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

发布时间：2018-02-09 04:36

本文关键词： 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　出处：《山东大学学报(理学版)》2016年05期 　论文类型：期刊论文

【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。
[Abstract]:The cytotoxicity and cell and molecular mechanism of ofloxacin OFX, a commonly used ophthalmic antibiotic, were studied in human corneal epithelial cell line (HCEP) in vitro. HCEP cells cultured in vitro were treated with different concentrations of OFX. Cell cycle arrest and apoptosis were detected by flow cytometry, acridine orange / ethidium bromide double staining DNA gel electrophoresis and transmission electron microscopy. The activation of cystatin, the content of mitochondrial specific apoptosis-activating protein and the breakdown of mitochondrial transmembrane potential (MTP) were identified by Western blot and flow cytometry. The results showed that OFX was more than 0.375 g / L in mass concentration. In a dose-and time-dependent manner, HCEP cells showed cytopathic effects, decreased activity and increased plasma membrane permeability, and resulted in cell cycle arrest in S phase, fragmentation of phosphatidyl serine valgus DNA and formation of apoptotic corpuscles. The activation of cystatin 2 and 3, the up-regulation of cytochrome c and apoptosis-inducing factor in cytoplasm, and the disintegration of MTP. Cell cycle arrest and apoptosis can induce cytotoxicity to HCEP cells, which is regulated by mitochondrial dependent signaling pathway mediated by death receptor.
【作者单位】：中国海洋大学海洋生命学院角膜组织工程实验室;
【基金】：国家高技术研究发展计划(863计划)资助项目(2006AA02A132)
【分类号】：R772.2

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