摘要
目的采用葡萄糖氧化酶(glucose oxidase,GO)作用于体外培养的血管纹边缘细胞(marginal strial cells,MC)建立大鼠MC的氧化应激模型,评价该模型的可靠性。方法提取幼年大鼠耳蜗MC,原代培养并鉴定后通过CCK8法检测GO对MC损伤的半抑制浓度(half maximal inhibilory concentration,IC50);然后用IC50浓度的GO处理MC作为实验组,不加GO处理的MC作为对照组,各处理24小时后,比较各组结果。流式细胞术和免疫荧光检测两组细胞中活性氧(ROS)的含量,WST试剂盒检测两组超氧化物歧化酶(SOD)的活性,Westernblot检测两组细胞聚腺苷酸二磷酸核糖转移酶-1(PARP1)和凋亡蛋白cleaved-caspase-3的表达,流式细胞术检测细胞凋亡率,用荧光试剂盒Hoechst/PI法观测凋亡细胞的形态学改变,比较两组结果。结果 GO对MC损伤的IC50为32.9mU/ml;与对照组相比较,实验组MC内ROS含量升高、SOD活降低,cleaved-caspase-3和PARP1表达升高,MC凋亡率增多,细胞核发生皱缩、碎裂。结论 GO作用于体外培养的大鼠MC可成功制作稳定可靠的氧化应激损伤模型,促使MC发生凋亡。
Objective To establish an oxidative stress model of rat cochlear marginal strial cells（MC）using glucose oxidase（GO）in vitro,and evaluate its stability and reliability.Methods The IC50 value of GO damage to MC cells,which was extracted from the cochlea of juvenile rats,was detected by CCK8 method,and the MC cells treated with GO in half maximal inhibitory concentration（IC50）were used as the experimental group,and untreated MC cells were used as the control group,and the results were compared after 24 hours.The content of reactive oxygen species（ROS）was investigated byflow cytometry and immunofluorescence,and the activity of superoxide dismutase（SOD）was detectedbyWST kit;western-blot was adopted to detected the expression of PARP1 and cleaved-caspase-3 in each group,and apoptosis rate was detected by flowcytometry,and morphological changes were observed by immunofluorescence.Results The content of intracellular ROS increased and the activity of SOD decreased in the GO group.The IC50 in GO group was 32.9 mU/ml.Compared with those of in the control group,the expressions of cleaved-caspase-3 and PARP1 increased.Conclusion GO can be used to construct stable and reliable MC oxidative stress model in rat.
作者
胡泠影
张圆圆
曾峰
华清泉
Hu Lingying;Zhang Yuanyuan;Zeng Feng;Hua Qingquan(Department of Otolaryngology Head and Neck Surgery,Renming Hospital,Wuhan University,Wuhan,430060,Chin)
出处
《听力学及言语疾病杂志》
CAS
CSCD
北大核心
2018年第3期298-302,共5页
Journal of Audiology and Speech Pathology
基金
国家自然科学基金应急管理项目(81541001)
国家自然科学基金青年基金(81700916)
湖北省自然科学青年基金(2017CFB242)
湖北省卫生和计划生育委员会青年人才项目(WJ2017Q005)资助
关键词
氧化应激
葡萄糖氧化酶
过氧化损伤
Oxidative stress
Glucose oxidase（GO）
Peroxidative injury
