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Mechanisms of Bacterial Endotoxin-induced Cell Activation by a Serum-independent Pathway.

细菌内毒素通过血清非依赖性途径诱导细胞激活的机制。

基本信息

批准号：
08670315
负责人：
KIRIKAE Teruo
金额：
$ 1.47万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670315/
关键词：
ENDOTOXIN LPS CD14 ST2 CELLS IL-6 モノクロナール抗体 ST2細胞 IL-6

项目摘要

The present study was done to elucidate the biological properties of LPS receptor (s) and its signal transduction system, and to identify some molecules of them. A cell line, ST2, derived from murine bone marrow stroma, was mainly used in the present study. We examined the following points ; a) responsiveness to LPS antagonists and LPS agonists, b) detection of LPS-binding proteins on the ST2 cells and production of polyclonal antisera against these proteins, c) detection of tyrosine-phosphorylated proteins on the ST2 cells. We identified some molecules which is related to LPS-induced activation of ST2 cells.1) Adaptation of CD14-negative marrow stromal ST2 cells in serum-free medium and LPS-responsiveness of ST2 cellsTo exclude completely the effects of CD14 and serum components on LPS responsiveness, we established a subclone of CD14-negative marrow stromal ST2 cells growing under serum-free conditions.2) Detection of LPS-binding proteins on ST2 sells growing under serum-free conditions.A ligand-botting method and a cross-linker method using radiolabelled LPS demonstrated a number of LPS-binding proteins on ST2 cells.3) Production of polyclonal antisera against membrane fraction containing LPS-binding proteins on ST2 cells.Rabbits were immunized with partial purified LPS-binding proteins on ST2 cells and polyclonal antisera were collected. The biological immunological properties were determined.4) Detection of tyrosine-phosphorylated proteins on ST2 sells growing under serum-free conditions.A immunoprecipitation and western blotting methods revealed tyrosine-phosphorylated proteins on ST2 sells stimulated with LPS.5) Production of monoclonal antibodies against LPS-binding proteins and their associated proteins on ST2 cells.On based our previous finding, we produced monoclonal antibodies against LPS-binding proteins and their associated proteins on ST2 cells.

本研究旨在阐明LPS受体及其信号转导系统的生物学特性，并对其中部分分子进行鉴定。本研究主要采用小鼠骨髓基质细胞株ST 2。我们检查了以下几点：a）对LPS拮抗剂和LPS激动剂的反应性，B）检测ST 2细胞上的LPS结合蛋白并产生针对这些蛋白的多克隆抗血清，c）检测ST 2细胞上的酪氨酸磷酸化蛋白。本研究鉴定了与LPS诱导的ST 2细胞活化有关的分子：1）CD 14阴性的骨髓基质ST 2细胞在无血清培养基中的适应性和ST 2细胞对LPS的反应性为了完全排除CD 14和血清成分对LPS反应性的影响，我们建立了在无血清条件下生长的CD 14阴性骨髓基质ST 2细胞的亚克隆。用放射性标记的脂多糖（LPS）标记的ST 2细胞膜上的LPS结合蛋白，通过配体印迹法和交联剂法证实了ST 2细胞膜上存在大量的LPS结合蛋白; 3）制备抗ST 2细胞膜上LPS结合蛋白的多克隆抗血清。收集ST 2细胞上的结合蛋白和多克隆抗血清。4）检测无血清培养条件下培养的ST 2细胞表面酪氨酸磷酸化蛋白的表达，免疫沉淀法和Western blotting法检测到ST 2细胞表面酪氨酸磷酸化蛋白的表达; 5）制备抗ST 2细胞表面LPS结合蛋白及其相关蛋白的单克隆抗体。我们在ST 2细胞上制备了抗LPS结合蛋白及其相关蛋白的单克隆抗体。