CHARACTERIZATION OF MURINE ACIDIC FGF GENE

这项医师科学家奖助金申请的目标是 候选人(1)获得深入的基础科学学习经验 强调一门新学科(分子生物学)，同时(2)指导 在赞助商的直接监督下进行的研究 在这方面的经验。随后，应聘者将申请 这些研究工具使他目前在第二阶段下的研究兴趣 与咨询一起继续监督他的赞助人 委员会审议阶段。 第一阶段的项目将专注于表征小鼠的酸性 成纤维细胞生长因子基因。具体目标是：(1)确定 小鼠成纤维细胞生长因子的基因结构。研究计划包括隔离和 通过对小鼠基因组DNA克隆测序来鉴定aFGF. 转录起始点(S)的鉴定将由 引物延伸和核糖核酸酶保护试验。潜力分析 幼年和老年小鼠基因表达的差异将通过 Northern印迹和免疫印迹法检测年轻细胞和老年细胞中aFGF的产生 杂交。编码基因调控机制的确定 A成纤维细胞生长因子，将涉及通过CAT实验鉴定功能启动子区域， 缺失作图和定点突变识别关键基因 启动子/增强子区域的核苷酸。描述小鼠的特征 为了更好地研究生物学问题，基因是必要的。 与炎症和肿瘤发生有关的；以及[2]启动对 小鼠系统中的定点基因打靶。在第二阶段，研究 这种生长因子和其他生长因子将被应用于小鼠的 自身免疫性疾病，MRL LPR/LPR小鼠。我们假设在狼疮患者中 细胞因子(HBGFs、IL-1和PDGF)和/或 巨噬细胞对细胞因子的反应性导致异常 免疫功能。进一步建议，通过系统地 分析巨噬细胞产生这些细胞因子，我们希望能更好地 了解它们在自身免疫表达中可能起到的作用。vbl.使用 狼疮的小鼠模型，这些研究试图筛选产生 自身免疫小鼠和正常小鼠巨噬细胞中aFGF、bFGF、IL-1和PDGF的表达； 对所提出的感兴趣的细胞因子进行生化表征；评估 目的蛋白(S)的生物学特性，即； 有丝分裂活性、趋化活性；检查培养效果 在这些细胞因子对质膜重组的作用下，前列腺素和 白三烯的产生；并研究其调节机制 控制这些因素的生产。研究这些因素将 让我们更好地了解它们在促进炎症方面的作用 在这种情况下。

The objectives of this Physician Scientist Grant Application are for the candidate to (1) obtain an in depth basic science learning experience emphasizing a new discipline (Molecular Biology) while (2) conducting research under the direct supervision of a sponsor with extensive experience in this discipline. Subsequently, the candidate will apply these research tools to his current research interest in Phase II under the continuing supervision of his sponsor in conjunction with the advisory committee. The Phase I project will focus on characterization of murine acidic fibroblast growth factor gene. The specific aims are: (1) to determine the gene structure of mouse aFGF. The research plan involves isolation and characterization through sequencing of mouse genomic DNA clones for aFGF. Identification of the transcription initiation site(s) will be done by primer extension and ribonuclease protection assay. Analysis of potential differences in gene expression in young and old mice will proceed by examining aFGF production in young and old cells by Northern blotting and hybridization. Determination of regulatory mechanisms of the gene encoding aFGF, will involve identifying the functional promoter region by CAT assay, deletional mapping and site directed mutagenesis to identify critical nucleotides in the promoter/enhancer region. Characterization of the mouse gene is necessary in order to: [1] better study biologic questions pertaining to inflammation and tumorigenesis; and [2] initiate studies of site-directed gene targeting in mouse systems. During phase II, studies of this and other growth factors will be applied to a murine model of autoimmune disease, the MRL lpr/lpr mouse. We hypothesize that in lupus a disturbance in the production of cytokines (HBGFs), IL-1, and PDGF) and/or in the responsiveness of macrophages to cytokines results in anomalous immunological function. It is suggested further that by systematically analyzing macrophages for production of these cytokines, we hope to better understand the role they may have in the expression of autoimmunity. Using murine models of lupus, these studies seek to screen for the production of aFGF, bFGF, IL-1 and PDGF in macrophages from autoimmune and normal mice; to biochemically characterize the proposed cytokines in interest; to assess the biological characteristics of the protein(s) of interest i.e.; mitogenic activity, chemotactic activity; to examine the effects of culture with these cytokines on plasma membrane reorganization, prostanoid and leukotriene production; and to examine the regulatory mechanisms controlling the production of these factors. Examining these factors will allow us to better understand the role they have in promoting inflammation in this setting.