REGULATION OF CD14 GENE EXPRESSION IN VIVO

描述：(改编自申请人的摘要)身份证明 负责识别和传递信号的细胞表面分子 转导和细胞激活对于理解 感染性休克的病理生理学。CD14是一种细胞表面 内毒素结合糖蛋白参与内毒素的介导 信号转导；它也以一种可溶性血浆蛋白的形式存在 增强细胞对内毒素的反应。这一点的主要假设是 建议是CD14在决定 髓系和非髓系(如上皮)细胞对内毒素的反应。 具体的目标是识别组织、细胞和信号 在体内调节CD14的表达和抗原的处理。老鼠会 内毒素或其他激动剂治疗及其对CD14基因表达的影响 对蛋白质产量进行评估。为了测量CD14 mRNA的表达， 定量逆转录聚合酶链式反应 将开发化验方法；这些将由现场补充。 杂交以确定负责细胞的特定表型。一个 ELISA、SDS PAGE、Western印迹、免疫化学和 将开发用于分析的Triton X 114相分离技术 血浆和组织中膜结合和可溶性CD14蛋白的表达。在……里面 单独研究，中和TNFa，IL-1和IL-6，以及 涉及内毒素低反应小鼠品系C3H/HeJ的实验将 以确定这些介体在细胞特异性中的作用 脂多糖对CD14的诱导作用。十二烷基硫酸钠-PAGE和相分离研究将 膜结合的CD14与可溶性CD14的比例 各种条件下的组织。这些对生物合成的分析 CD14的检测有助于阐明血浆CD14的来源。

DESCRIPTION: (Adapted from the applicant's abstract) Identification of cell surface molecules responsible for LPS recognition, signal transduction, and cellular activation is important to understanding the pathophysiology of septic shock. CD14 is a cell surface lipopolysaccharide (LPS) binding glycoprotein involved in LPS mediated signal transduction; it also exists as a soluble plasma protein which potentiates cellular responses to LPS. The primary hypothesis of this proposal is that CD14 is of central importance in determining the responses of myeloid and nonmyeloid (e.g., epithelial) cells to LPS. The specific objective is to identify the tissues, cells, and signals that regulate CD14 expression and antigen processing in vivo. Mice will be treated with LPS or other agonists and their effect on CD14 mRNA and protein production evaluated. To measure CD14 mRNA expression, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays will be developed; these will be complemented by in situ hybridization to identify responsible cell specific phenotypes. A combination of ELISA, SDS PAGE, Western blot, immunochemistry, and Triton X 114 phase separation techniques will be developed for analysis of membrane bound and soluble CD14 protein in plasma and tissues. In separate studies, neutralization of TNFa, IL 1, and IL 6, and experiments involving the LPS hyporesponsive mouse strain C3H/HeJ will be performed to determine the role of these mediators in cell specific induction of CD14 by LPS. SDS PAGE and phase separation studies will characterize the proportion of membrane bound versus soluble CD14 in tissues under various conditions. These analyses of the biosynthesis and processing of CD14 should elucidate the origin of plasma CD14.