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Identification of FX-activating molecules of convulsion-inducing lipoprotein purified from egg granulomas in Schistosoma mansoni-infected mice and of its inducing mechanism

曼氏血吸虫感染小鼠卵肉芽肿中纯化的惊厥诱导脂蛋白FX激活分子的鉴定及其诱导机制

基本信息

批准号：
15590374
负责人：
TANABE Masanobu
金额：
$ 1.47万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Biochemical studies has been performed to identify the factor-X(FX)-activating molecules of convulsion-inducing lipoprotein (CILIP) purified from hepatic egg granulomas in Schistosoma mansoni-infected mice, the inducing mechanism of CILIP by macrophages and dendritic cells, and the signal transduction pathway involved in CILIP induction.1.In vitro analysis demonstrated that macrophages and dendritic cells could produce CILIP(≒FX-activating PCA) when stimulated with several ligands for Toll-like receptors such as LPS, LTA, peptidoglycan, poly IC and CpGDNA, with splenic DX5+ NK cells and/or CD4+ T cell sensitized with SWAP antigen, or with cytokines such as IFNγ and GM-CSF.2.The induction of macrophage FX-activating PCA by stimulation with SWAP sensitized lymphocytes required de novo RNA and protein synthesis and activation of PTK and PKC, but not MAPK activation, calmodulin or increase of intracellular Ca++ concentration. Moreover, the induction of macrophage FX-activating PCAwas down-regulated by increment of intracellular cAMP.3.Two kinds of monoclonal antibodies against CILIP were prepared from BALB/c mice and Wister rats. Rat's MoAb appeared to be highly specific and sensitive to native CILIP, and inhibited dose-dependently FX-activating activity of CILIP, but did not react with denatured CILIP treated with detergents. Sandwich ELISA system using two kinds of MoAbs against CILIP was developed and confirmed its usefulness when applied for quantitative determination of CILIP.4.Although various biochemical analysis using MoAb against tissue factor and specific antibodies against various coagulation factors have been conducted to identify the EX-activating molecules of CILIP, conclusive evidence has not been obtained yet.

从曼氏血吸虫感染小鼠肝卵肉芽肿中纯化的惊厥诱导脂蛋白（CILIP）的因子- x （FX）激活分子，巨噬细胞和树突状细胞诱导CILIP的机制，以及诱导CILIP的信号转导途径进行了生化研究。体外分析表明，巨噬细胞和树突状细胞在LPS、LTA、肽聚糖、poly IC和CpGDNA等toll样受体配体的刺激下，在SWAP抗原致敏的脾DX5+ NK细胞和/或CD4+ T细胞，或IFNγ和GM-CSF.2等细胞因子的刺激下，可以产生CILIP（≒FX-activating PCA）。通过SWAP致敏淋巴细胞刺激诱导巨噬细胞fx激活PCA，需要从头合成RNA和蛋白质，激活PTK和PKC，但不需要激活MAPK、钙调素或增加细胞内ca2 ++浓度。此外，通过增加细胞内camp，可下调诱导巨噬细胞激活fx的pca3。从BALB/c小鼠和Wister大鼠制备了两种抗CILIP的单克隆抗体。大鼠的MoAb对天然CILIP具有高度特异性和敏感性，并且抑制了CILIP的剂量依赖性fx激活活性，但与洗涤剂处理的变性CILIP不发生反应。建立了两种MoAbs对CILIP的夹心ELISA系统，并将其应用于CILIP.4的定量测定。虽然已经进行了各种针对组织因子的MoAb生化分析和针对各种凝血因子的特异性抗体来鉴定CILIP的ex激活分子，但尚未获得确凿的证据。