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目的探讨炎性环境中内皮微粒(EMPs)在单核细胞参与的促内皮细胞增殖中的作用,并探讨EMPs对单核细胞表达内皮细胞标志物的影响。方法体外培养人脐静脉内皮细胞(HUVECs),炎性因子肿瘤坏死因子α(TNF-α)刺激内皮细胞,超速离心法获取EMPs;用透射电镜观察其形态和结构;运用酶联免疫吸附法(ELISA)检测经EMPs激活的单核细胞株THP-1对血管内皮生长因子(VEGF)中VEGF-A、VEGF-C的表达情况;将HUVECs和THP-1细胞或者经EMPs诱导的THP-1细胞共培养,检测HUVECs的增殖情况;运用RT-PCR和免疫荧光细胞化学技术,观察经EMPs激活的THP-1细胞对内皮细胞标志物vWF和血管内皮生长因子受体2(VEGFR-2)的表达情况。结果内皮细胞经炎性因子刺激后释放EMPs,EMPs刺激的THP-1细胞上清中VEGF-A和VEGF-C蛋白水平明显升高;HUVECs细胞增殖实验中,与THP-1细胞或者经EMPs诱导的THP-1共培养的两种情况下,HUVECs均显著增多;与EMPs共培养3 d后,THP-1中vWF和VEGFR-2蛋白表达为阳性,基因表达为阴性,共培养8 d后,vWF和VEGFR-2的蛋白和基因表达均为阴性。结论炎性环境中内皮细胞释放EMPs可以促使单核细胞分泌VEGF-A和VEGF-C,后两者可使HUVECs增殖,EMPs仅能使单核细胞一过性呈现内皮表型,而不能将其转分化为内皮细胞。
Objective To investigate the role of endothelial progenitor cells (EMPs) in the proliferation of monocytes-mediated endothelial cells in inflammatory environment and to explore the effect of EMPs on the expression of endothelial cell markers in monocytes. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in vitro. Endothelial cells were stimulated with tumor necrosis factor-α (TNF-α), and EMPs were obtained by ultracentrifugation. Morphology and structure were observed by transmission electron microscopy. ELISA was used to detect the expression of VEGF-A and VEGF-C in vascular endothelial growth factor (VEGF) by EMPs-stimulated monocytic cell line THP-1. HUVECs and THP-1 cells or THP-1 cells induced by EMPs The proliferation of HUVECs was detected by RT-PCR and immunocytochemistry. The expression of vWF and VEGFR-2 in THP-1 cells activated by EMPs was observed by RT-PCR and immunocytochemistry Happening. Results EMPs were released from endothelial cells stimulated by inflammatory cytokines, and the levels of VEGF-A and VEGF-C in EMPs-stimulated THP-1 cells were significantly increased. In HUVECs, THP-1 cells or induced by EMPs THP-1 co-cultured in both cases, HUVECs were significantly increased; co-cultured with EMPs 3 d, THP-1 vWF and VEGFR-2 protein expression was positive, gene expression was negative, co-cultured for 8 days, vWF and VEGFR-2 protein and gene expression were negative. Conclusion The release of EMPs by endothelial cells in inflammatory environment can induce monocytes to secrete VEGF-A and VEGF-C. The latter two can proliferate HUVECs. EMPs can only make monocytes transiently express endothelial phenotype, Transdifferentiation into endothelial cells.