Department of Cardiovascular Disease, Fourth Affiliated Hospital, Nanchang University, China.
Cell Prolif. 2011 Aug;44(4):352-9. doi: 10.1111/j.1365-2184.2011.00764.x.
The aim of this investigation was to determine whether tumour necrosis factor-alpha (TNF-α) has any effect on endothelial progenitor cells (EPCs). Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with tumour necrosis factor-α (final concentrations: 0, 10, 20, 50 and 100 mg/l) for 0, 6, 12, 24 and 48 h. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding, by direct fluorescence staining. EPC proliferation and migration were assayed using the MTT assay and modified Boyden chamber assay, respectively. EPC adhesion assay was performed by re-plating those cells on fibronectin-coated dishes, and adherent cells were counted. Tube formation activity was assayed using a tube formation kit. Levels of apoptosis were revealed using an annexin V apoptosis detection kit. Vascular endothelial growth factor Receptor-1 (VEGF-R1) and stromal derived factor-1 (SDF-1) mRNA, assessed by real-time RT-PCR inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were assayed by western blot analysis. Incubation of EPCs with tumour necrosis factor-α reduced EPC proliferation, migration, adhesion, tube formation capacity, iNOS and eNOS in concentration- and time-dependent manners. Tumour necrosis factor-α reduced proliferation, migration, adhesion and tube formation capacity of EPCs. TNF-α increased EPC apoptosis level, reduced VEGF-R1 and SDF-1 mRNA expression; tumour necrosis factor-α also reduced iNOS and eNOS in the EPCs.
本研究旨在探讨肿瘤坏死因子-α（TNF-α）对内皮祖细胞（EPCs）是否有影响。采用 Ficoll 密度梯度离心法从外周血中分离出总单个核细胞，然后将细胞铺在纤维连接蛋白包被的培养皿上。培养 7 天后，用 TNF-α（终浓度：0、10、20、50 和 100mg/L）刺激贴壁细胞 0、6、12、24 和 48 小时。通过直接荧光染色，将双阳性摄取 DiLDL 和结合凝集素的贴壁细胞鉴定为 EPCs。采用 MTT 法和改良 Boyden 室法分别检测 EPC 增殖和迁移。通过将这些细胞重新铺在纤维连接蛋白包被的培养皿上进行 EPC 黏附实验，并计数黏附细胞。采用管形成试剂盒检测管形成活性。采用 Annexin V 凋亡检测试剂盒检测细胞凋亡水平。通过实时 RT-PCR 检测血管内皮生长因子受体-1（VEGF-R1）和基质衍生因子-1（SDF-1）mRNA，通过 Western blot 分析检测诱导型一氧化氮合酶（iNOS）和内皮型一氧化氮合酶（eNOS）。结果显示，TNF-α呈浓度和时间依赖性地降低 EPC 的增殖、迁移、黏附、管形成能力、iNOS 和 eNOS。TNF-α降低 EPC 的增殖、迁移、黏附和管形成能力。TNF-α增加 EPC 的凋亡水平，降低 VEGF-R1 和 SDF-1mRNA 表达；TNF-α还降低了 EPC 中的 iNOS 和 eNOS。
