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目的 研究细菌脂多糖 (lipopolysaccharide,LPS)刺激时 ,血管内皮细胞 (vascularen dothelialcells,VECs)激活cAMP反应元件结合蛋白 (cAMPresponseelementbindingprotein ,CREB)。 方法 用 10 0ng/mlLPS刺激VECs至预定时相点 (0 ,15 ,30 ,6 0 ,12 0 ,180min) ,然后用免疫细胞化学和Westernblotting方法研究CREB的磷酸化 ,用凝胶电泳迁移分析法 (EMSA)研究CREB与DNA的结合活性 ;用不同浓度的LPS(0 ,0 .0 1,0 .1,1.0 ,10 ,10 0 ,10 0 0ng/ml)刺激VECs30min ,然后用凝胶电泳迁移分析法研究CREB与DNA的结合活性。 结果 10 0ng/mlLPS刺激VECs后 15～ 12 0min ,均可检测到CREB的磷酸化及与DNA结合活性的增强 ,30～ 6 0min时增强最为明显 ,180min后恢复至正常水平 ;LPS刺激VECs可以诱导CREB -DNA结合活性的增强 ,且随着LPS刺激浓度增加 ,结合活性逐渐增强 ,10 0ng/ml及 10 0 0ng/ml浓度组具有最强的结合活性。 结论 LPS刺激VECs能激活CREB转录因子
Objective To investigate the effects of lipopolysaccharide (LPS) on activation of cAMP response element binding protein (CREB) by vascular endothelial cells (VECs). Methods VECs were stimulated with 10 0 ng / ml LPS for a predetermined time (0, 15, 30, 60, 120, 180 min). The phosphorylation of CREB was studied by immunocytochemistry and Western blotting. (EMSA) was used to study the binding activity of CREB to DNA. VECs were stimulated with different concentrations of LPS (0, 0.01, 1.0, 1.0, 10, 10 0, 10 ng / ml for 30 min and then electrophoresed by gel electrophoresis Analytical method to study the binding activity of CREB to DNA. Results After stimulated with 10ng / ml LPS for 15-12Omin, the phosphorylation and DNA binding activity of CREB were detected. The enhancement was most obvious at 30-60min and returned to normal level after 180min. VECs stimulated by LPS could induce CREB-DNA binding activity increased, and with the increasing concentration of LPS stimulation, binding activity gradually increased, 10 0ng / ml and 100ng / ml concentration group has the strongest binding activity. Conclusion LPS stimulates VECs to activate CREB transcription factor