CAJ | 학술논문

目的采用不同浓度的肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)体外刺激人脐静脉内皮细胞,观察TNF-α所致内皮细胞损伤及其培养上清中CD62E+、CD31+内皮微颗粒(Endothelial micro-particles,EMPs)释放水平,并初步探讨内皮细胞内的活性氧(Reactive oxygen species,ROS)在内皮微颗粒形成中的可能机制.方法倒置光学显微镜和共聚焦荧光显微镜下观察不同浓度TNF-α作用于内皮细胞24h后细胞形态和核的改变;流式细胞仪检测培养上清中CD62E+EMPs、CD31+EMPs的释放水平;荧光探针DCFH-DA法检测内皮细胞内活性氧的生成情况.结果 TNF-α可致内皮细胞形态和细胞核改变;随着TNF-α刺激浓度的增加,其培养上清中CD62E+EMPs释放水平越明显,与对照组相比,差异具有显著性(P<0.01),上清中CD31+EMPs释放水平也明显增高,与对照组相比,差异有统计学意义(P<0.01),同时CD62E+ EMPs /CD31+EMPs比值明显增大(P<0.01);随着TNF-α刺激浓度的增加,细胞内活性氧的水平升高越明显,与对照组相比,差异有统计学意义(P<0.05).结论不同浓度的TNF-α致损内皮细胞的程度不同,随着TNF-α刺激浓度的增加,内皮细胞的损伤程度更明显,CD62E+EMPs和CD31+EMPs释放量也相应增加,尤以CD62E+EMPs增加为甚,推测CD62E+EMPs释放水平可用来预测内皮损伤程度;推测ROS途径可能参与了内皮微颗粒形成与释放的过程.
목적채용불동농도적종류배사인자-α(Tumor necrosis factor-α,TNF-α)체외자격인제정맥내피세포,관찰TNF-α소치내피세포손상급기배양상청중CD62E+、CD31+내피미과립(Endothelial micro-particles,EMPs)석방수평,병초보탐토내피세포내적활성양(Reactive oxygen species,ROS)재내피미과립형성중적가능궤제.방법도치광학현미경화공취초형광현미경하관찰불동농도TNF-α작용우내피세포24h후세포형태화핵적개변;류식세포의검측배양상청중CD62E+EMPs、CD31+EMPs적석방수평;형광탐침DCFH-DA법검측내피세포내활성양적생성정황.결과 TNF-α가치내피세포형태화세포핵개변;수착TNF-α자격농도적증가,기배양상청중CD62E+EMPs석방수평월명현,여대조조상비,차이구유현저성(P<0.01),상청중CD31+EMPs석방수평야명현증고,여대조조상비,차이유통계학의의(P<0.01),동시CD62E+ EMPs /CD31+EMPs비치명현증대(P<0.01);수착TNF-α자격농도적증가,세포내활성양적수평승고월명현,여대조조상비,차이유통계학의의(P<0.05).결론불동농도적TNF-α치손내피세포적정도불동,수착TNF-α자격농도적증가,내피세포적손상정도경명현,CD62E+EMPs화CD31+EMPs석방량야상응증가,우이CD62E+EMPs증가위심,추측CD62E+EMPs석방수평가용래예측내피손상정도;추측ROS도경가능삼여료내피미과립형성여석방적과정.
Objective To observe the injury of endothelial cell stimulated by tumor necrosis factor(TNF-α),to compare different types and levels of endothelial micro-particles(EMPs) released in culture supernatant,and then to discuss whether the ROS path is one of formation mechanism of EMPs.Methods Through observing the morphologic change of human umbilical venous endothelial cells(HUVEC) by light microscope,and detecting the effect of TNF-α on the cell nucleus by Hoechst;the number of EMPs(CD31 positive,CD62E positive EMPs) in culture supernatant were measured by flow cytometry.The level of ROS was tested by DCFH-DA assay.Result TNF-α can induce the change of cell morphology and cell nucleus.The levels of CD62E positive,CD31 positive EMPs were significantly elevated in TNF-α stimulated cells compared with controls(P<0.01).We also observed that the ratio of CD62E positive EMPs to CD31 positive EMPs was remarkably increased compared with the controls(P<0.01).The levels of intracellular ROS were also significantly increased in TNF-α stimulated cells(P<0.05).Conclusion TNF-α can induce activation of HUVEC and promote release of CD62E positive and CD31 positive EMPs which may be considered one of surrogate marker for endothelial injury and dysfunction.ROS path is one of important formation mechanism of EMPs.