A2E损伤溶酶体及线粒体膜介导蓝光致人RPE细胞凋亡

Age-related macular degeneration and retinal degeneration diseases are closely related to human retinal pigment epithelium cells (RPE) apoptosis. At present, the blue light-induced apoptosis mechanism of human RPE cells is still many unknown and has important clinical significance for the research. Early research found that blue light could cause RPE cells mitochondrial membrane structure damage, intracellular calcium ion concentration increased, RPE cells apoptosis. Preliminary studies suggested that N-retinylidene-N-retinylethanol-amine (A2E) and/or blue light could cause lysosomal membrane permeability increased in RPE cells. This project is based on the model that blue light irradiation induces apoptosis of retinal pigment epithelium cells loaded A2E, a laser confocal microscope, flow cytometry, high performance liquid chromatography - mass spectrometry, and the methods of radioactive nuclide are used to detect intracellular calcium ion concentration and membrane permeability, etc, confirme that A2E may influence intracellular calcium ion concentration by changing the lysosomal and mitochondrial membrane permeability of RPE cells after blue light irradiation, which affect the Ca2+-PKC signaling pathways and change intracellular ROS concentration, promote apoptosis. This research will understand the molecular biology mechanism of blue light-induced apoptosis in human RPE cells, enrich and supply the understanding of the biological functions in retinal light damage of A2E, for clinical prevention and treatment of age-related macular degeneration, and for new therapeutic targets to find the theoretical basis.

RPE细胞凋亡与年龄相关性黄斑变性及视网膜变性类疾病密切相关，蓝光致人RPE细胞凋亡的机理目前仍有许多未知，对其研究具有重要临床意义。前期发现蓝光可致RPE细胞内线粒体膜结构损伤、胞内钙离子浓度增加,细胞凋亡。预实验提示脂褐素中主要荧光物质A2E和/或蓝光光照可导致RPE细胞溶酶体膜通透性增加。本项目拟采用蓝光照射负载A2E的人RPE细胞，用激光共聚焦显微镜、流式细胞术、高效液相色谱-质谱联用，非放射性核素等方法，检测胞内Ca2+浓度及膜通透性等指标，证实蓝光照射后A2E可能通过改变RPE细胞内溶酶体及线粒体膜的通透性，影响细胞内Ca2+浓度，进而影响Ca2+-PKC信号通路并改变细胞内ROS的浓度，促进细胞凋亡这一假设。本研究将深入认识蓝光致人RPE细胞凋亡的分子生物学机理，并丰富和补充对A2E在视网膜光损伤中生物学功能的认识，为临床防治年龄相关性黄斑变性、寻找新的治疗靶点奠定理论基础。

目的：探索蓝光照射人RPE细胞后，A2E在细胞中的分布规律、A2E是否影响溶酶体膜与线粒体膜通透性，对溶酶体及线粒体摄取、释放Ca2+功能，以及对Ca2+-PKC信号通路有何影响。.方法：采用Annexin V及TUNEL法检测细胞凋亡，吖啶橙染色观察细胞内溶酶体膜通透性。用流式细胞法检测各组细胞胞浆内ROS浓度及A2E荧光强度。采用激光扫描共聚焦显微镜（LSCM）观察各组RPE细胞钙离子荧光强度，酶联免疫吸附试验（ELISA）检测各组细胞内三磷酸肌醇（IP3）和二酰甘油（DAG）浓度，非放射性核素法测定各组细胞内PKC活性。采用激光扫描共聚焦显微镜观察各组 RPE 细胞胞浆、线粒体及溶酶体钙离子荧光强度。.结果：（1）光照组、A2E负载组、A2E负载+光照组与对照组比较红色颗粒状荧光均减弱，胞浆的绿色荧光增强。光照组、A2E负载组及A2E负载+光照组红色荧光强度均低于对照组。（2）与对照组相比，各组胞浆内ROS浓度升高，胞浆及溶酶体内A2E荧光强度增强。（3）各组RPE细胞内钙离子荧光强度高于对照组，细胞内IP3及DAG浓度高于对照组，PKC活性高于对照组。（4）各组人 RPE 细胞线粒体内钙离子荧光强度变化，蓝光光照组高于对照组，负载 A2E+蓝光光照组低于对照组。（5）各组 RPE 细胞溶酶体内钙离子荧光强度变化，蓝光光照组荧光强度高于对照组，负载 A2E+蓝光光照组低于对照组。（6）各组 RPE 细胞线粒体膜电位变化，对照组膜电位水平高于蓝光光照组、负载 A2E 组及负载 A2E+蓝光光照组。.结论：（1）蓝光光照、A2E联合蓝光光照可导致RPE细胞凋亡，且两者具有协同作用。（2）A2E、蓝光光照、A2E联合蓝光光照可导致RPE细胞内溶酶体膜通透性增加。（3）蓝光光照、A2E负载可致人RPE细胞胞浆内ROS增加, 胞浆内钙离子浓度增加，PKC活性增强，细胞胞浆内IP3、DAG浓度升高，且两者具有协同作用。（4）A2E和（或）蓝光照射可损伤溶酶体膜，使溶酶体膜通透性增加、破裂，从而使A2E可从溶酶体释放入胞浆。（5）蓝光光照可导致 RPE 细胞胞浆、溶酶体及线粒体内 Ca2+ 浓度升高，不影响溶酶体及线粒体摄取 Ca2+ 功能。（6）A2E通过损伤溶酶体及线粒体膜致二者释放 Ca2+ 进入胞浆。（7）蓝光光照、A2E 负载均可使线粒体膜电位降低，且二者具有协同