摘要
目的研究肺腺癌细胞来源的外泌体对巨噬细胞极化的作用及受外泌体作用后的巨噬细胞对肺癌细胞生物学行为的影响。方法提取A549和H1299肺腺癌细胞外泌体,Western blot法检测其表面标志物CD9、CD63进行鉴定。100 ng/mL佛波酯(PMA)处理人THP-1细胞48 h,实时定量PCR检测细胞CD68的mRNA水平。200μg/mL外泌体处理贴壁巨噬细胞24 h后,实时定量PCR检测诱导型一氧化氮合酶(iNOS)、转化生长因子β(TGF-β)、肿瘤坏死因子α(TNF-α)、CD163的mRNA水平,IMMULITE 1000化学发光免疫分析仪检测白细胞介素6(IL-6)、IL-8、IL-10的蛋白水平;将外泌体处理后的巨噬细胞分别与A549细胞和H1299细胞共培养,通过TranswellTM实验检测肺腺癌细胞侵袭能力,实时定量PCR检测基质金属蛋白酶2(MMP2)、MMP9的mRNA水平。结果CD9、CD63在外泌体高表达,PMA诱导后的巨噬细胞高表达CD68,外泌体处理后的巨噬细胞iNOS表达降低,CD163、TNF-α、IL-6、IL-8、IL-10表达水平均增加;外泌体处理后的巨噬细胞与肺腺癌细胞共培养,可增强肺腺癌细胞的侵袭能力并促进MMP2、MMP9的表达。结论肺腺癌细胞来源的外泌体可以诱导巨噬细胞极化,表现为M1/M2的混合表型,进而增强肺癌细胞的自身侵袭能力。
Objective To examine the effect of exosomes derived from lung adenocarcinoma cells on macrophage polarization and the change of cytobiological behaviors in lung cancer cells induced by activated macrophages.Methods Exosomes derived from lung adenocarcinoma cells were extracted by exosomes extraction kit.The expression of exosomal markers including CD9 and CD63 was detected by Western blot analysis.After THP-1 cells were treated with 100 ng/mL phorbol ester(PMA)for 48 hours,the macrophage marker of CD68 was detected by real-time quantitative PCR(RT-qPCR).Following 24-hour treatment of macrophages with the exosomes(200μg/mL),the mRNA levels of transforming growth factorβ(TGF-β),tumor necrosis factorα(TNF-α),inducible nitric oxide synthase(iNOS)and CD163 were detected by RT-qPCR,and the protein levels of IL-6,IL-8 and IL-10 were measured by IMMULITE 1000.The macrophages after exosome treatment were co-cultured with A549 or H1299 cells.The invasion of lung adenocarcinoma cells was tested by TranswellTM assay and the mRNA levels of MMP9,MMP2 in lung adenocarcinoma cells were detected by RT-qPCR.Results CD9 and CD63 were highly expressed in exosomes.The THP-1 cells after PMA induction produced a high level of CD68.After the macrophages were treated with exosomes,the expression of iNOS decreased and the expression of CD163,TNF-α,IL-6,IL-8 and IL-10 significantly increased in the macrophages.The co-culture of macrophages with A549 and H1299 after exosome treatment enhanced significantly the invasion ability of lung adenocarcinoma cells and increased the levels of MMP2 and MMP9.Conclusion The exosomes derived from lung adenocarcinoma cells can activate macrophages to exhibit a mixed M1/M2 phenotype,thus promot the invasion of lung cancer cells.
作者
陈良远
杨阳
黄春莉
曹鹏驹
吴秋妹
陈少婷
陈发林
CHEN Liangyuan;YANG Yang;HUANG Chunli;CAO Pengju;WU Qiumei;CHEN Shaoting;CHEN Falin(Department of Clinical Laboratory,Fujian Provincial Hospital,Shengli Clinical Medical College of Fujian Medical University,Fuzhou 350001;Clinical Laboratory Center,Gansu Provincial People's Hospital,Lanzhou 730000,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2019年第11期967-972,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
福建省自然科学基金(2017J0105)
福建省卫生计生委中青年骨干人才培养项目(2018-ZQN-08)。
