ERK1/2信号通路在小鼠库普弗细胞-内毒素反应中的活化规律及其作用  

A study on activation and role of ERK1/2 signal cascade in LPS-induced response of mice Kupffer cells

作　　者：张宇[1] 蒋建新[1] 王正国[1] 朱佩芳[1] 吉善和[1] 单佑安[1] 周继红[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所第四研究室,重庆400042

出　　处：《中华创伤杂志》2002年第6期364-366,共3页Chinese Journal of Trauma

基　　金：国家重点基础研究发展规划资助项目(G19990 5 42 0 3 );军队医药卫生杰出中青年基金资助项目

摘　　要：目的　研究内毒素诱导下小鼠库普弗细胞 (KC)ERK1 2活化规律、肿瘤坏死因子α(TNFα)分泌规律以及ERK1 2信号通路在TNFα分泌中的作用 ,探讨防治内毒素血症的新方法。方法　磷酸化ERK时效组以含有 10 0ng mlLPS的培养基分别培养KC 0 ,5 ,10 ,2 0 ,30 ,4 5 ,6 0 ,12 0min ;磷酸化ERK量效组分别用含有 0 ,0 .1,1,10 ,10 0 ,10 0 0 ,10 0 0 0ng mlLPS的培养基孵育KC 30min ,免疫印迹杂交检测内毒素诱导KCERK1 2活化规律 ,酶联免疫分析检测内毒素诱导KC释放TNFα规律。以 0 ,0 .5 ,1,10 ,2 5 ,5 0 μmol LPD980 5 9(ERK信号通路特异性阻断剂 )预处理KC 1h ,酶联免疫分析检测PD980 5 9对LPS诱导TNFα分泌的抑制作用。　结果　 10 0ng ml内毒素刺激后 ,KC内ERK1 2被迅速活化 ,30min达到高峰 ,2h后基本恢复至正常水平 ;在 10pg ml～ 10 0ng ml范围内 ,内毒素对ERK1 2激酶具有剂量依赖性的激活作用 ;内毒素诱导KCTNFα释放显著增加 ,呈显著的剂量依赖性关系 ;随着PD980 5 9剂量的增加 ,其对LPS诱导KCTNFα分泌的抑制作用越大。　结论内毒素可诱导KCERK1 2活化和TNFα分泌 ,ERK1 2对内毒素诱导KC分泌TNFα具有显著的调节作用 ,ERK1Objective To study the rule of Kupffer cell (KC) ERK1/2 activation, TNFα secretion and regulation effect of ERK1/2 signal cascade on releasing of TNFα in order to explore the novel methods for preventing and treating clinical patients with endotoxemia. Methods After overnight culture, KCs were stimulated with 100 ng/ml LPS for 0, 5, 10, 20, 30, 45, 60, 120 minutes, respectively. In another experiment, KCs were stimulated with LPS of 0, 0.1, 1, 10, 100, 1 000, 10 000 ng/ml for 20 minutes. In the experiment of inhibition effect of PD98059 to TNFα releasing, KCs were pretreated with 0, 0.5, 1, 10, 25, 50 μmol/L PD98059 for 1 hour, and then stimulated with 100 ng/ml LPS for 4 hours. And western blotting analysis was used to detect the activation of ERK1/2 in mice KC stimulated by LPS, ELISA used to study the LPS induced TNFα releasing and effect of ERK1/2 signaling cascade on secretion of TNFα. Results In mice KC, LPS treatment activation of ERK1/2, with maximal value at 30 minutes and a return near to baseline within two hours, and LPS induced ERK1/2 activation from LPS concentration of 10 pg/ml to the top activation at 100 ng/ml . No activation was observed in unstimulated KC. LPS markedly increased releasing of TNFα and caused a dose dependent increase of TNFα. Inhibition of the ERK1/2 pathway using the MEK inhibitor PD98059 caused a marked, concentration dependent reduction of TNFα production. Conclusions LPS can markedly induce ERK1/2 activation and TNFα secretion in mice KC. ERK1/2 signal cascade has a significant regulative effect on the endotoxin inducing TNFα secretion. ERK1/2 may be the novel target to prevent and treat clinical patients with endotoxemia.

关 键 词：ERK1/2信号通路 小鼠 库普弗细胞-内毒素 脂多糖类 肿瘤坏死因子α 内毒素血症 细胞外信号调节激酶类 

分 类 号：R363[医药卫生—病理学]