REGULATION OF CD14 GENE EXPRESSION IN VIVO

描述：（改编自申请人摘要） 负责LPS识别的细胞表面分子，信号 转导和细胞活化对于理解 败血性休克的病理生理学CD 14是一种细胞表面 脂多糖（LPS）结合糖蛋白参与LPS介导的 信号转导;它也作为可溶性血浆蛋白存在， 增强细胞对LPS的反应。这个问题的主要假设是 建议是，CD 14是至关重要的决定， 骨髓和非骨髓的反应（例如，上皮）细胞对LPS的反应。 具体目标是识别组织、细胞和信号 其在体内调节CD 14表达和抗原加工。小鼠将 用LPS或其他激动剂处理，以及它们对CD 14 mRNA的作用， 评价蛋白质产量。为了测量CD 14 mRNA表达， 定量逆转录聚合酶链反应（RT-PCR） 将开发检测方法;这些方法将通过现场 杂交以鉴定责任细胞特异性表型。一 ELISA、SDS PAGE、蛋白质印迹、免疫化学和 将开发Triton X 114相分离技术进行分析 膜结合和可溶性CD 14蛋白在血浆和组织中的表达。在 单独的研究，TNF α、IL 1和IL 6的中和，以及 涉及LPS低反应小鼠品系C3 H/HeJ的实验将 以确定这些介质在细胞特异性 LPS诱导CD 14。SDS PAGE和相分离研究将 表征细胞中膜结合与可溶性CD 14的比例， 各种条件下的组织。这些生物合成的分析 和加工的CD 14应该阐明血浆CD 14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.