【摘要】 目的:研究三阴性乳腺癌(triple-negative breast cancer, TNBC)细胞来源的外泌体对巨噬细胞极化作用以及经外泌体活化的巨噬细胞对TNBC细胞迁移和侵袭能力的影响及作用机制。方法:提取TNBC细胞BT-549和MDA-MB-231外泌体，透射电镜、纳米颗粒跟踪分析(nanoparticle tracking analysis, NTA)、蛋白质印记(Western blot)鉴定外泌体；流式细胞术检测经外泌体处理后巨噬细胞分子标志物CD206、CD80的表达；实时荧光定量PCR检测单核细胞THP-1、巨噬细胞M0及外泌体处理后巨噬细胞中CD163、转化生长因子β(transforming growth factorβ，TGF-β)、一氧化氮合酶(nitric oxide synthase,iNOS)、肿瘤坏死因子α(tumor necrosis factorα，TNF-α)的mRNA水平；Transwell实验分析外泌体活化的巨噬细胞对TNBC细胞迁移和侵袭的影响，Western blot方法检测MMP-2、MMP-9、N-cadherin、E-cadherin的蛋白水平。结果：透射电镜下BT-549和MDA-MB-231细胞的外泌体具有膜结构囊状小泡并表达外泌体的分子标记物CD81、CD63;BT-549细胞来源的外泌体直径分布为80～150 nm,MDA-MB-231细胞来源的外泌体直径分布为100～200 nm；经BT-549和MDA-MB-231细胞源性外泌体处理的巨噬细胞与M0相比，CD206均有稳定表达(P<0.05)，而CD80表达未见明显差异；外泌体处理后巨噬细胞的CD163、TGF-βmRNA表达水平均增加(P<0.01),iNOS mRNA表达水平降低(P<0.05),TNF-αmRNA表达水平未见明显变化；外泌体处理后的巨噬细胞使得TNBC细胞的迁移、侵袭穿膜数目增加(P<0.01),N-cadherin、MMP-2和MMP-9蛋白表达增加(P<0.05),E-cadherin蛋白表达降低(P<0.05)。结论：TNBC细胞分泌的外泌体可以诱导巨噬细胞向M2极化，进而促进TNBC细胞间质转化，增强TNBC细胞的迁移和侵袭能力。更多还原

【Abstract】 Objective:To study the effects of exosomes derived from triple-negative breast cancer(TNBC) cells on the polarization of macrophages and the effects of exosome-activated macrophages on the migration and invasion of TNBC cells and the mechanism of action.Methods:Exosomes of TNBC cells BT-549 and MDA-MB-231 were extracted and identified by transmission electron microscopy, nanoparticle tracking analysis(NTA) and Western blot.The expression of CD206 and CD80 in macrophages after exosomal treatment was detected by flow cytometry.The mRNA levels of CD163,transforming growth factor β(TGF-β),nitric oxide synthase(iNOS) and tumor necrosis factor α(TNF-α) in monocytes THP-1,macrophages M0 and macrophages treated with exosomes were detected by real-time fluorescence quantitative PCR.Transwell assay was used to analyze the effects of exosomal-activated macrophages on the migration and invasion of TNBC cells, and Western blot was used to detect the protein levels of MMP-2,MMP-9,N-cadherin and E-cadherin.Results:Under transmission electron microscopy, the exosomes of BT-549 and MDA-MB-231 cells had membrane-like vesicles and expressed the molecular markers CD81 and CD63 of exosomes.The diameter distribution of exosomes derived from BT-549 cells was 80～150 nm, and that from MDA-MB-231 cells was 100～200 nm.Compared with M0,CD206 expression was stable in macrophages treated with BT-549 and MDA-MB-231 exosomes(P<0.05),but there was no significant difference in CD80 expression.After exosome treatment, the mRNA expression levels of CD163 and TGF-β in macrophages were increased(P<0.01),the mRNA expression level of iNOS was decreased(P<0.05),and the mRNA expression level of TNF-α was not significantly changed.Exosomal-treated macrophages increased the number of TNBC cell migration and invasion transmembrane(P<0.01),increased the expression of N-cadherin, MMP-2 and MMP-9 proteins(P<0.05),and decreased the expression of E-cadherin protein(P<0.05).Conclusion:The exosomes secreted by TNBC cells can induce the polarization of macrophages to M2,thus promoting the interstitial transformation of TNBC cells and enhancing the migration and invasion ability of TNBC cells.更多还原