摘要
目的对比分析泛素对正常状态下的人脐静脉内皮细胞(HUVECs)和巨噬细胞的影响及其差别,并评价泛素是否对脂多糖(LPS)诱导的HUVECs损伤具有保护作用。方法在显微镜下观察不同浓度泛素干预后HUVECs细胞形态学的变化。将体外培养的HUVECs和人巨噬细胞分为空白对照组和4个泛素干预组(0.01μg/m L组、0.1μg/m L组、1μg/m L组和10μg/m L组),ELISA法测定干预后24 h各组细胞上清肿瘤坏死因子α(TNF-α)和血管细胞粘附分子1(VCAM-1)水平,Western blot法测定细胞核因子κB(NF-κB)蛋白水平。用0.1μg/m L泛素干预10μg/m L LPS诱导的HUVECs,ELISA法测定8、16和24 h泛素+LPS组和LPS组细胞上清TNF-α和VCAM-1水平。结果随着泛素干预浓度的增加,HUVECs损伤的程度逐步加重,当泛素浓度达到50μg/m L时,细胞开始呈气球样变并大量死亡。随着泛素干预浓度的升高,HUVECs细胞上清TNF-α和VCAM-1水平呈先升高后降低的变化趋势,而人巨噬细胞的TNF-α和VCAM-1水平则是呈逐步升高的趋势;HUVECs和人巨噬细胞NF-κB蛋白水平在泛素浓度为0.1μg/m L和1μg/m L时明显增高,而在0.01μg/m L和10μg/m L时增高不明显,甚至有减低的趋势,其中HUVECs减低的趋势更为明显。0.1μg/m L泛素干预10μg/m L LPS诱导的HUVECs,各时间点LPS组和泛素+LPS组的TNF-α和VCAM-1水平相似,差异无统计学意义。结论较低浓度的泛素对HUVECs即有明显的损伤作用,而人巨噬细胞则需要较高的泛素浓度才出现损伤。泛素对体外培养的LPS诱导的HUVECs损伤不具有明显的保护作用。
Objective To compare the different effects of ubiquitin( UB) on human umbilical vein endothelial cells( HUVECs) and macrophages under normal circumstances,and analyze whether UB could protect HUVECs from lipopolysaccharide( LPS) induced injury. Methods The morphologic changes of HUVECs in vitro with up-rising concentrations of UB interventions were observed. HUVECs and human macrophages in vitro were divided into 4 groups according to UB concentration( 0. 01 μg / m L,0. 1 μg / m L,1 μg / m L,and 10 μg / m L). Supernatant and cells of each group were collected in 24 h after UB intervention. The levels of TNF-α and VCAM-1 in supernatant were measured by ELISA while NF-κB protein level in cells was detected by Western blot. HUVECs were divided into a LPS group( LPS10 μg / m L) and an UB + LPS group( UB 0. 1 μg / m L,LPS 10 μg / m L). The supernatant of the two groups were collected in 8,16 and 24 h after LPS and UB intervention. The levels of TNF-α and VCAM-1 in supernatant were measured by ELISA. Results The injury of HUVECs got worse with the ascending concentrations of UB. At the concentration of 50 μg / m L,UB induced HUVECs got ballooned and died massively. With the increase of UB concentration,the levels of TNF-α and VCAM-1 in HUVECs 'supernatant ascended firstly and then descended,while those in human macrophages' supernatant ascended gradually. z However,the tendency of the NF-κB protein level in the two kinds of cells was similar when the concentration of UB increased. At the consentration of 0. 1 μg / m L or 1 μg / m L,ubiquitin induced NF-κB protein level obviously increased. At the concentration of 0. 01 μg / m L or 10 μg / m L,UB induced the protein level was similar with those of the control group and even decreased slightly. There was no significant difference in TNF-α or VCAM-1 levels at each time point between the LPS group and the UB + LPS group.Conclusions UB injuries HUVECs obviously at a low concentration but injuires human macrophages at much higher concentraton. UB can not protect HUVECs from LPS-induced injury in vitro.
出处
《中国呼吸与危重监护杂志》
CAS
北大核心
2015年第2期173-177,共5页
Chinese Journal of Respiratory and Critical Care Medicine
基金
南京医科大学科技发展基金面上项目(编号:2011NJMU076)
关键词
泛素
人脐静脉内皮细胞
人巨噬细胞
脂多糖
Ubiquitin
Human umbilical vein endothelial cells
Human macrophages
Lipopolysaccharide
