机械牵张肺上皮细胞通过促进巨噬细胞极化诱导上皮细胞间质转分化  

Mechanical stretching of lung epithelial cells induces mesenchymal transition of epithelial cells by promoting macrophages polarization

作　　者：郑永信 杨媛媛 黄勇波 杨宝欣 陈康勰 毛璞 张海波 黎毅敏 ZHENG Yongxin;YANG Yuanyuan;HUANG Yongbo;YANG Baoxin;CHEN Kangxie;MAO Pu;ZHANG Haibo;LI Yimin(Department of Respiratory and Critical Care Medicine,The First Affiliated Hospital of Guangzhou Medical University,Guangzhou Institute of Respiratory Health,State Key Laboratory of Respiratory Diseases,Guangzhou,Guangdong 510120,P.R.China)

机构地区：[1]呼吸疾病国家重点实验室,广州呼吸健康研究院,广州医科大学附属第一医院呼吸与危重症医学科,广东广州510120

出　　处：《中国呼吸与危重监护杂志》2021年第12期870-876,共7页Chinese Journal of Respiratory and Critical Care Medicine

基　　金：广东省自然科学基金项目(2017A030313781);国家自然科学基金(81870069、81770077)。

摘　　要：目的探究机械牵张人肺上皮细胞上清对巨噬细胞极化的影响以及极化的巨噬细胞促进人肺上皮细胞发生上皮–间质转分化(EMT)。方法选用肺上皮细胞BEAS-2B构建机械牵张模型,通过酶联免疫吸附法(ELISA)检测静止培养和机械牵张的BEAS-2B细胞上清γ干扰素、粒细胞–巨噬细胞集落刺激因子、肿瘤坏死因子-α、白细胞介素(IL)-4、IL-6、IL-10等细胞因子水平变化。应用佛波酯诱导人急性单核细胞白血病THP-1细胞分化为M0巨噬细胞,使用牵张或静止的BEAS-2B细胞上清分别刺激M0巨噬细胞,通过流式细胞仪检测M1巨噬细胞(CD197)及M2巨噬细胞(CD206)表面标志物表达。建立牵张或静止BEAS-2B细胞上清刺激M0巨噬细胞和BEAS-2B细胞共培养体系,并加入血小板生长因子受体抑制剂(PDGFRi),使用蛋白印迹法检测EMT标志蛋白表达。结果机械牵张BEAS-2B细胞后,牵张上清的促M1/M2极化的细胞因子均显著升高。通过机械牵张的BEAS-2B细胞上清培养M0巨噬细胞后,可以显著升高M1巨噬细胞的标志物CD197和M2巨噬细胞的标志物CD206表达水平。机械牵张的BEAS-2B细胞上清促进M0巨噬细胞和BEAS-2B细胞共培养体系中的BEAS-2B细胞发生EMT,表现为上皮标志物细胞角蛋白8和E-钙黏素表达下降,间质标志物α-平滑肌肌动蛋白、N-钙黏素和波形蛋白表达增高,并且共培养体系上清的血小板生长因子(PDGF)的显著升高。加入PDGFRi后可阻断EMT的发生。结论机械牵张诱导的BEAS-2B细胞上清可以促进M0巨噬细胞向M1和M2极化,而极化的巨噬细胞通过释放PDGF促进BEAS-2B细胞间质转分化,通过阻断PDGF通路可以显著减轻巨噬细胞介导的BEAS-2B细胞间质转分化。Objective To study the effect of mechanical stretch on the microenvironment of BEAS-2 B on macrophage polarization and the role of polarized macrophages in the epithelial-mesenchymal transition(EMT) of BEAS-2 B.Methods Using enzyme linked immunosorbent assay to detect the changes in the levels of cytokines such as interferon-γ,granulocyte-macrophage colony stimulating factor, tumor necrosis factor-α, interleukin(IL)-4, IL-6, IL-10 in the supernatant of lung epithelial cells cultured statically and mechanically stretched. The M0 macrophages(derived from THP-1) were stimulated by stretch/static conditioned medium of BEAS-2 B. The surface markers of M1(CD197)/M2(CD206) macrophages were detected by flow cytometer. Stretch/static conditioned medium were used to stimulate the co-culture system of macrophages and BEAS-2 B in the presence or absence of platelet-derived growth factor receptor inhibitor(PDGFRi), then the protein expression level of EMT makers was examined by Western blot. Results Exposure of BEAS-2 B to mechanical stretch resulted in significantly higher production of the pro-M1/M2 polarized factor. The EMT of the co-culture system of M0 and BEAS-2 B could be induced by stretch conditioned medium, epithelial marker cytokeratin(CK)-8 and E-cadherin were decreased, while mesenchymal marker α-smooth muscle actin, N-cadherin and vimentin were increased in stretch conditioned medium group. The expression of platelet-derived growth factor(PDGF)was significantly higher in stretch conditioned medium group. The PDGFRi can block the EMT in stretch conditioned medium group. Conclusions The lung epithelial cell supernatant induced by mechanical stretch can promote the polarization of macrophages to M1 and M2. Polarized macrophages promote EMT in human lung epithelial cells via PDGF, and blocking PDGF might attenuate the VILI-associated lung fibrosis.

关 键 词：急性呼吸窘迫综合征 巨噬细胞 血小板生长因子 上皮–间质转分化 机械牵张 

分 类 号：R563.8[医药卫生—呼吸系统]