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  

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

作　　者：李建明[1] 蔡黔[1] 周红[1] 肖光夏[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所,重庆400038

出　　处：《中华烧伤杂志》2002年第2期78-80,共3页Chinese Journal of Burns

基　　金：国家重点基础研究规划资助项目 (G19990 5 42 0 2 )

摘　　要：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 -ECVObjective 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 ×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 TNFα and IL 6 levels of the supernatant of the cultured cells were determined at 4, 8, 12 and 24 hours after culturation. Results The TNFα production of cultured pure ECV 304 exhibited no remarkable change when stimulated by different concentrations of LPS. But the TNFα production of the ECV 304 increased significantly when co cultured with PMNs at 4 hr and stimulated by LPS in concentration of 10 μg/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 μg/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. [

关 键 词：内毒素 中性粒细胞 血管内皮细胞 共培养 体外研究 烧伤 炎症反应 

分 类 号：R644[医药卫生—外科学]