脂褐素成分A2E诱导人视网膜色素上皮细胞自噬及损伤的研究  

Research on autophagy and injury of human retinal pigment epithelial cells induced by lipofuscincomponent A2E

作　　者：张晶晶 陈云珍 黄旅珍[2] 陈莉[3] ZHANG Jingjing;CHEN Yunzhen;HUANG Lüzhen;CHEN Li(Department of Ophthalmology,Daxing Teaching Hospital Affiliated to Capital Medical University,Beijing 102600,China;Department of Ophthalmology,Peking University People’s Hospital,Beijing 100044,China;Department of Ophthalmology,Beijing Chaoyang Hospital,Capital Medical University,Beijing 100020,China)

机构地区：[1]首都医科大学大兴教学医院眼科,北京市102600 [2]北京大学人民医院眼科,北京市100044 [3]首都医科大学附属北京朝阳医院眼科,北京市100020

出　　处：《眼科新进展》2021年第7期608-614,共7页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助项目(编号81470649,81670870)。

摘　　要：目的探讨N-亚视黄基-N-视黄基-乙醇胺(A2E)能否诱导人视网膜色素上皮细胞(ARPE-19细胞)的自噬及损伤反应,并从自噬的角度探索其与年龄相关性黄斑变性(AMD)发病相关的分子机制。方法CCK-8法筛选A2E作用于ARPE-19细胞的最佳浓度用于后续实验。采用多重细胞因子检测技术检测A2E作用于ARPE-19细胞后对细胞间黏附分子(ICAM)、白细胞介素(IL)-1β、IL-6、IL-8、IL-10、促血管生成素-2(Ang-2)、胎盘生长因子(PIGF)、胰岛素样生长因子1(IGF-1)、转化生长因子-β(TGF-β)以及血管内皮生长因子A(VEGFA)表达的影响,透射电子显微镜观察ARPE-19细胞超微结构的变化,免疫荧光标记检测细胞内自噬相关蛋白LC3的表达变化,Western blot检测自噬相关蛋白Beclin-1和p62的表达变化。结果依据CCK-8法检测结果,选择20μmol·L^(-1)的A2E作用于ARPE-19细胞用于后续实验。20μmol·L^(-1)的A2E作用于ARPE-19细胞后,细胞因子ICAM、IL-1β、IL-6、IL-8、IL-10、Ang-2、IGF-1、TGF-β的表达水平自6 h起均有所升高,PIGF和VEGFA的表达水平在A2E作用12 h后也明显升高,与未给予A2E处理的对照相比差异均有统计学意义(均为P<0.05)。透射电子显微镜下可见,20μmol·L^(-1)的A2E作用于ARPE-19细胞1 h后细胞质内可见碗状、尚未收口的双层膜结构的“吞噬泡”,作用3 h、6 h、12 h时出现自噬体结构以及自噬溶酶体结构,自噬囊泡在A2E作用24 h时开始出现内容物减少,体积逐渐缩小。荧光显微镜下可见,与未给予A2E处理的对照相比,20μmol·L^(-1)的A2E作用于ARPE-19细胞6 h起即可见细胞质内LC3荧光呈现点状聚集,至12 h时荧光斑点数量达到最多、颗粒也最大、差异最明显,24 h时绿色荧光斑点数量开始下降,荧光强度也较前减弱。Western blot检测结果显示,与未给予A2E处理的对照相比,ARPE-19细胞中Beclin-1蛋白的表达在A2E作用1 h、3 h、6 h、12 h及24 h时均显著升高,p62蛋白的表达�Objective To investigate whether N-retinylidene-N-retinylethanolamine(A2E)can induce autophagy and injury response of human adult retinal pigment epithelium-19(ARPE-19)cells,and to study the molecular mechanism of A2E related to age-related macular degeneration(AMD)from the perspective of autophagy.Methods CCK-8 methods was used to screen the optimal concentration of A2E on ARPE-19 cells for subsequent experiments.The effect of A2E on the expression levels of intercellular adhesion molecule(ICAM),interleukin(IL)-1β,IL-6,IL-8,IL-10,angiopoietin-2(Ang-2),placental growth factor(PIGF),insulin-like growth factor-1(IGF-1),transforming growth factor-β(TGF-β)and vascular endothelial growth factor A(VEGFA)in ARPE-19 cells were detected by multiple cytokine assay.The ultrastructural changes in ARPE-19 cells were observed by transmission electron microscopy,the expression of autophagy related protein LC3 was detected by immunofluorescence labeling,and the expression of autophagy related protein Beclin-1 and p62 was detected by Western blot.Results According to the CCK-8 results,20μmol·L^(-1) A2E on ARPE-19 cells was selected in the following experiments.After treated with 20μmol·L^(-1) A2E on ARPE-19 cells,the expression levels of ICAM,IL-1β,IL-6,IL-8,IL-10,Ang-2,IGF-1 and TGF-βwere increased from 6 h,and the expression of PIGF and VEGFA were also increased after 12 h.The differences were statistically significant between ARPE-19 cells treated with A2E and the control without A2E treatment(all P<0.05).By transmission electron microscopy,after 20μmol·L^(-1) A2E acting on ARPE-19 cells for 1 h,a bowl-shaped,unclosed double-layer membrane structure began to appear in the cytoplasm called“phagophores”.Autophagosomes and autophagolysosomes appeared at 3 h,6 h and 12 h,the content of autophagic vesicles decreased at 24 h and the volume of autophagic vesicles decreased gradually,indicating the degeneration and degradation of autophagic lysosomes.By fluorescence microscope,compared with the controls without A2E trea

关 键 词：N-亚视黄基-N-视黄基-乙醇胺(A2E) 视网膜色素上皮细胞 自噬 老年性黄斑变性 

分 类 号：R774[医药卫生—眼科]