内毒素诱导共培养中性粒细胞——血管内皮细胞活化的体外研究

目的　建立人外周血中性粒细胞与人脐静脉血管内皮细胞系ECV 30 4体外共培养模型 ,研究内毒素对共培养中性粒细胞 血管内皮细胞活化的作用。　方法　将ECV 30 4细胞接种培养 ,待细胞接近融合 ,加入 2× 10 6/ml即时分离纯化的人外周血中性粒细胞 ,按不同的分组加入不同浓度的内毒素 (lipopolysaccharide ,LPS) ,在倒置显微镜下观察细胞形态学的改变 ,于 4、8、12、2 4h测定细胞上清中TNFα及IL 6水平的变化。　结果　不同浓度的LPS刺激内皮细胞TNFα产生没有明显的变化 ,而 1μg/mlLPS刺激共培养中性粒细胞 -ECV 30 4 ,在 4h其TNFα水平明显上升 ,10 μg/mlLPS刺激共培养中性粒细胞 -ECV 30 4 ,其TNFα水平逐渐上升 ,8h后比较明显 (P <0 .0 5 ) ,12、2 4h仍维持较高水平 ;对于单纯内毒素刺激ECV 30 4细胞 ,随着LPS浓度的增加 ,IL 6生成明显增加。 1μg/mlLPS刺激ECV 30 4IL 6自 4h后即明显上升 ,8、12h一直维持在高水平 ,直到 2 4h才明显回落。对于共培养的中性粒细胞和血管内皮细胞 ,单纯共培养中性粒细胞 -ECV 30 4IL 6无明显变化 ,而较低浓度10 0ng/mlLPS刺激共培养中性粒细胞 -ECV 30 4IL 6与 1μg/mlLPS刺激ECV 30 4相当 ,2 4h仍维持在高水平。 1μg/ml及 10 μg/ml刺激共培养中性粒细胞 -ECV 30

A preliminary in vitro study on the activation of polymorphonuclear cells and vascular endothelial cells co-cultured with and stimulated by lipopolysaccharide

OBJECTIVE: To explore the activation of polymorphonuclear cells (PMNs) and vascular endothelial cells co-cultured with and stimulated by lipopolysaccharide. METHODS: PMNs in concentration of 2 x 10(6)/ml isolated from healthy volunteers by Percoll gradient were added to monolayer of ECV-304 cells grown to confluency, then different groups were prepared according to final concentration of lipopolysaccharide. The morphological change was observed under invert microscope. The changes in TNFalpha and IL-6 levels of the supernatant of the cultured cells were determined at 4, 8, 12 and 24 hours after culturation. RESULTS: The TNFalpha production of cultured pure ECV-304 exhibited no remarkable change when stimulated by different concentrations of LPS. But the TNFalpha production of the ECV-304 increased significantly when co-cultured with PMNs at 4 hr and stimulated by LPS in concentration of 10 micro g/ml, and increased at 8 hours and lasted up to 24 hours of culturation in higher levels (P < 0.05). The IL-6 production of cultured pure ECV-304 increased obviously along with the increase of LPS concentration, and it showed no change when PMNs co-cultured with ECV-304. While the IL-6 level in the supernatant of co-cultured ECV-304 with PMNs increased sharply when stimulated by both low (100 ng/ml) and high (1 micro g/ml) concentrations of LPS and maintained at high levels up to 24 hours of culturation. The higher the concentration of LPS was, the quicker the IL-6 level increased. CONCLUSION: Co-cultured PMNs and endothelial cells could be activated and activation state could be maitained by low concentrations of LPS.