蓝光所致人视网膜色素上皮细胞损伤与细胞内钙离子含量变化的关系  

The association of blue light-induced human retinal pigment epithelium cell damage with intercellular freeCa^2＋ change in vitro

作　　者：宿罡[1] 宫鑫[1] 蔡善君[1] 李海辉[1] 

机构地区：[1]遵义医学院附属医院眼科,563003

出　　处：《中华实验眼科杂志》2013年第8期734-738,共5页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81060079）;贵州省教育厅重点项目[（黔教科）2008034]

摘　　要：背景 对蓝光照射所致视网膜色素上皮（RPE）细胞损伤与细胞内钙离子含量变化关系的深入研究可能对探讨视网膜变性类疾病的发生机制及防治具有重要意义. 目的 建立人RPE细胞蓝光损伤模型,探讨RPE细胞损伤与细胞内钙离子含量变化的关系.方法 将人RPE细胞系培养传代,锥虫蓝染色评估细胞活力.第4代人RPE细胞分别用（2000±500） lx蓝光照射3、6、9、12h,终止细胞培养24 h后,采用TUNEL法检测不同时间点细胞的凋亡情况,确定最适宜的光照度.然后将RPE细胞分为无光照组、光照组、硝苯地平组、光照＋硝苯地平组、（-）BayK8644组、光照＋（-）BayK8644组.除无光照组外,其他各组细胞均用蓝光照射6h,终止细胞培养24h,采用激光扫描共焦显微镜检测细胞内游离钙离子含量,激发光波长为488 nm,发射光波长为505 nm,每组随机选取30个细胞,扫描细胞图像,采用分析软件分析各组细胞内游离钙离子的荧光强度.结果 锥虫蓝染色证实RPE细胞的活力均在90％以上,TUNEL染色和免疫组织化学染色证实光照3h组未见细胞凋亡,而光照后6、9、12h发现不同数量的凋亡细胞.硝苯地平组RPE细胞内游离钙离子荧光强度比无光照组和光照＋硝苯地平组低,差异均有统计学意义（均P=0.000）；光照组、（-）BayK8644组、光照＋（-）BayK8644组RPE细胞内钙离子荧光强度高于无光照组,差异均有统计学意义（均P=0.000）；光照＋硝苯地平组与无光照组相比较,（-）BayK8644组与光照＋（-）BayK8644组比较,差异均无统计学意义（P=0.339、0.410）. 结论 光照度（2000±500）1x、光照6h、终止培养24 h为建立蓝光照射致人RPE细胞损伤模型的最适合条件.蓝光照射所致的人RPE细胞损伤与细胞内游离钙离子含量增加有关.Background Investigating the association of blue light-induced damage of retinal pigmenepithelial （RPE） cellwith intracellulaCa2＋ conteniimportanfounderstanding the mechanism of retinal disorders.Objective Thistudy wato establish blue light-induced damage model of human RPE celland explore the relationship between the damage of RPE cell and intracellulaCa2＋ content.MethodHuman RPE cellwere cultured and passaged.Cell vitality waassayed by trypan blue staining.Fourth-generation cellwere used in these experiments.The cellwere exposed to blue lighwith an intensity of （2000±500）lx fo3,6,9 o12 hours,and the rate of apoptosiwaassayed by TUNEL to assesthe optimal irradiation time focellcultured.The cellwere then randomized into the withouirradiation group,irradiation only group,nifedipine group,ligh＋ nifedipine group,（-） BayK8644 group and ligh＋ （-） BayK8644 group.The laseconfocal microscope waused to determine the fluorescence intensity of intracellulafree Ca2＋ aan excitation wavelength of 488 nm and an emission wavelength of 505 nm.The cell imagewere analyzed using computesoftware.The differenceof fluorescence intensity among the differengroupwere compared by one-way analysiof variance.ResultTrypan blue staining showed thathe viability of RPE cellwamore than 90% afteculturing and passaging.No apoptoticell waseen aftelighexposure fo3 hours.However,differennumberof apoptoticellappeared aftelighexposure fo6,9 and 12 hours.The fluorescence intensity of intracellulafree Ca2＋ in the nifedipine group wasignificantly lowethan thaof the withouirradiation group othe ligh＋ nifedipine group（both aP=0.000）.Lasescanning confocal microscopy showed thathe fluorescence intensitieof intracellulafree Ca2＋ in the irradiation only group,（-） BayK8644 group,ligh＋ （-）BayK8644 group were highethan thaof the withouirradiation group,with statistical significancebetween them（all P=0.000）.No significandifferencewere found in the fluorescence intensity of intracellulafree Ca2＋ between the ligh＋

关 键 词：光 损伤 视网膜色素上皮 钙离子 细胞内 游离 拮抗剂 激动剂 凋亡 

分 类 号：R774.1[医药卫生—眼科]