脂褐质荧光基团N-亚视黄基-N-视黄基乙醇胺在人视网膜色素上皮细胞蓝光损伤中的作用研究  

Blue light-induced damage to haman retinal pigmented epithelial cells mediated by A2E

作　　者：生晖[1] 卢奕[1] 卿风翎[2] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院眼科,上海200031 [2]中国科学院上海有机化学研究所

出　　处：《中华眼科杂志》2007年第11期1017-1021,共5页Chinese Journal of Ophthalmology

摘　　要：目的探讨成人视网膜色素上皮（hRPE）细胞对脂褐质荧光基团N-亚视黄基-N-视黄基乙醇胺（A2E）的摄取及A2E在hRPE细胞蓝光损伤中的作用。方法全反式视黄醛和乙醇胺混合，体外合成A2E。体外培养hRPE细胞。将A2E加入hRPE细胞培养液中，使A2E的浓度分别为25、50、100μmoL／L。观察hRPE细胞对A2E的摄取。用蓝光（450nm）照射hRPE细胞20min，光照强度70mW／mm^2，CCK-8检测细胞活力，Hoechst 33342荧光素染色、流式细胞仪检测细胞凋亡情况。结果全反式视黄醛（100．0mg）和乙醇胺（9．5mg）混合后生成53．8mg的A2E。吞噬A2E的hRPE细胞中能够检测到自发荧光，主要分布在细胞核周围。蓝光照射后，含有A2E的hRPE细胞活力下降，下降程度随A2E浓度增加而增强。不含A2E的hRPE细胞，经蓝光照射后，细胞活力未见明显下降。蓝光照射含有A2E的hRPE细胞，细胞核中出现浓染的碎块状荧光，流式细胞仪检测可见凋亡细胞特有的亚二倍体峰，加入25μmoL／L的A2E，光照后12、24、36及48h，细胞凋亡率分别为（12．11±2．32）％、（31．21±3．72）％、（64．23±3．53）％及（58．71±3．48）％。仅接受光照不加入A2E、不接受光照（加入或不加入A2E）的hRPE细胞凋亡率在光照后各时间点的平均值均小于5％。结论在hRPE细胞蓝光损伤的过程中，单独的光照射不会造成明显的细胞损伤或凋亡，A2E的存在是蓝光损伤的因素之一。提示在老年人中，含有较多脂褐质的hRPE细胞容易受到蓝光损伤。（中华眼科杂志．2007，43：1017-1021）Objective To observe the internalization of A2E by human retinal pigmented epithelial （hRPE） cells and study whether the lipofuscin fluorophore A2E （N-retinylidene-N-retinylethanolamine） participates in blue light-induced damage to hRPE cells. Methods A mixture of all-trans-retinal and ethanolamine was used to produce A2E in one step. A2E granules were delivered to medium of cultured hRPE cells for internalization. Confluent cultures were subsequently exposed to 450 nm （blue） light for 20 minutes with or without A2E （25,50,100 μmol/L）. The light intensity was 70 mW/mm^2. Phototoxicity was quantified at 12, 24, 36, and 48 h after exposure by CCK-8 of viable cells. Apoptosis of cells was detected by Hoechst 33342 DNA staining and flow cytometry. Results The reaction of all-trans-retinal （100 mg） and ethanolamine （9.5 mg） produced 53.8 mg A2E in one step. When A2E was delivered to hRPE cells in culture, it accumulated intracellularly. Internalized A2E presented as autofluorescent granules having a perinuclear distribution. As shown by CCK-8 analysis, the A2E-fed hRPE cell viability reduced with duration after 450 nm light exposure. Conversely, blue light-exposed hRPE cells that did not contain A2E showed less loss of cell viability. The percentage of hRPE cell apoptosis with 25 μmol/L A2E 12, 24, 36 and 48 h after blue light exposure was（ 12.11 ± 2.32）%, （31.21 ± 3.72）%, （64.23 ± 3.53）% and （58.71 ± 3. 48）% respectively. Conversely, the apoptosis was less than 5% in other hRPE cells. Conclusions A2E is essential to blue light-induced hRPE cell damage. Only blue light exposure and without A2E lead to little cell injury, hRPE cells in old people which contain much lipofuscin are sensitive to blue light injury. （Chin J Ophthalmol , 2007,43：1017-1021 ）

关 键 词：色素上皮 眼 荧光 维生素A酸类 细胞凋亡 

分 类 号：R686[医药卫生—骨科学]