Effect of brain-derived microvesicles on cytoskeleton of human umbilical vein endothelial cells

Q4 Medicine

中华神经医学杂志 Pub Date : 2020-01-15 DOI:10.3760/CMA.J.ISSN.1671-8925.2020.01.003

Ji-wei Wang, Yingang Wu, Qi-feng Li, Yalong Gao, Yuan Zhou, Guili Yang, Jian-ning Zhang

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Abstract

Objective To observe the effect of brain-derived microvesicles (BDMVs) on cytoskeleton in human umbilical vein endothelial cells (HUVECs). Methods BDMVs were prepared in vitro and identified by transmission electron microscopy and particle size identification. HUVECs were co-cultured with PKH26-labeled BDMVs for 0.5, 1, and 2 h; flow cytometry was used to detect the phagocytosis of HUVECs for BDMVs at different time points. HUVECs cultured in vitro were divided into control group, BDMVs treatment group and nimodipine treatment group; cells in the BDMVs treatment group were given 1.5×107/mL BDMVs; cells in the nimodipine treatment group were pretreated with 2 μg nimodipine (0.2 mg/mL) for 10 min, and then, given 1.5×107/mL BDMVs. After being stained with rhodamine-labeled phalloidin, the fluorescence intensity and number of stress fibers of fibroactin in HUVECs were observed by laser confocal microscopy. Results BDMVs had complete membrane structure with a diameter of 100-1000 nm under transmission electron microscopy.The proportion of cells phagocytizing BDMVs increased significantly with prolonged incubation time, enjoying significant differences (0.5 h: 22.7%±1.2%; 1 h: 52.3%±1.3%; 2 h: 71.6%±1.9%, P<0.05). Laser confocal microscopy showed that, as compared with the control group, the fluorescence intensity of cytoskeletal protein was obviously increased and the number of stress fibers increased was obviously larger in the BDMVs treatment group. As compared with those in the BDMVs treatment group, the fluorescence intensity of cytoskeletal protein was decreased and the number of stress fibers was obviously smaller in the nimodipine group. Conclusion The role of BDMVs in phagocytosis of HUVECs becomes stronger as time being prolonged, and BDMVs phagocytosis leads to cytoskeletal remodeling, which can be partially blocked by nimodipine. Key words: Brain-derived microvesicle; Human umbilical vein endothelial cell; Cytoskeleton

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脑源性微泡对人脐静脉内皮细胞骨架的影响

目的观察脑源性微泡(BDMVs)对人脐静脉内皮细胞(HUVECs)细胞骨架的影响。方法体外制备bdmv，采用透射电镜和粒度鉴定方法对其进行鉴定。huvec与pkh26标记的bdmv共培养0.5、1和2小时;流式细胞术检测不同时间点HUVECs对bdmv的吞噬作用。体外培养HUVECs分为对照组、BDMVs治疗组和尼莫地平治疗组;BDMVs治疗组给予1.5×107/mL BDMVs;尼莫地平治疗组用2 μg尼莫地平(0.2 mg/mL)预处理细胞10 min，然后给予1.5×107/mL BDMVs。用罗丹明标记的phalloidin染色后，用激光共聚焦显微镜观察HUVECs中纤维肌动蛋白的荧光强度和应力纤维的数量。结果bdmv具有完整的膜结构，透射电镜下直径为100 ~ 1000 nm。随着培养时间的延长，吞噬BDMVs的细胞比例显著增加，差异有统计学意义(0.5 h: 22.7%±1.2%;1 h: 52.3%±1.3%;2 h: 71.6%±1.9%，P<0.05)。激光共聚焦显微镜观察显示，与对照组相比，BDMVs处理组细胞骨架蛋白荧光强度明显增强，增加的应力纤维数量明显增多。与BDMVs处理组比较，尼莫地平组细胞骨架蛋白荧光强度降低，应力纤维数量明显减少。结论BDMVs对HUVECs的吞噬作用随着时间的延长而增强，BDMVs的吞噬作用导致细胞骨架重塑，尼莫地平可部分阻断BDMVs的吞噬作用。关键词:脑源性微泡;人脐静脉内皮细胞;细胞骨架

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