蓝光诱导的人视网膜色素上皮细胞的氧化损伤及其线粒体机制  

Oxidative damage of human retinal pigment epithelium cells induced by blue light irradiation and mitochondria-participated mechanism

作　　者：邹秀兰[1] 俞永珍 徐哲[1] 张楚[1] 王观峰[3] 邹玉平[1] 

机构地区：[1]广州军区广州总医院眼科,510010 [2]广州中医药大学,510405 [3]广州医科大学第三附属医院眼科,510150

出　　处：《中华实验眼科杂志》2015年第2期129-134,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：广东省科技计划项目（20118031800202、20128031800419）;广东省自然科学基金项目（S2013010012045）;广州市科技计划项目（2014J4100035）

摘　　要：背景 研究表明,线粒体及氧化应激反应在蓝光导致的视网膜光化学损伤机制中具有重要作用,但蓝光对人视网膜色素上皮（RPE）细胞的损伤与照射时间的关系研究较少. 目的 从供体眼球中分离和培养人RPE细胞,研究蓝光诱导的人RPE细胞氧化损伤的可能机制. 方法 从供体眼球中分离和培养RPE细胞,将培养的细胞分为正常对照组（0h组）、光照0.5、1、2、3、4、5、6、12和24 h组.正常对照组细胞培养在暗环境中且不给予蓝光照射,光照组细胞给予辐射强度为（4.0±0.5） mW/cm2的蓝光照射,按分组分别照射相应时间.采用MTT法检测各组RPE细胞的活性,透射电子显微镜下观察各组细胞超微结构的变化;采用流式细胞术检测RPE细胞中活性氧簇（ROS）的生成量以评价各组细胞的氧化应激反应情况;采用实时荧光定量PCR技术检测RPE细胞线粒体中烟酰胺腺嘌呤二核苷酸磷酸（NADPH）及环氧合酶1（COX1） mRNA的相对表达量,评价RPE细胞中线粒体的功能.结果 正常对照组的细胞存活率为（100.00±20.00）％,光照1、2、3、4、5、6、12、24 h组人PRE细胞存活率分别为（95.73±0.89）％、（94.67±2.56）％、（84.23±0.16）％、（78.57±3.09）％、（75.43±2.18）％、（66.13±1.42）％、（53.43±1.91）％和（47.97±1.36）％,各组细胞存活率的总体比较差异有统计学意义（F=172.270,P=0.000）,其中光照3、4、5、6、12、24 h组细胞存活率与正常对照组比较均显著下降,差异均有统计学意义（均P＜0.05）.透射电子显微镜下可见光照6、12、24 h组RPE细胞空泡样变、线粒体肿胀、微绒毛减少甚至消失等改变.流式细胞术检测发现,正常对照组、光照0.5、1、2、3、4、5、6h组人RPE细胞中ROS的相对量分别为（14.75±2.49）％、（19.04±1.02）％、（22.81±3.20）％、（28.75±2.15）％、（33.06±0.96）％、（40.64±2.11）％、（48.25±2.50）％和（60.44�Background Researches showed that mitochondria and oxidative stress play a crucial role in retinal photochemical injury,but the relationship between the damage of human retinal pigment epithelium （RPE） cell-induced by blue light and light-irradiated time is less studied.Objective The aim of this study was to research the possible mechanism of RPE oxidative damage induced by blue light in vitro.Methods Human RPE cells were isolated from healthy donors and cultured.The cells were divided into the normal control group and the light exposure group.The cells of light exposure group were irradiated using the blue light of （4.0±0.5） mW/cm2 for 0.5,1,2,3,4,5,6,12 and 24 hours,respectively,and the cells of the normal control group were cultured in dark environment.Cellular viability was detected by MTT method,and the ultrastructure change of subcellular organelles in RPE cells was examined under the transmission electron microscope （TEM）.The content of reactive oxygen species （ROS） was assayed by flow cytometry for the assessment of oxidative stress reaction.The relative expressions of nicotinamide adenine dinucleotide phosphate （NADPH） mRNA and cyclooxygenase 1 （COX1） mRNA in the cells were detected by real-time fluorescence quantitative PCR to evaluate the mitochondria function.Results The percentages of cellular viability were （100.00±20.00） %,（95.73±0.89） %,（94.67±2.56） %,（84.23±0.16） %,（78.57±3.09）%,（75.43±2.18）%,（66.13±1.42）%,（53.43±1.91）% and （47.97±1.36）% in the normal control group and light exposure for 1-hour,2-hour,3-hour,4-hour,5-hour,6-hour,12-hour and 24-hour groups,respectively,showing a significant difference among the groups （F =172.270,P =0.000）,and the percentages of light exposure for the more than 3 hours groups were significantly lower than those of the normal control group （all at P〈 0.05）.The vacuoles-like degeneration,mitochondrial swelling,decreased microvilli were seen under the TEM.The contents of ROS in RPE cells

关 键 词：氧化应激 活性氧簇 人视网膜色素上皮细胞 

分 类 号：R774.1[医药卫生—眼科]