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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 effects of ubiquitin on human umbilical vein endothelial cells (HUVECs) and macrophages under normal conditions and to determine whether ubiquitin has a protective effect on lipopolysaccharide (LPS) -induced HUVECs injury. Methods The morphological changes of HUVECs were observed under the microscope with different concentrations of ubiquitin. HUVECs and human macrophages cultured in vitro were divided into blank control group and 4 ubiquitin intervention groups (0.01μg / m L group, 0.1μg / m L group, 1μg / m L group and 10μg / m L group) The levels of tumor necrosis factor-alpha (TNF-α) and vascular cell adhesion molecule-1 (VCAM-1) in the supernatants were measured by ELISA at 24 h after intervention, and the level of NF-κB protein was determined by Western blot. HUVECs induced by 10μg / mL LPS were treated with 0.1μg / mL Ubiquitin, and the levels of TNF-α and VCAM-1 in the supernatant of ubiquitin + LPS and 8h groups were determined by ELISA. Results With the increase of ubiquitin concentration, the degree of injury of HUVECs gradually increased. When the concentration of ubiquitin reached 50 μg / m L, the cells started to balloon and died in large numbers. The levels of TNF-α and VCAM-1 in the supernatant of HUVECs increased first and then decreased with the increase of ubiquitin concentration, while the levels of TNF-α and VCAM-1 in human macrophages were gradually increased The levels of NF-κB in HUVECs and human macrophages were significantly increased at ubiquitin concentrations of 0.1μg / ml and 1μg / ml, but not at 0.01μg / ml and 10μg / ml Obviously, there is even a trend of decrease, in which the trend of decrease of HUVECs is more obvious. 0.1μg / mL Ubiquitin intervention 10μg / m L LPS-induced HUVECs, TNF-α and VCAM-1 levels in LPS group and ubiquitin + LPS group were similar at any time point, the difference was not statistically significant. Conclusions Ubiquitin at a lower concentration has obvious damage to HUVECs, while human macrophages require higher concentrations of ubiquitin to induce damage. Ubiquitin has no significant protective effect on LPS-induced HUVECs injury in vitro.