Sheng Hui, Lu Yi, Qing Feng-ling
Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China.
Zhonghua Yan Ke Za Zhi. 2007 Nov;43(11):1017-21.
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.
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 micromol/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.
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 micromol/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.
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.
观察人视网膜色素上皮（hRPE）细胞对A2E的摄取，并研究脂褐素荧光团A2E（N - 视黄叉基 - N - 视黄基乙醇胺）是否参与蓝光诱导的hRPE细胞损伤。
采用全反式视黄醛和乙醇胺的混合物一步法制备A2E。将A2E颗粒加入培养的hRPE细胞培养基中使其摄取。汇合培养的细胞随后在有或无A2E（25、50、100 μmol/L）的情况下暴露于450 nm（蓝光）下20分钟。光强度为70 mW/mm²。在暴露后12、24、36和48小时，通过CCK - 8检测活细胞数量来定量光毒性。通过Hoechst 33342 DNA染色和流式细胞术检测细胞凋亡。
全反式视黄醛（100 mg）与乙醇胺（9.5 mg）反应一步生成53.8 mg A2E。当将A2E加入培养的hRPE细胞中时，它在细胞内积累。摄取的A2E呈现为具有核周分布的自发荧光颗粒。如CCK - 8分析所示，摄取A2E的hRPE细胞活力在450 nm光照后随时间降低。相反，未含A2E的蓝光照射hRPE细胞显示出较少的细胞活力丧失。蓝光照射后12、24、36和48小时，25 μmol/L A2E处理的hRPE细胞凋亡率分别为（12.11±2.32）%、（31.21±3.72）%、（64.23±3.53）%和（58.71±3.48）%。相反，其他hRPE细胞的凋亡率小于5%。
A2E对蓝光诱导的hRPE细胞损伤至关重要。仅蓝光照射而无A2E导致的细胞损伤很小。含有大量脂褐素的老年人hRPE细胞对蓝光损伤敏感。
