^(60)Coγ射线照射的小鼠肺上皮MLE-12细胞分泌的外泌体对T淋巴细胞的活化作用  

Exosome-mediated T cell activation by mouse lung epithelial MLE-12 cells irradiated with^(60)Coγray

作　　者：王志鑫 王美玉 郭浩鑫 杜丽 王易龙 杨陟华 朱茂祥 Wang Zhixin;Wang Meiyu;Guo Haoxin;Du Li;Wang Yilong;Yang Zhihua;Zhu Maoxiang(Institute of Radiation Medicine,Academy of Military Medicine,Academy of Military Sciences,BeijingKey Laboratory for Radiobiology,Beijing 100850,China;College of Life Sciences,Hebei University,Baoding 071002,China;School of Public Health,University of South China,Hengyang 421001,China)

机构地区：[1]军事科学院军事医学研究院辐射医学研究所,北京市放射生物学重点实验室,北京100850 [2]河北大学生命科学院,保定071002 [3]南华大学公共卫生学院,衡阳421001

出　　处：《国际放射医学核医学杂志》2023年第4期220-228,共9页International Journal of Radiation Medicine and Nuclear Medicine

摘　　要：目的探讨小鼠肺上皮MLE-12细胞(简称MLE-12细胞)受到^(60)Coγ射线照射后分泌的外泌体介导的T细胞活化。方法将MLE-12细胞分为对照组和^(60)Coγ射线照射组(2、4、6和8 Gy),采用超速离心法分别从其培养液的上清液中提取外泌体,应用透射电子显微镜和纳米颗粒跟踪分析技术确定外泌体的形态结构和数量特征,采用蛋白质印迹法(WB)检测外泌体中溶酶体相关膜蛋白3(CD63)、四次跨膜蛋白28(CD81)、肿瘤易感基因101蛋白(TSG101)、Ⅰ型内质网膜蛋白(Calnexin)的表达,采用流式细胞术(FCM)检测外泌体表面Ⅰ类主要组织相容性复合体(MHCⅠ)、Ⅱ类主要组织相容性复合体(MHCⅡ)、免疫调节蛋白B7-1(CD80)和免疫调节蛋白B7-2(CD86)的表达水平。将从小鼠脾脏中分离出来的初始T细胞分别与对照组MLE-12细胞(简称NC MLE-12)分泌的外泌体(简称exo/NC-MLE)、6 Gyγ射线照射组的MLE-12细胞(简称IR MLE-12)分泌的外泌体(简称exo/IR-MLE)共培养,采用FCM检测T细胞亚群CD3^(+)、CD4^(+)和CD8^(+)及其活化指标T细胞特定表面糖蛋白CD28和早期活化抗原1(CD69)的变化;将初始T细胞分别与NC MLE-12、IR MLE-12和外泌体抑制剂GW4869处理组的MLE-12细胞共培养,采用FCM检测T细胞亚群CD3^(+)、CD4^(+)和CD8^(+)及其活化指标CD28和CD69的变化。2组间比较采用两独立样本t检验,多组间比较采用方差分析法,组间两两比较采用Bonferroni调整法。结果MLE-12细胞分泌的外泌体显示出典型的一面凹陷的茶托样结构,粒径为30~150 nm;WB结果显示,与MLE-12细胞相比,其外泌体中特异性标志物CD63、CD81和TSG101高表达,而阴性标志物Calnexin低表达。与对照组相比,在6 Gyγ射线照射后不同时间,单个MLE-12细胞分泌的外泌体数量于24、48 h时均增加(t=5.36、6.66,均P<0.05);在不同剂量γ射线照射后24 h,单个MLE-12细胞分泌的外泌体数量增加的现象具有剂量-效应关系,在照射剂量为6、8 Gy时,�Objective To evaluate T cell activation driven by exosomes from mouse lung epithelial MLE-12 cells(MLE-12 cells)irradiated with ^(60)Coγray.Methods MLE-12 cells were divided into a control group and a ^(60)Coγirradiation group(2,4,6,and 8 Gy),and exosomes were extracted from the supernatant of their culture medium by using ultracentrifugation.Nanoparticle tracking analysis and transmission electron microscope were used to determine the morphological structure and quantity of exosomes.The expression of lysosomal associated membrane protein(CD63),tetraspanin(CD81),tumor susceptibility gene(TSG101),and typeⅠendoplasmic reticulum protein(Calnexin)in exosomes were identified by Western blot(WB).Flow cytometry(FCM)was used to detect the expression of major histocompatibility complex classⅠ(MHCⅠ),major histocompatibility complex classⅡ(MHCⅡ),immune regulatory protein B7-1(CD80),and immune regulatory protein B7-2(CD86)on the surface of exosomes.Naive T cells isolated from mouse spleens were cocultured with exosomes(exo/NC MLE)secreted by MLE-12 cells in the control group(NC MLE-12)and exosomes(exo/IR MLE)secreted by MLE-12 cells in the 6 Gy ^(60)Coγirradiation group(IR MLE-12),respectively.FCM was used to detect the changes of T cell subsets CD3^(+),CD4^(+),and CD8^(+)and their activated proliferation indicators T cell specific surface glycoprotein CD28 and early activation antigen 1(CD69).Naive T cells were incubated with NC MLE-12,IR MLE-12,and MLE-12 cells from exosome inhibitor GW4869-treated groups,respectively.FCM was used to detect the changes of T cell subsets CD3^(+),CD4^(+),and CD8^(+)and their activation indicators CD28 and CD69.Independent samples t-test was used for comparison between two groups.Analysis of variance was used to compare multiple groups.Bonferroni adjustment was applied for pairwise comparison between two groups.Results The exosomes produced from MLE-12 cells showed a typical saucer-like structure,with a particle size of 30-150 nm.WB results showed that the exosomes specific marke

关 键 词：辐射 肺泡上皮细胞 外泌体 T淋巴细胞 抗原呈递 

分 类 号：R818[医药卫生—放射医学]