肿瘤相关成纤维细胞外泌体对乏氧条件下肺腺癌细胞自噬的影响及其机制研究  

Effect of tumor-associated fibroblast-derived exosomes on autophagy of lung adenocarcinoma cells under hypoxic condition and its mechanism

作　　者：麻佑锋 韩利红[1] 王梦丽 郑其帆 MA Youfeng;HAN Lihong;WANG Mengli;ZHENG Qifan(Department of Respiratory and Critical Care Medicine,Luoyang Central Hospital,Luoyang,Henan 471009,China;Xinxiang Medical University,Xinxiang,Henan 453003,China;Zhengzhou University,Zhengzhou,Henan 450001,China)

机构地区：[1]洛阳市中心医院呼吸与危重症医学科,河南洛阳471009 [2]新乡医学院,河南新乡453003 [3]郑州大学,河南郑州450001

出　　处：《临床肺科杂志》2024年第8期1206-1212,共7页Journal of Clinical Pulmonary Medicine

基　　金：河洛青年人才托举工程项目(No.2022HLTJ20);河南省科技攻关联合共建项目(No.LHGJ20210850);洛阳市科技局立项(No.2101047A)。

摘　　要：目的 探讨肿瘤相关成纤维细胞外泌体对乏氧条件下肺腺癌A549细胞自噬的影响及其机制。方法 选取手术的肺癌标本及癌旁组织标本,进行肿瘤相关成纤维细胞(CAFs)和成纤维细胞(NFs)的原代分离和鉴定;收集CAFs和NFs细胞培养上清,利用差速离心法从细胞培养上清液中分离出外泌体(exo),并通过电子显微镜和Western Blot对分离出的外泌体进行鉴定;采用CoCL_(2)体外构建乏氧微环境,将A549细胞复苏并传代后,把外泌体与A549细胞共培养,共分为4组:常氧A549组、乏氧A549组、乏氧A549+CAFs-exo组、乏氧A549+NFs-exo组;利用Western印迹法检测不同细胞组中LC3Ⅱ、LC3Ⅰ和LC3Ⅱ/LC3Ⅰ的表达变化,并通过Western Blot确定各组中AMPK、p-AMPK、mTOR和p-mTOR的表达水平;采用CCK8法、划痕实验、Transwell法,研究外泌体在不同环境中与细胞共同培养后对细胞增殖、迁移和侵袭能力的影响。结果 Western Blot显示CAFs中FSP-1、FAP、a-SMA蛋白高表达,透射电镜观察到提取的外泌体具备典型的结构特征。与常氧A549组相比,乏氧A549各组细胞增殖、迁移、侵袭能力显著降低(P<0.001),LC3Ⅱ/LC3Ⅰ,p-AMPK/AMPK比值上升,p-mTOR/mTOR比值下降(P<0.001);与乏氧A549组相比,乏氧A549+NFs-exo组细胞增殖、迁移、侵袭能力无明显变化,LC3Ⅱ/LC3Ⅰ、p-AMPK/AMPK、p-mTOR/mTOR比值均无明显变化;与乏氧A549组相比,乏氧A549+CAFs-exo组细胞增殖、迁移、侵袭能力明显强于乏氧A549组且LC3Ⅱ/LC3Ⅰ,p-AMPK/AMPK比值上升,p-mTOR/mTOR比值下降。结论 在乏氧微环境下,肿瘤相关成纤维细胞外泌体基于AMPK/mTOR信号通路,增强A549细胞自噬,可能是A549细胞抵抗乏氧环境、维持细胞增殖的机制之一。Objective To explore the effect of tumor-associated fibroblast-derived exosomes on autophagy of lung adenocarcinoma cells under hypoxic condition and its mechanism.Methods Lung cancer tissue samples and post-tissue samples were selected to identify and isolate primary CAFs and rod cells(NFs).Then,CAFs and NFs cell culture supernatants were collected to extract exosomes(exo)using differential centrifugation,respectively.The extracted exo were then subjected to identification by electron microscopy and Western blot.Furthermore,under CoCL_(2)-induced hypoxic microenvironment in vitro,A549 cells were resuscitated and subcultured,followed by co-culture with exo and establishment of four groups,including the normoxic A549 group,the hypoxic A549 group,the hypoxic A549+CAFs-exo group,and the hypoxic A549+NFs-exo group.Western blot was employed to detect the expression changes of LC3Ⅱ,LC3Ⅰ,and LC3Ⅱ/LC3Ⅰin different groups.Results The results of Western blot showed high protein expressions of FSP-1,FAP and a-SMA in CAFs,indicating the isolation of CAFs.Identification by transmission electron microscopy revealed that the structure of the extracted exo conformed to corresponding characteristics.Furthermore,Compared with the normoxic A549 group,the proliferation,migration,and invasion abilities of cells in the hypoxic A549 group were significantly reduced(P<0.001),LC3Ⅱ/LC3Ⅰ,and p-AMPK/AMPK ratio increased.The p-mTOR/mTOR ratio decreased(P<0.001).Compared with the hypoxic A549 group,the hypoxic A549+NFs exo group showed no significant change in cell proliferation,migration,and invasion ability,LC3Ⅱ/LC3Ⅰ,p-AMPK/AMPK,and p-mTOR/mTOR ratio.Compared with the hypoxic A549 group,the hypoxic A549+CAFs exo group had stronger cell proliferation,migration,and invasion ability,the ratio of LC3Ⅱ/LC3Ⅰand p-AMPK/AMPK increased,and the p-mTOR/mTOR ratio decreased.Conclusion CAFs-derived exo can enhance the autophagy of A549 cells via the AMPK/mTOR signaling pathway under hypoxic conditions,which may explain the resistance

关 键 词：肿瘤相关成纤维细胞 外泌体 非小细胞肺癌 

分 类 号：R734.2[医药卫生—肿瘤]