葡萄糖氧化酶诱导大鼠血管纹边缘细胞氧化应激模型的建立与评估

Establishment and Evaluation of Glucose Oxidase-induced Oxidative Stress Model in Rat Cochlear Marginal Strial Cells

胡泠影;张园园;曾峰;华清泉;

1:武汉大学人民医院耳鼻咽喉头颈外科

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。
关键词
氧化应激;葡萄糖氧化酶;过氧化损伤
基金项目(Foundation):
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
作者
胡泠影;张园园;曾峰;华清泉;
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