Objective:To investigate the effect of macrophage on cardiac fibroblasts (CFs) transformation in 3-dimensional nano gels co-culture with Angiotension Ⅱ (Ang Ⅱ) treatment.Studying the role of macrophage in regulating CFs transformation with Ang Ⅱt treatment will provide important evidence regarding development of novel targeted therapies for preventing hypertension-induced cardiac fibrosis.Methods:Bone marrowderived macrophages (BMDMs) were isolated from tibias and femurs of adult mice,CFs from ventricle of heart of neonatal mice were cultured by differential anchoring velocity method.We cocultured BMDMs with mouse CFs in 3-dimensional peptide gels,as an in vitro model of macrophage-mediated fibroblast-to-myofibroblast differentiation.Cells were divided into CFs group,CFs + macrophage group,CFs + Ang Ⅱ group and CFs + macrophage + Ang Ⅱ group.Identification of macrophages by detecting F4/80 by immunofluorescence.Identification of fibroblasts by detecting vimentin by immunofluorescence.The morphology of 3-dimensional nano gels co-culture was assess by phase contrast microscopy.The expression levels of α-SMA were analyzed by immunofluorescence staining.In addition,the mRNA levels of α-SMA,Collagen Ⅰ,TGF-β and CTGF were measured by Real-time PCR.Results:In 3-dimensional peptide gels,compared with CFs or CFs + macrophage group,the protein and mRNA levels of α-SMA were markedly increased in co-culture with macrophages in response to Ang Ⅱ.Moreover,the mRNA levels of Collagen Ⅰ and profibrotic factors(TGF-β,CTGF)were also significantly upregulated in co-culture with macrophages in response to Ang Ⅱ compared with that in CFs or CF + macrophage control.Conclusion:Macrophages promote cardiac fibroblast-to-myofibroblast transformation in response to Ang Ⅱ,which suggests that macrophages play the pivotal roles in Ang Ⅱ-induced cardiac fibrosis.%目的:探讨血管紧张素Ⅱ(angiotensionⅡ,AngⅡ)作用下,细胞三维共培养体系中巨噬细胞对心脏成纤维细胞(cardiac fibroblasts,CFs)表型转化的影响.为观察细胞之间的相互作用,探讨高血压导致心脏纤维化的分子机制及防治策略提供了新思路.方法:分离、培养原代小鼠骨髓巨噬细胞及乳鼠CFs,建立巨噬细胞和CFs多肽纳米凝胶三维共培养体系.实验分为CFs组,巨噬细胞与CFs共培养组,CFs+AngⅡ处理组,巨噬细胞与CFs共培养+AngⅡ处理组.通过免疫荧光染色鉴定培养的巨噬细胞(巨噬细胞标志物F4/80)及成纤维细胞(波形蛋白Vimentin)；采用倒置相差显微镜观察三维培养细胞形态；免疫荧光染色检测α平滑肌肌动蛋白(α-SMA)表达；实时定量PCR检测α-SMA、Ⅰ型胶原(Collagen Ⅰ)及促纤维化因子[转化生长因子β(TGF-β)、结缔组织生长因子(CTGF)]的表达.结果:在无AngⅡ作用下,巨噬细胞与CFs共培养组与单独培养的CFs组比较,α-SMA mRNA和蛋白表达,并且Collagen Ⅰ、TGF-β3和CTGF mRNA的水平差异无统计学意义.巨噬细胞与CFs共培养组经AngⅡ处理后,可明显诱导α-SMA mRNA和蛋白高表达,并且上调Collagen Ⅰ、TGF-β3和CTGF mRNA的水平.结论:在AngⅡ作用下,三维培养体系中巨噬细胞可促进CFs向肌成纤维细胞表型转化,提示巨噬细胞在AngⅡ导致心脏纤维化的病理过程中发挥关键作用.
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