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山东大学学报（理学版） ›› 2016, Vol. 51 ›› Issue (5): 11-17.doi: 10.6040/j.issn.1671-9352.0.2015.635

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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

王德平,樊文艺,温茜,樊廷俊*   

  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
  • 收稿日期:2015-12-29 出版日期:2016-05-20 发布日期:2016-05-16
  • 通讯作者: 樊廷俊(1964— ),男,博士,教授,博士生导师,研究方向为动物细胞工程与细胞分化.E-mail:tjfan@ouc.edu.cn E-mail:wdplovef@163.com
  • 作者简介:王德平(1989— ),女,硕士,研究方向为细胞工程.E-mail:wdplovef@163.com
  • 基金资助:
    国家高技术研究发展计划(863计划)资助项目(2006AA02A132)

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

WANG De-ping, FAN Wen-yi, WEN Qian, FAN Ting-jun*   

  1. Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong, China
  • Received:2015-12-29 Online:2016-05-20 Published:2016-05-16

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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MTT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。

关键词: 氧氟沙星, 细胞毒性, 线粒体依赖性信号途径, 人角膜上皮细胞, 细胞凋亡

Abstract: 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 flow cytometry(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 concentration 山 东 大 学 学 报 (理 学 版)第51卷 - 第5期王德平,等: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 \=-has 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.

Key words: cytotoxicity, apoptosis, mitochondrion-dependent signaling pathway, ofloxacin, human cornel epithelial cells

中图分类号: 

  • R772.2
[1] WHITCHER J P, SRINIVASAN M, UPADHYAY M P. Corneal blindness:a global perspective[J]. Bull World Health Organ, 2001, 79(3):214-221.
[2] KINOSHITA S, ADACHI W, SOTOZONO C, et al. Characteristics of the human ocular surface epithelium[J]. Prog Retin Eye Res, 2001, 20(5):639-673.
[3] LOMA P, GUZMAN-ARANGUEZ A, PÉREZ DE LARA M J, et al. Diadenosine tetraphosphate induces tight junction disassembly thus increasing corneal epithelial permeability[J]. Br J Pharmacol, 2015, 172(4):1045-1058.
[4] DAJCS J J, MOREAU J M, THIBODEAUX B A, et al. Effectiveness of ciprofloxacin and ofloxacin in a prophylaxis model of Staphylococcus keratitis[J]. Cornea, 2001, 20(8):878-880.
[5] KOVOOR T A, KIM A S, MCCULLEY J P, et al. Evaluation of the corneal effects of topical ophthalmic fluoroquinolones using in vivo confocal microscopy[J]. Eye Contact Lens, 2004, 30(2):90-94.
[6] SOSA A B, EPSTEIN S P, ASBELL P A. Evaluation of toxicity of commercial ophthalmic fluoroquinolone antibiotics as assessed on immortalized corneal and conjunctival epithelial cells[J]. Cornea, 2008, 27(8):930-934.
[7] CASTRO-MUÑOZLEDO F. Corneal epithelial cell cultures as a tool for research, drug screening and testing[J]. Exp Eye Res, 2008, 86(3):459-469.
[8] FAN T J, XU B, ZHAO J, et al. Establishment of an untransfected human corneal epithelial cell line and its biocompatibility with denuded amniotic membrane[J]. Int J Ophthalmol, 2011, 4(3):228-234.
[9] CASTRO-MUÑOZLEDO F. Corneal epithelial cell cultures as a tool for research, drug screening and testing[J]. Exp Eye Res, 2008, 86(3):459-469.
[10] 孙倩,樊廷俊,邱月,等. 贝特舒对人角膜上皮细胞凋亡诱导作用的实验研究[J]. 山东大学学报(理学版), 2013, 48(7):14-19. SUN Qian, FAN Tingjun, QIU Yue, et al. Experimental studies of the apoptosis-inducing effect of betaxolol on human corneal endothelial cells[J].Journal of Shandong University(Natural Science), 2013, 48(7):14-19.
[11] AYAKI M, IWASAWA A, YAGUCHI S, et al. In vitro assessment of the cytotoxicity of anti-allergic eye drops using 5 cultured corneal and conjunctival cell lines[J]. J Oleo Sci, 2011, 60(3):139-144.
[12] BRUSTMANN H. Apoptotic bodies as a morphological feature in serous ovarian carcinoma:correlation with nuclear grade, Ki-67 and mitotic indices[J]. Pathol Res Pract, 2002, 198(2):85-90.
[13] LEITE M, QUINTA-COSTA M, LEITE P S, et al. Critical evaluation of techniques to detect and measure cell death-study in a model of UV radiation of the leukaemic cell line HL60[J]. Anal Cell Pathol, 1999, 19(3/4):139-151.
[14] OBERHAMMER F, WILSON J W, DIVE C, et al. Apoptotic death in epithelial cells:cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation[J]. EMBO J, 1993, 12(9):3679-3684.
[15] VERMES I, HAANEN C, STEFFENS-NAKKEN H, et al. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine early apoptotic cells using fluorescein labeled expression on Annexin V[J]. J Immunol Methods, 1995, 184(1):39-51.
[16] 苗莹,王瑞鑫,于昊泽,等. 利多卡因对人角膜上皮细胞的毒性作用及其机理研究[J]. 山东大学学报(理学版), 2014, 49(1):8-14. MIAO Ying, WANG Ruixin, YU Haoze, et al. Cytotoxic effects of lidocaine on human corneal epithelial cells and its underlying mechanisms[J].Journal of Shandong University(Natural Science), 2014, 49(1):8-14.
[17] ZHANG F T, DING Y, SHAH Z, et al. TNF/TNFR1 pathway and endoplasmic reticulum stress are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes[J]. Toxicol Appl Pharmacol, 2014, 276(2):121-128.
[18] FAN T J, HAN L H, CONG R S, et al. Caspase family proteases and apoptosis[J]. Acta Biochim Biophys Sin(Shanghai), 2005, 37(11):719-727.
[19] SHENG Z, CAO X, PENG S, et al. Ofloxacin induces apoptosis in microencapsulated juvenile rabbit chondrocytes by caspase-8-dependent mitochondrial pathway[J]. Toxicol Appl Pharmacol, 2008, 226(2):119-127.
[20] SUN X M, MACFARLANE M, ZHUANG J, et al. Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis[J]. J Biol Chem, 1999, 274(8):5053-5060.
[21] ADAMS J M, CORY S. The Bcl-2 protein family:arbiters of cell survival[J]. Science, 1998, 281(5381):1322-1326.
[22] HAJJI N, WALLENBORG K, VLACHOS P, et al. Combinatorial action of the HDAC inhibitor trichostatin A and etoposide induces caspase-mediated AIF-dependent apoptotic cell death in non-small cell lung carcinoma cells[J]. Oncogene, 2008, 27(22):3134-3144.
[23] BRENNER C, KROEMER G. Apoptosis. Mitochondria-the death signal integrators[J]. Science, 2000, 289(5482):1150-1151.
[1] 苗莹,王瑞鑫,于昊泽,于苗苗,葛源,樊廷俊*. 利多卡因对人角膜上皮细胞的毒性#br# 作用及其机理研究[J]. 山东大学学报（理学版）, 2014, 49(1): 8-14.
[2] 孙倩，樊廷俊*，邱月，葛源，于苗苗. 贝特舒对人角膜上皮细胞凋亡诱导作用的实验研究[J]. J4, 2013, 48(7): 14-19.
[3] 徐晓辉,樊廷俊*，景毅,姜国建,杨秀霞,葛源. 氯化镉对条斑星鲽卵巢细胞的毒性作用及其机理研究[J]. J4, 2013, 48(11): 1-6.
[4] 李秀娥1,高素莲2,张秋3*. 功能化多壁碳纳米管对NHFB细胞的毒性研究[J]. J4, 2010, 45(5): 6-11.
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