DISSECTING THE HEMATOPOIETIC GROWTH FACTOR NETWORK

造血调控涉及到一个复杂的相互作用网络 基因表达层次结构中的细胞和细胞因子 复杂的自分泌和旁分泌放大机制。每个人 细胞因子表现出多种生物活性，其中一些是 与其他细胞因子有协同作用。在一个这样的系统中 复杂性，一种抑制基因表达的实验技术 特定的基因将极大地促进对 功能、联系和因果关系。为此，我们已经证明了 与GM-CSF或G-CSF mRNA互补的反义寡核苷酸 抑制IL-1诱导的相应生长因子的表达 内皮细胞或成纤维细胞以序列特定的方式。在这 提议，我们将审查效率的决定因素 反义寡核苷酸对基因表达的抑制作用 调节这种抑制的细胞和分子机制。这 这种方法在两个实验环境中特别有用：(1) 长期骨髓培养(在这种情况下很难交付 干扰旁分泌机制的抗体)和(2)对 IL-1信号转导机制(完全是细胞内的 且不受抗体操纵)。因此，我们将 应用反义寡核苷酸直接分析GM-2的作用 脑脊液、粒细胞集落刺激因子、白介素6和干细胞因子(KIT配体)在启动中的作用 在长期的骨髓培养中维持造血。我们 将阐明c-fos、c-jun和c-raf-1在IL-1诱导中的作用 利用反义寡核苷酸表达造血生长因子 来特别抑制它们的表达。

Hematopoietic regulation involves an elaborate network of interacting cells and cytokines in which hierarchies of gene expression are complicated by autocrine and paracrine amplification mechanisms. Each cytokine exhibits a variety of biological activities, some of which are synergistic with those of other cytokines. In a system of such complexity, an experimental technique which inhibits the expression of specific genes would greatly facilitate the analysis of questions of function, linkage, and causality. To this end, we have demonstrated that antisense oligodeoxynucleotides complementary to GM-CSF or G-CSF MRNA inhibit expression of the corresponding growth factor in IL-1-induced endothelial cells or fibroblasts in a sequence-specific fashion. In this proposal, we will examine the determinants of the efficiency of inhibition of gene expression by antisense oligonucleotides and the cellular and molecular mechanisms which mediate this inhibition. This approach would be particularly useful in two experimental settings: (1) long-term bone marrow culture (in which it is difficult to deliver antibodies that interrupt juxtacrine mechanisms) and (2) studies on mechanisms of IL-1 signal transduction (these are entirely intracellular and not amenable to manipulation with antibodies). Accordingly, we will employ antisense oligonucleotides to analyze directly the role of GM- CSF, G-CSF, IL-6, and stem cell factor (kit ligand) in the initiation and maintenance of hematopoiesis in long-term bone marrow culture. We will clarify the role of c-fos, c-jun, and c-raf-1 in IL-1-induced hematopoietic growth factor expression using antisense oligonucleotides to specifically inhibit their expression.