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.

本研究的目的是阐明LPS受体及其信号转导系统的生物学特性，并鉴定其某些分子。本研究主要使用源自鼠骨髓基质的细胞系ST2。我们检查了以下几点； a）对LPS拮抗剂和LPS激动剂的反应，b）在ST2细胞上检测LPS结合蛋白，并对这些蛋白质的多克隆抗血清产生，c）检测ST2细胞上酪氨酸磷酸化蛋白的检测。 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 2））在ST2销售中检测LPS结合蛋白在无血清条件下生长的销售。一种使用放射性标记LPS的配体刺激方法和一种交联方法，证明了许多LPS结合蛋白在ST2细胞上的LPS结合蛋白。收集了ST2细胞和多克隆抗血清上的LPS结合蛋白。确定了生物免疫学特性。4）在无血清条件下销售酪氨酸磷酸化蛋白的检测。一种免疫沉淀和蛋白质印迹方法揭示了酪氨酸磷酸化的蛋白质在ST2上的酪氨酸磷酸化蛋白在ST2上的销售刺激了LPS。发现，我们在ST2细胞上产生了针对LPS结合蛋白及其相关蛋白的单克隆抗体。