PDF(Pubmed)
Abstract：
EVs released by adipose derived stem cells (ADSCs) have shown promise as a therapeutic for tissue repair because of their purported immune-regulatory properties. Extracellular vesicles (EVs) from ADSCs could be beneficial in improving graft retention rates for autologous fat grafting (AFG) post-mastectomy as, currently, grafted tissue rates are variable. Enriching grafted tissue with ADSC-EVs may improve retention rates by modulating macrophages resident within both the breast and lipoaspirate. We aimed to identify key macrophage phenotypes that are modulated by ADSC-EVs in vitro. ADSCs were isolated from lipoaspirates of women undergoing AFG and characterised by flow cytometry and differentiation potential. ADSC-EVs were isolated from culture media and characterised by tuneable resistive pulse sensing, transmission electron microscopy and Western blot. Primary monocyte-derived macrophages were polarized to an M1-like (GM-CSF, IFNγ), M2-like phenotype (M-CSF, IL-4) or maintained (M0-like; M-CSF) and ADSC-EVs were co-cultured with macrophages for 48 h. Flow cytometry and high-dimensional analysis clustered macrophages post co-culture. A manual gating strategy was generated to recapitulate these clusters and was applied to a repeat experimental run. Both runs were analysed to examine the prevalence of each cluster, representing a unique macrophage phenotype, with and without ADSC-EVs. Following the addition of ADSC-EVs, M0-like macrophages demonstrated a reciprocal shift of cell distribution from a cluster with a \'high inflammatory profile\' (CD36+++CD206+++CD86+++; 16.5 ± 7.0%; p < 0.0001) to a cluster with a \'lower inflammatory profile\' (CD36+CD206+CD86+; 35 ± 21.5%; p < 0.05). M1-like macrophages shifted from a cluster with a \'high inflammatory profile\' (CD206++CD11b++CD36++CD163++; 26.1 ± 9.4%; p = 0.0024) to a \'lower inflammatory profile\' (CD206+CD11b+CD36+CD163+; 72.8 ± 8.7%; p = 0.0007). There was no shift in M2-like clusters following ADSC-EV treatment. ADSC-EVs are complex regulators of macrophage phenotype that can shift macrophages away from a heightened pro-inflammatory state.
摘要：
由脂肪来源的干细胞（ADSC）释放的EV由于其所谓的免疫调节特性，已显示出有望作为组织修复的治疗剂。来自ADSC的细胞外囊泡（EV）可能有益于提高自体脂肪移植（AFG）乳房切除术后的移植物保留率，目前,移植组织率是可变的。用ADSC-EV富集移植组织可以通过调节驻留在乳房和脂肪抽吸物中的巨噬细胞来提高保留率。我们的目的是确定ADSC-EV在体外调节的关键巨噬细胞表型。从接受AFG的女性的脂肪抽吸物中分离ADSC,并通过流式细胞术和分化潜能进行表征。ADSC-EV从培养基中分离，并通过可调电阻脉冲感应进行表征，透射电镜和蛋白质印迹。原代单核细胞衍生的巨噬细胞极化为M1样(GM-CSF，IFNγ),M2样表型（M-CSF，IL-4）或维持的（M0样；M-CSF）和ADSC-EV与巨噬细胞共培养48小时。流式细胞术和高维分析在共培养后聚集的巨噬细胞。生成手动门控策略以概括这些簇，并将其应用于重复的实验运行。对两次运行进行了分析，以检查每个集群的患病率，代表一种独特的巨噬细胞表型，有和没有ADSC-EV。在添加ADSC-EV之后，M0样巨噬细胞显示细胞分布从具有高炎症谱的簇（CD36++CD206++CD86++；16.5±7.0%；p<0.0001）到具有较低炎症谱的簇（CD36CD206CD86；35±21.5%；p<0.05）的相互转移。M1样巨噬细胞从“高炎症谱”(CD206++CD11b++CD36++CD163++;26.1±9.4%;p=0.0024)转移到“低炎症谱”(CD206+CD11b+CD36+CD163+;72.8±8.7%;p=0.0007)。ADSC-EV治疗后M2样簇没有变化。ADSC-EV是巨噬细胞表型的复杂调节因子，可以使巨噬细胞远离高度的促炎状态。
