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

本研究旨在阐明内毒素受体(S)及其信号转导系统的生物学特性，并对其部分分子进行鉴定。本研究以小鼠骨髓基质来源的细胞株ST2为研究对象。我们检测了以下几点：a)对内毒素拮抗剂和内毒素激动剂的反应性，b)在ST2细胞上检测到内毒素结合蛋白并产生针对这些蛋白的多克隆抗血清，c)在ST2细胞上检测到酪氨酸磷酸化蛋白。1)CD14阴性的骨髓基质ST2细胞在无血清培养液中的适应和ST2细胞的内毒素应答完全排除CD14和血清成分对内毒素应答的影响。我们建立了无血清条件下生长的CD14阴性骨髓基质ST2细胞的亚克隆。2)检测无血清条件下生长的ST2细胞上的内毒素结合蛋白。配基引导法和交联法显示ST2细胞上有大量的内毒素结合蛋白。3)制备针对ST2细胞上含有内毒素结合蛋白的膜部分的多克隆抗血清。4)检测在无血清条件下生长的ST2细胞的酪氨酸磷酸化蛋白。免疫沉淀和免疫印迹法检测到在LPS刺激下的ST2细胞上的酪氨酸磷酸化蛋白。5)在ST2细胞上产生抗脂多糖结合蛋白及其相关蛋白的单抗。

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.