低氧条件下食管癌来源外泌体调控巨噬细胞M2型分化与自噬的机制研究  

Hypoxia-induced esophageal cancer-derived exosomes regulating differentiationand autophagy of M2 macrophages

作　　者：尹星[1] 邢艳丽[1] 常欢 周阳 付民 尹先哲[1] YIN Xing;XING Yanli;CHANG Huan;ZHOU Yang;FU Min;YIN Xianzhe(First Ward,Oncology Department,Second People's Hospital of Nanyang City,Nanyang 473000,China)

机构地区：[1]南阳市第二人民医院肿瘤科一病区,南阳473000

出　　处：《中国免疫学杂志》2024年第2期299-304,311,共7页Chinese Journal of Immunology

基　　金：河南省科技计划发展项目(202102310125)。

摘　　要：目的:探究低氧条件下食管癌外泌体对巨噬细胞分化和自噬的影响及机制。方法:50 ng/ml PMA诱导THP-1分化为M0型巨噬细胞,超速离心法提取并鉴定HEEC、Eca-109、缺氧诱导的Eca-109细胞分泌的外泌体(记为HEEC-exo、NomEca-109-exo、Hypo-Eca-109-exo),激光共聚焦显微镜观察巨噬细胞对外泌体的摄取,将PBS、HEEC exo、Nom-Eca-109-exo、HypoEca-109-exo分别与M0型巨噬细胞共孵育,流式细胞术检测各组细胞CD14和CD206表达,ELISA检测细胞因子IL-1β、IL-12、TNF-α、Arg1、IL-10和TGF-β,qRT-PCR检测各组细胞CCR7、TLR2、CD206和CD14表达,Transwell检测各组细胞迁移和侵袭,Western blot检测各组细胞自噬相关蛋白LC3-Ⅰ、LC3-Ⅱ、Beclin-1和p62表达及PI3K、Akt、mTOR磷酸化水平。结果:提取的外泌体符合结构和生物学特征。相比于PBS组,与Nom-Eca-109-exo、Hypo-Eca-109-exo孵育后,各组M0型巨噬细胞CCR7、TLR2表达显著降低、CD206和CD14表达显著增加,IL-1β、IL-12、TNF-α分泌显著减少,Arg1、IL-10和TGF-β分泌显著增加,LC3-Ⅱ、Beclin-1表达显著增加,p62表达显著降低,细胞迁移和侵袭数显著增加,PI3K、Akt、mTOR磷酸化水平显著升高。相比于NomEca-109-exo组,Hypo-Eca-109-exo组细胞CD206和CD14表达显著增加,Arg1、IL-10和TGF-β分泌显著增加,LC3-Ⅱ、Beclin-1表达显著增加,p62表达显著降低,细胞迁移和侵袭数显著增加,PI3K、Akt、mTOR的磷酸化水平显著升高。结论:低氧诱导的食管癌细胞外泌体可显著促进M2型巨噬细胞分化和自噬,进而促进肿瘤迁移和侵袭,其机制为激活PI3K/Akt/mTOR信号通路。Objective:To explore effect and mechanism of esophageal cancer exosomes on differentiation and autophagy of macrophages under hypoxic conditions.Methods:50 ng/ml PMA induced THP-1 differentiate into M0 type macrophages,and exo-somes secreted by HEEC,Eca-109 and hypoxia-induced Eca-109 cells(HEEC-exo,Nom-Eca-109-exo,Hypo-Eca-109-exo)were extracted and identified by ultracentrifugation.Laser confocal microscope was used to observe uptake of exosomes.PBS,HEEC exo,Nom-Eca-109-exo,Hypo-Eca-109-exo were incubated with M0 macrophages,flow cytometry was used to detect expressions of CD14 and CD206 of cells in each group.ELISA was used to detect cytokines IL-1β,IL-12,TNF-α,Arg1,IL-10,IL-10 and TGF-β,expressions of CCR7,TLR2,CD206 and CD14 in each group were detected by qRT-PCR,migration and invasion of each group were detected by Transwell,and expressions of autophagy-related proteins LC3-Ⅰ,LC3-Ⅱ,Beclin-1 and p62,phosphorylation levels of PI3K,Akt,mTOR in each group were detected by Western blot.Results:Exosomes extracted conformed to their structural and biologi-cal characteristics.Compared with PBS group,after incubating with Nom-Eca-109-exo and Hypo-Eca-109-exo,expressions of CCR7 and TLR2 were significantly reduced,expressions of CD206 and CD14 were significantly increased,secretions of IL-1β,IL-12 and TNF-αwere significantly decreased,secretions of Arg1,IL-10 and TGF-βwere significantly increased,and expressions of LC3-Ⅱand Beclin-1 were significantly increased,expression of p62 was significantly reduced,numbers of cell migration and invasion were significantly increased,and phosphorylation levels of PI3K,Akt and mTOR were significantly increased.Compared with Nom-Eca-109-exo group,expressions of CD206 and CD14 in Hypo-Eca-109-exo group were increased significantly,secretions of Arg1,IL-10 and TGF-βwere increased significantly,and expressions of LC3-Ⅱand Beclin-1 were increased significantly,expression of p62 was significantly reduced,numbers of cell migration and invasion were increased significantly,

关 键 词：低氧 食管癌 外泌体 巨噬细胞分化 自噬 机制 

分 类 号：R73[医药卫生—肿瘤]