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.
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)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
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)
