脂多糖刺激巨噬细胞获得的外泌体促进TGF-β1诱导的人A549细胞上皮间质转化  被引量:7

Exosomes derived from LPS-stimulated macrophages promote TGF-β1-induced epithelial-mesenchymal transition of human type Ⅱ alveolar epithelial A549 cells

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作  者:尤学红 郭媛媛 刘晓明 马春燕 YOU Xuehong;GUO Yuanyuan;LIU Xiaoming;MA Chunyan(College of Life Sciences,Ningxia University,Ministry-of-Education Key Laboratory of Conservation and Utilization of Western Biological Resources,Yinchuan 750021,China)

机构地区:[1]宁夏大学生命科学学院西部特色生物资源保护与利用教育部重点实验室

出  处:《细胞与分子免疫学杂志》2019年第8期673-681,共9页Chinese Journal of Cellular and Molecular Immunology

基  金:国家自然科学基金(31660255);宁夏回族自治区重点研发计划项目(2017BN04)

摘  要:目的探索炎性环境下巨噬细胞来源外泌体对转化生长因子β1(TGF-β1)诱导的肺脏上皮间质转化(EMT)的影响。方法采用透射电镜鉴定外泌体形态,Western blot法检测外泌体特异性标志物肿瘤易感基因101(TSG101)、附属蛋白质凋亡关联基因α相互作用蛋白X(ALIX)、CD81、CD9蛋白及外泌体不含有的钙连蛋白(calnexin)对THP-1巨噬细胞来源的外泌体进行鉴定。利用TGF-β1诱导肺泡上皮A549细胞发生EMT,比较脂多糖(LPS)刺激和未刺激THP-1细胞来源外泌体对A549细胞间质化特征的影响。结果成功建立TGF-β1诱导A549细胞发生EMT模型和获得THP-1巨噬细胞来源的外泌体。与未经LPS处理巨噬细胞来源外泌体比较,LPS刺激THP-1细胞来源的外泌体能显著促进TGF-β1诱导A549细胞EMT,包括显著下调上皮钙黏蛋白(E-cadherin)表达水平,上调波形蛋白(vimentin)、α平滑肌肌动蛋白(α-SMA)、TGF-β1/SMAD家族成员2/3(Smad2/3)通路Smad2/3蛋白和磷酸化的Smad2/3(p-Smad2/3)和1型胶原蛋白(Col1)等细胞间质化相关的信号蛋白表达。结论LPS刺激巨噬细胞来源的外泌体通过激活TGF-β/Smad2/3信号,上调A549细胞中vimentin、α-SMA和Col1等细胞间质化相关蛋白的表达,促进A549细胞发生EMT。Objective To investigate the impact of the macrophage-derived exosomes on transforming growth factor-β1(TGF-β1)-induced epithelial-mesenchymal transition(EMT) of lung epithelial cells in an inflammatory environment. MethodsThe morphology of exosomes derived from THP-1 macrophages was evaluated by transmission electron microscopy, and the biochemistry properties of exosomes were identified by accessing exosome-specific markers including tumor susceptibility gene 101(TSG101), accessory protein ALG-2 interacting protein X(ALIX), CD81 and CD9 protein, and the calnexin, a negative control marker absent in exosomes, using an immunoblotting assay. The EMT of alveolar epithelial A549 cells was induced by TGF-β1, and the impacts of exosomes on the EMT of A549 cells was ascertained by comparing cells treated with exsomes derived from LPS-primed THP-1 macrophages and na?ve THP-1 cells. Results We successfully established an A549 cell EMT model by TGF-β1 induction and isolated exosomes derived from THP-1 macrophages. In comparison with the exosomes derived from untreated na?ve THP-1 macrophages, exosomes derived from LPS-primed THP-1 cells exhibited an ability to significantly promote TGF-β-induced EMT of A549 cells, as determined by a significantly down-regulated E-cadherin, and an dramatically increased expression of proteins in EMT-related signaling including vimentin, alpha smooth muscle actin(α-SMA), TGF-β1/Smad2/3 signaling proteins Smad2/3 protein and phosphorylated Smad2/3(p-Smad2/3) and type 1 collagen(Col1). Conclusion Exosomes derived from LPS-stimulated macrophages are able to activate TGF-β/Smad2/3 signaling, which in turn increase the expression of EMT-related proteins vimentin, α-SMA and Col1 in A549 cells, and subsequently promote EMT in A549 cells.

关 键 词:巨噬细胞 外泌体(exosome) 肺脏上皮细胞 上皮间质转化(EMT) 转化生长因子β1 

分 类 号:R392.12[医药卫生—免疫学] R392-33[医药卫生—基础医学]

 

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