丙型肝炎病毒核心蛋白细胞内信号传导途径的探讨  

Exploration of intracellular signal transduction pathway of hepatitis C virus core protein

作　　者：朱峰[1] 钱家鸣[1] 孙钢[1] 杨晓鸥[1] 陆星华[1] 

机构地区：[1]中国医学科学院中国协和医科大学北京协和医院消化内科,100730

出　　处：《肝脏》2003年第1期6-8,共3页Chinese Hepatology

摘　　要：目的　研究丙型肝炎病毒核心蛋白致病作用的细胞内信号传导途径 (STPs)。方法　在前期工作已证实HCV核心蛋白激活NF κB介导的细胞内STPs这一结论的基础上 ,对核心蛋白活化NF κB的机制和作用位点进行了测定。用 0 .4μgpCXN2 core与 pNF κB共转染Hela细胞 3h后分别加入 5mmolIKKβ的抑制剂乙酰水杨酸和 2 5 μmol消炎痛 (作为对照 )各 0 .5ml ,继续转染 3 3h ,测定荧光素酶比活性 ,以此检测乙酰水杨酸对NF κB介导的STPs的抑制作用。以 pCXN2 core转染Cos 7细胞 3 6h ,收获细胞溶解液 ,用Western印迹分析检测IκBα表达 ,确定IκBα的降解情况。用pCXN2 core、pMEKK△ (表达非活性MEKK的质粒 )、pNF κB共转染Hela细胞 3 6h ,测定荧光素酶比活性 ,确定HCV核心蛋白是否通过MEKK活化NF κB介导的STPs。结果　 ( 1)HCV核心蛋白活化NF κB介导的STPs被乙酰水杨酸抑制 ;( 2 )pCXN2 core转染后的细胞溶解液中IκBα的量较pCXN2 转染后的少 ;( 3 )HCV核心蛋白活化NF κB介导的STPs的作用不被MEKK△阻断。结论　 ( 1)乙酰水杨酸通过抑制IKK的IκBα磷酸化作用 ,抑制IκB降解 ,阻断NF κB的活化 ,表明核心蛋白作用位点可能在IKK或其上游某个部位 ;( 2 )在表达HCV核心蛋白的细胞存在IκBα降解的增加 。Objective To investigate the intracellular signal tranduction pathway of HCV core protein.Methods On the basis of early works which confirmed HCV core protein activated NF κB mediated intracellular STPs, the mechanism and acting site were determined. 1) Hela cells were transfected with pCXN 2 core 0.4μg and pNF κB for 3 hours. 5mmol IKKβ inhibitor, aspirin, or 25μmol mezolin (as control), 0.5ml each, were then added and transfection proceeded for 33 hours. Fluorescein enzyme specific activities were assayed and the inhibitory effect of aspirin on NF κB mediated STPs was estimated. 2) Cos 7 cells were transfected with pCXN 2 core for 36 hours, then the cells lysate was collected. Expression of IκBα was assayed by Western blot to determine the degradation of IκBα. 3) Hela cells were co transfected with pCXN 2 core, pMEKK△(plasmid that expressed non active MEKK), and pNF κB for 36 hours. Fluorescein enzyme specific activities were assayed to determine if HCV core protein activating NF κB mediated STPs by MEKK.Results 1) the HCV core protein activation of NF κB mediated STPs was inhibited by aspirin. 2) The IκBα dosage in the lysate of Cos 7 cells transfection with pCXN 2 core was less than that of the pCXN 2. 3) the HCV core protein activation of NF κB mediated STPs were not blocked by MEKK△.Conclusion 1) Aspirin inhibits IKK phosphorylation of IκBα. The IκBα degradation is inhibited and activation of NF κB is blocked. The acting site is probably at IKK or some upstream point. 2) In cells that expressed HCV core protein, IκBα degradation increases and leads to activation of NF κB. 3) The acting site of HCV core protein is at the downstream of MEKK point. 4) It is a possible STPs of HCV core protein that HCV core protein acts directly on the process of IKK→IKK phosphorylation of IκBα→IκBα degradation→NF κB escaped from inhibition and transferred from the cytoplasm into the nucleus, where it binds with DNA and initiates its action.

关 键 词：丙型肝炎病毒核心蛋白 核转录因子ΚB κB抑制因子 信号传导途径 

分 类 号：R373[医药卫生—病原生物学]