自噬抑制剂3-甲基腺嘌呤(3-MA)对A2E诱导视网膜色素上皮细胞自噬的调节以及相关细胞因子表达的影响  

Regulation of autophagy inhibitor 3-methyladenine on the autophagy of retinal pigment epithelial cells induced by N-retinyl-N-retinylidene ethanolamine and its impact on the expression of related cytokines

作　　者：陈莉[1] 张晶晶 陈云珍 黄旅珍[3] CHEN Li;ZHANG Jingjing;CHEN Yunzhen;HUANG Lüzhen(Department of Ophthalmology,Beijing Chaoyang Hospital of Capital Medical University,Beijing 100020,China;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)

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

出　　处：《眼科新进展》2022年第3期179-183,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助项目(编号:81470694,81670870)。

摘　　要：目的探讨自噬抑制剂3-甲基腺嘌呤(3-MA)对N-亚视黄基-N-视黄基乙醇胺(A2E)诱导视网膜色素上皮(RPE)细胞自噬的调节以及相关细胞因子表达的影响。方法取人RPE细胞(ARPE-19细胞系)进行培养。将细胞分为4组。对照组:仅使用正常DMEM-F12完全培养基孵育ARPE-19细胞;A2E组:将ARPE-19细胞置于含20μmol·L^(-1) A2E的DMEM-F12完全培养基中孵育12 h;3-MA组:使用含10 mmol·L^(-1)3-MA(美国Sigma-Aldrich公司)的DMEM-F12完全培养基预处理细胞1 h后,再用正常DMEM-F12完全培养基孵育细胞12 h;3-MA联合A2E组:先使用10 mmol·L^(-1)3-MA预处理细胞1 h,再将细胞放入含20μmol·L^(-1) A2E的DMEM-F12完全培养基孵育12 h。使用CCK\|8法检测各组ARPE-19细胞增殖情况,透射电镜观察各组ARPE-19细胞超微结构,Western blot法检测各组ARPE-19细胞Beclin-1和P62自噬相关蛋白表达,免疫荧光染色法检测各组ARPE-19细胞自噬标志物LC3蛋白的表达,使用Procarta细胞因子分析试剂盒检测各组细胞培养上清液10种细胞因子表达情况,10种细胞因子包括:细胞间黏附分子、白细胞介素(IL)-1β、IL-6、IL-8、IL^(-1)0、血管生成素2、血小板衍生生长因子、胰岛素样生长因子、转化生长因子和VEGFA。结果与A2E组相比,3-MA联合A2E组细胞生存率下降了27.16%(P<0.01)。与A2E组相比,3-MA联合A2E组细胞培养上清液10种细胞因子含量均明显升高(均为P<0.01)。与对照组相比,3-MA组ARPE-19细胞超微结构无明显变化,而A2E组ARPE-19细胞胞浆内出现大量自噬泡;3-MA联合A2E组ARPE-19细胞自噬泡数量较A2E组明显减少。3-MA联合A2E组ARPE-19细胞胞浆内可见荧光斑点亮度较A2E组减弱,绿色荧光斑点数量也明显减少。与对照组相比,A2E组ARPE-19细胞Beclin-l蛋白相对表达量显著升高(P<0.001),P62蛋白的相对表达量显著降低(P<0.001)。与A2E组相比,3-MA联合A2E组ARPE-19细胞中Beclin-l蛋白相对表达量显著降低(P<0.01,),而P62蛋白相Objective To investigate the regulation of autophagy inhibitor 3-methyladenine(3-MA)on the autophagy of retinal pigment epithelial(RPE)cells induced by N-retinyl-N-retinylidene ethanolamine(A2E)and its impact on the expression of related cytokines.Methods Human RPE cells(ARPE-19 cell line)were divided into the control group(ARPE-19 cells were cultured in normal DMEM-F12 complete medium),A2E group(ARPE-19 cells were cultured in DMEM-F12 complete medium containing 20μmol·L^(-1) A2E for 12 h),3-MA group[ARPE-19 cells were first pretreated in DMEM-F12 complete medium containing 10 mmol·L^(-1)3-MA(American Sigma-Aldrich Company)for 1 h and then cultured in normal DMEM-F12 complete medium for 12 h],and 3-MA+A2E group(ARPE-19 cells were first pretreated in DMEM-F12 complete medium containing 10 mmol·L^(-1)3-MA for 1 h and then cultured in DMEM-F12 complete medium containing 20μmol·L^(-1) A2E for 12 h).CCK\|8 assay was used to determine the proliferation of ARPE-19 cells in each group.The transmission electron microscope was used to observe the ultrastructural changes of ARPE-19 cells in each group.The expression of autophagy-related proteins Beclin-1 and P62 in ARPE-19 cells was detected by Western blot,and the expression of autophagy marker protein LC3 was detected by immunofluorescence assay.Procarta cytokine assay kit was used to measure the expression of 10 cytokines in the cell culture supernatant of each group,including intercellular adhesion molecules,interleukin(IL)-1β,IL-6,IL-8,IL^(-1)0,angiopoietin 2,placental growth factor,insulin-like growth factor-1,transforming growth factor,and vascular endothelial growth factor A.Results Compared with the A2E group,the survival rate of ARPE-19 cells significantly reduced by 27.16%and the levels of 10 cytokines in the cell culture supernatant significantly increased in the 3-MA+A2E group(all P<0.01).Compared with the control group,the ultrastructure of ARPE-19 cells in the 3-MA group did not change significantly,while a great number of autophagic vacuoles were foun

关 键 词：N-亚视黄基-N-视黄基乙醇胺 3-甲基腺嘌呤 视网膜色素上皮细胞 自噬 年龄相关性黄斑变性 

分 类 号：R774.1[医药卫生—眼科]