HEMATOPOIETIC STEM CELLS--MECHANISMS OF SELF REPLICATION

这个项目的总体目标是识别细胞外信号和 参与造血干细胞维持的特异性基因 (HSC)。这些研究的一个重要实践目标是建立 在体外产生HSC的条件。取得进展的主要障碍 这一区域一直无法净化生物定义的 HSC的亚群，再加上识别 HSC自我复制相关基因的表达(或抑制)， 特别是考虑到可供研究的HSC数量非常少 (通常是每个定义的HSC亚群的几千个细胞)。 然而，我们的团队最近开发了一种流式细胞仪方法， 使用顺序Hoechst 33342/罗丹明123方法进行纯化 适合于细胞研究的HSC不同亚群 和分子水平。具体地说，我们计划：a)界定具体的 生长条件[例如，生长因子、生长抑制剂、粘附力 分子/配体(“粘附素”)]，控制维持 长期重新填充Hoechst 33342/罗丹明123的“严酷”-选择 肝星状细胞体外单细胞培养及增殖力分析 体外重克隆或体内检测HSC子代细胞的潜能 使用适当的长期或短期人口回流分析；b)确定 新分离的Hoechst表达(或抑制)的独特基因 33342/罗丹明123-选择的造血干细胞亚群可以 分离的并且在生物学上是不同的(基于短期与 两个亚群在体内的长期再繁殖能力) 新的基于聚合酶链式反应的消减克隆方法； 定义后代在体外表达(或抑制)的独特基因 长期重新繁殖Hoechst产生的子代细胞 33342/罗丹明123--暴露于特定生长因子的精选肝星状细胞，+/- 增殖抑制药和/或粘附素。

The overall aim of this project is to identify extracellular signals and specific genes involved in the maintenance of hematopoietic stem cells (HSC). An important practical goal of these studies is the establishment of conditions that generate HSC in vitro. Major obstacles to progress in this area have been the inability to purify biologically defined subpopulations of HSC, coupled with the difficulties of identifying the expression (or suppression) of genes involved in HSC self-replication, especially given the very small numbers of HSC available for study (typically a few thousand cells of each defined HSC subpopulation). However, our group has recently developed a flow cytometric method that utilizes a sequential Hoechst 33342/Rhodamine 123 approach to purify distinct subpopulations of HSC suitable for investigations at the cellular and molecular levels. Specifically, we plan: a) to define the specific growth conditions [e.g., growth factors, growth inhibitors, adhesion molecules/ligands ("adhesins")] that control the maintenance of "sternness" of long-term repopulating Hoechst 33342/Rhodamine 123-selected HSC in vitro using single cell cultures and analysis of the proliferative potential of HSC daughter cells by recloning in vitro or assaying in vivo using suitable long-term or short-term repopulation assays; b) to define unique genes expressed (or suppressed) by freshly isolated Hoechst 33342/Rhodamine 123-selected HSC subpopulations that can be physically separated and are biologically distinct (based on the short-term versus long-term repopulating ability in vivo of two subpopulations) using a novel polymerase chain reaction-based subtractive cloning method; c) to define unique genes expressed (or suppressed) in vitro by descendant daughter cells generated from long-term repopulating Hoechst 33342/Rhodamine 123-selected HSC exposed to specific growth factors, + /- proliferation inhibitors and/or adhesins.