中华耳鼻咽喉头颈外科杂志
中華耳鼻嚥喉頭頸外科雜誌
중화이비인후두경외과잡지
CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
2014年
2期
145-150
,共6页
姜振东%钟诚%李太军%向召兰%张学渊
薑振東%鐘誠%李太軍%嚮召蘭%張學淵
강진동%종성%리태군%향소란%장학연
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
螺鏇神經節%RNA,小分子榦擾%DNA-(無嘌呤或無嘧啶位點)裂閤酶%氧化性應激%過氧化氫
라선신경절%RNA,소분자간우%DNA-(무표령혹무밀정위점)렬합매%양화성응격%과양화경
Spiral ganglion%RNA,small interfering%DNA-(apurinic or apyrimidinic site) lyase%Oxidative stress%Hydrogen peroxide
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.
目的 通過轉染pGenesil-APE/Ref1-shRNA抑製體外培養大鼠耳蝸螺鏇神經節細胞(spiral ganglion cell,SGC)中無嘌呤無嘧啶覈痠內切酶/氧化還原因子1(apurinic/apyrimidimic endonuclase/redox factor 1,APE/Ref1)的錶達,觀察SGC在過氧化氫(H2O2)所緻氧化應激環境中的活性及凋亡情況,探討APE/Ref1在SGC抗氧化損傷過程中的作用.方法 體外培養大鼠SGC,加入pGenesil-APE/Ref1-shRNA轉染72 h,更換培養基後加入不同濃度的H2O2 (0、10、25、50、100、300μmol/L)榦預1h,更換正常培養基後繼續培養24h.通過免疫印跡、分光光度檢測法、原位缺口末耑標記法(TUNEL)、四甲基偶氮唑藍法(MTT)分彆檢測SGC APE/Refl、燐痠化組蛋白H2AX的錶達、caspase3活性、細胞活性以及凋亡情況.結果 pGenesil-APE/Ref1-shRNA能明顯抑製APE/Ref1的錶達,H2O2損傷濃度在50 ～ 300 μmol/L時,與對照組相比,pGenesil-APE/Ref1-shRNA組燐痠化組蛋白H2AX錶達增加,caspase 3活性增加,細胞凋亡率增高,細胞活性降低,差異具有統計學意義(P值均＜0.05).結論 pGenesil-APE/Ref1-shRNA抑製APE/Refl錶達後,大鼠體外培養SGC抗氧化損傷能力降低;APE/Ref1可能通過DNA脩複功能,實現對SGC的保護作用.
목적 통과전염pGenesil-APE/Ref1-shRNA억제체외배양대서이와라선신경절세포(spiral ganglion cell,SGC)중무표령무밀정핵산내절매/양화환원인자1(apurinic/apyrimidimic endonuclase/redox factor 1,APE/Ref1)적표체,관찰SGC재과양화경(H2O2)소치양화응격배경중적활성급조망정황,탐토APE/Ref1재SGC항양화손상과정중적작용.방법 체외배양대서SGC,가입pGenesil-APE/Ref1-shRNA전염72 h,경환배양기후가입불동농도적H2O2 (0、10、25、50、100、300μmol/L)간예1h,경환정상배양기후계속배양24h.통과면역인적、분광광도검측법、원위결구말단표기법(TUNEL)、사갑기우담서람법(MTT)분별검측SGC APE/Refl、린산화조단백H2AX적표체、caspase3활성、세포활성이급조망정황.결과 pGenesil-APE/Ref1-shRNA능명현억제APE/Ref1적표체,H2O2손상농도재50 ～ 300 μmol/L시,여대조조상비,pGenesil-APE/Ref1-shRNA조린산화조단백H2AX표체증가,caspase 3활성증가,세포조망솔증고,세포활성강저,차이구유통계학의의(P치균＜0.05).결론 pGenesil-APE/Ref1-shRNA억제APE/Refl표체후,대서체외배양SGC항양화손상능력강저;APE/Ref1가능통과DNA수복공능,실현대SGC적보호작용.
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.
