Youn Hyun-Yi, Bantseev Vladimir, Bols Niels C, Cullen Anthony P, Sivak Jacob G
School of Optometry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1.
J Photochem Photobiol B. 2007 Jul 27;88(1):21-8. doi: 10.1016/j.jphotobiol.2007.04.012. Epub 2007 May 8.
The present study demonstrates broadband UV-B-induced damage of cultured human retinal pigment epithelial cells as an effort to develop an in vitro model that can be used, along with in vivo research and other in vitro efforts, to evaluate the need for retinal UV protection in humans after cataract removal. The human retinal pigment epithelial cell line, ARPE-19, was cultured in two groups: control and treated. Treated cells were irradiated with three broadband UVB radiations at energy levels of 0.05, 0.1 and 0.2J/cm(2). After irradiation, cells were incubated for 48h while cellular viability, morphology, and phagocytotic activity were analyzed using the Alamar blue assay, confocal microscopy, and fluorescent microspheres. Confocal analysis concentrated on the study of the cell nuclei and mitochondria. The Alamar blue assay of UV-B-exposed cells showed dose and time-dependent decreases in cellular viability in comparison to control cells. Loss of cell viability was measured at the two higher energy levels (0.2 and 0.1J/cm(2)), but the cell group exposed to 0.05J/cm(2) showed no significant viability change at 1-h time point. Morphological evaluation also showed dose and time-dependent degradation of mitochondria and nucleic acids. Cells exposed with 0.05J/cm(2) UVB did not show significant degradation of mitochondria and nucleic acids during the entire culture period. Phagocytotic activity assay data for UVB-exposed cells showed dose-dependent decreases in phagocytotic activity in comparison with the control cells. The control cells have significantly greater capacities for uptake than the 0.1 and 0.2J/cm(2) UV-B-exposed cells, while the 0.05J/cm(2) UV-B-exposed cell group showed no significant difference from the control cell group. The findings suggest that UVB radiation-induced cultured RPE cell damage can be evaluated by assays that probe cellular viability, morphological change, and phagocytotic activity, and that these assay methods together provide a valuable in vitro model for ultraviolet radiation-induced retinal toxicology research.
本研究证明了宽带UV-B对培养的人视网膜色素上皮细胞的损伤，旨在建立一种体外模型，该模型可与体内研究及其他体外研究相结合，用于评估白内障摘除术后人类视网膜紫外线防护的必要性。人视网膜色素上皮细胞系ARPE-19被分为两组培养：对照组和处理组。处理组细胞接受三种能量水平分别为0.05、0.1和0.2J/cm²的宽带UVB辐射。照射后，细胞孵育48小时，同时使用alamar蓝分析法、共聚焦显微镜和荧光微球分析细胞活力、形态和吞噬活性。共聚焦分析集中于细胞核和线粒体的研究。与对照细胞相比，UV-B照射细胞的alamar蓝分析法显示细胞活力呈剂量和时间依赖性下降。在两个较高能量水平（0.2和0.1J/cm²）下检测到细胞活力丧失，但暴露于0.05J/cm²的细胞组在1小时时间点未显示出显著的活力变化。形态学评估也显示线粒体和核酸呈剂量和时间依赖性降解。暴露于0.05J/cm²UVB的细胞在整个培养期间未显示出线粒体和核酸的显著降解。UVB照射细胞的吞噬活性分析数据显示，与对照细胞相比，吞噬活性呈剂量依赖性下降。对照细胞的摄取能力明显高于暴露于0.1和0.2J/cm²UV-B的细胞，而暴露于0.05J/cm²UV-B的细胞组与对照细胞组无显著差异。这些发现表明，UVB辐射诱导的培养RPE细胞损伤可通过探测细胞活力、形态变化和吞噬活性的分析方法进行评估，并且这些分析方法共同为紫外线辐射诱导的视网膜毒理学研究提供了一个有价值的体外模型。
