蓝光诱导人视网膜色素上皮细胞分泌外泌体与核苷酸结合寡聚化结构样受体蛋白炎性体的相关性研究  

Blue light damaged-retinal pigment epithelial cell derived-exosomes activate nod-like receptor protein inflammasome

作　　者：马映雪 陈松[1] 何广辉[1] 杨婧[1] 陈莉[1] 姜鉴洪 宋建[1] 

机构地区：[1]天津市眼科医院天津市眼科学与视觉科学重点实验室天津市眼科研究所天津医科大学眼科临床学院,300020 [2]天津市第一中心医院眼科

出　　处：《中华眼底病杂志》2017年第5期498-502,共5页Chinese Journal of Ocular Fundus Diseases

基　　金：天津市应用基础与前沿技术研究计划项目（14JCYBJC27400）;天津市卫生局科技基金（2015KZ073）

摘　　要：目的观察蓝光诱导人视网膜色素上皮（RPE）细胞外泌体对核苷酸结合寡聚化结构样受体蛋白（NLRP3）炎性体相关因子表达的影响。方法人RPE细胞株贴壁细胞分为实验组和对照组。实验组细胞以蓝光照射6h建立光损伤模型；对照组细胞常规培养。分级低温超速离心法获得两组细胞外泌体，透射电子显微镜观察其形态；蛋白免疫印迹法（Westernblot）检测两组细胞外泌体表面CD63及白细胞介素（IL）-113、IL-18、半胱氨酸天冬氨酸蛋白酶（caspase-1）蛋白相对表达水平。将两组细胞外泌体作用于正常RPE细胞，据此分为蓝光诱导细胞外泌体＋实验组、正常细胞外泌体＋对照组。培养24h后，Westernblot检测两组细胞外泌体中IL-113、IL．18、caspase．1蛋白表达；荧光实时定量聚合酶链反应检测两组细胞中NLRP3mRNA表达。结果实验组细胞发生损伤失去原有形态；外泌体均呈双凹托盘状，直径50~200nm；实验组细胞外泌体中IL-113（t=18．04）、IL-18（t=-12．55）、caspase-1（t=14．70）蛋白表达量明显高于对照组，差异有统计学意义（P〈0．001）。蓝光诱导细胞外泌体＋实验组细胞外泌体中IL．1B（t=-i8．59）、IL．18（t=23．95）、caspase．1（t-=35．27）蛋白表达量明显高于正常细胞外泌体＋对照组，差异均有统计学意义（P〈0．001）；蓝光诱导细胞外泌体＋实验组、正常细胞外泌体＋对照组细胞内NLRP3mRNA相对表达量分别为1．000±0．069、0．200±0．010，两者比较，差异有统计学意义（t=-12．20，P〈0．001）。结论蓝光诱导RPE细胞外泌体可使RPE细胞内NLRP3炎性体相关细胞因子IL．1B、IL．18、caspase-1蛋白和NLRP3mRNA表达上调。Objective To observe the effect of exosomes secreted by retinal pigment epithelial （RPE） cells which damaged by blue light to Nod-like receptor protein （NLRP3）. Methods Cultured ARPE-19 cells were divided into 2 groups; one group of RPE cells were exposed to blue light irradiation for 6 hours, the other group was cultured in routine environment. Total exosomes were extracted from the two groups by differential ultracentrifugation in low-temperature, and examined by transmission electron microscope to identify their forms. The exosomes were then incubated with normal ARPE-19 cells. The expression level of CD63, interleukin （IL）-113, IL-18 and caspase-1 on the exosome surface were measured by Western blotting. The expressions of NLRP3 mRNA in RPE cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction （RT-PCR）. Results Blue light damaged the cellular morphology. Transmission electron microscopy showed that the exosomes were 50-200nm in diameter and like doubleconcave disks. Blue light damaged cell-derived exosomes had significantly higher expression oflL-1β （t=-18.04）,IL-18 （t=12.55） and caspase-1 （t=14.70） than the control group （P〈0.001）. ARPE-19 cells cultured with blue light damaged cell-derived exosomes also had significantly higher expression of IL-113 （t=1 8.59）, IL-18 （t=23.95） and caspase-1 （t=35.27） than control exosomes （P〈0.001）. RT-PCR showed that the relative expression of NLRP3 mRNA of PRE cells in experimental group and control group were 1.000 4-0.069 and 0.2_ 0.01, respectively, the difference was significant （t=-12.20, P〈0.001）. Conelusion The expression IL-I[3, IL-18 and caspase-1 and NLRP3 mRNA were upregnlated by exosomes secreted by blue light damaged-RPE cells.

关 键 词：视网膜色素上皮/病理生理学 外泌体 炎性体 光刺激届0作用 

分 类 号：R774.1[医药卫生—眼科]