氧氟沙星对人角膜上皮细胞的毒性作用及其细胞与分子机理研究  

Cytotoxicity of ofloxacin to human corneal epithelial cells and its cellular and molecular mechanisms

作　　者：王德平[1] 樊文艺 温茜[1] 樊廷俊[1] 

机构地区：[1]中国海洋大学海洋生命学院角膜组织工程实验室,山东青岛266003

出　　处：《山东大学学报（理学版）》2016年第5期11-17,共7页Journal of Shandong University(Natural Science)

基　　金：国家高技术研究发展计划(863计划)资助项目(2006AA02A132)

摘　　要：以非转染人角膜上皮(HCEP)细胞系为体外实验模型,研究了眼科常用抗生素药物氧氟沙星(ofloxacin,OFX)的细胞毒性及其细胞与分子机理。体外培养的HCEP细胞用不同浓度OFX处理后,分别利用光镜和M TT方法测定细胞病变效应和细胞活力,用流式细胞术、吖啶橙/溴化乙锭双染色、DNA凝胶电泳和透射电镜检测细胞周期阻滞和细胞凋亡,使用ELISA、Western杂交和流式细胞术鉴定胱天蛋白酶的激活、胞质中线粒体特有凋亡激活蛋白的含量和线粒体跨膜电位(MTP)的崩解。结果显示,OFX在质量浓度大于0.375 g/L时,可剂量和时间依赖性地引起HCEP细胞出现细胞病变效应、活力下降和质膜通透性升高,并引起细胞周期阻滞在S期,磷脂酰丝氨酸外翻,DNA断片化,凋亡小体形成,胱天蛋白酶-2、-9和-3的激活,胞质中细胞色素c和凋亡诱导因子含量的上调,以及MTP崩解。可见,OFX在质量浓度为其临床治疗质量浓度的1/8以上时,能通过诱导细胞周期阻滞和细胞凋亡对HCEP细胞产生显著细胞毒性,其凋亡诱导作用要受到死亡受体介导的线粒体依赖性信号途径的调控。To define the cytotoxicity of ofloxacin( OFX),a widely used antibiotic drug in eye clinic,to human corneal epithelium and the possible cellular and molecular mechanisms,the cytotoxic effect,apoptosis-inducing effect and apoptosis-triggering pathways of OFX were investigated using an in vitro model of HCEP cells in this study. After in vitro cultured HCEP cells were treated with different concentrations of OFX for different time,the morphology,cytopathic effect( CPE) and viability were assessed by light microscopy and MTT assay,the cell cycle arrest and apoptosis were detected by flowcytometry( FCM),AO / EB double-staining,gel electrophoresis and transmission electron microscopy,and caspase activation,cytoplasmic amount of mitochondrion-specific apoptosis-triggering proteins and mitochondrial transmembrane potential( MTP) disruption were quantified using ELISA,Western blot and FCM. Our results revealed that OFX above concentrations of 0. 375 g / L induced morphological abnormality,CPE formation,viability decline and plasma membrane permeability elevation of HCEP cells in a dose-and time-dependent manner. Moreover,OFX could arrest the cells at S phase of the cell cycle and induce phosphatidylserine externalization,DNA fragmentation,apoptotic body formation,activations of caspase-2,-9,and-3,up-regulation of cytoplasmic cytochrome c and apoptosis inducing factor,and disruption of MTP. In conclusion,OFX above 1 /8 of its clinical therapeutic concentrationhas a significant cytotoxicity to HCEP cells by inducing cell cycle arrest and apoptosis which is regulated by a death receptor-mediated mitochondrion-dependent signaling pathway.

关 键 词：氧氟沙星 人角膜上皮细胞 细胞毒性 细胞凋亡 线粒体依赖性信号途径 

分 类 号：R772.2[医药卫生—眼科]