/Refl results in significantly decrease in spiral ganglion cells viability in oxidative stress.The repairing function of APE/Refl is necessary for optimal levels of neuronal rat spiral ganglion cells survival.

关 键 词：螺旋神经节 RNA 小分子干扰 DNA-(无嘌呤或无嘧啶位点)裂合酶 氧化性应激 过氧化氢 

分 类 号：R764.4[医药卫生—耳鼻咽喉科]