HEMATOPOIETIC STEM CELLS--MECHANISMS OF SELF REPLICATION

该项目的总体目标是识别细胞外信号， 维持造血干细胞的特定基因 （HSC）。这些研究的一个重要的实际目标是建立 在体外产生HSC的条件。取得进展的主要障碍 这一地区一直无法净化生物定义 HSC的亚群，再加上识别 参与HSC自我复制的基因的表达（或抑制）， 特别是考虑到可供研究的HSC数量非常少， （通常每个限定的HSC亚群的几千个细胞）。 然而，我们的小组最近开发了一种流式细胞术方法， 利用顺序Hoechst 33342/罗丹明123方法纯化 适合细胞研究的不同HSC亚群 和分子水平。具体而言，我们计划：a）确定具体的 生长条件[例如，生长因子，生长抑制剂，粘附 分子/配体（“粘附素”）]，其控制 Hoechst 33342/罗丹明123-选择的长期再填充的“干性” HSC体外单细胞培养及增殖活性分析 通过体外再克隆或体内测定HSC子细胞的潜力 使用合适的长期或短期再增殖测定; B）确定 新鲜分离的Hoechst表达（或抑制）的独特基因 33342/罗丹明123-选择的HSC亚群，可以物理地 分离，生物学上是不同的（基于短期与 两个亚群的体内长期再增殖能力）， 新的基于聚合酶链反应的消减克隆方法; c） 定义后代在体外表达（或抑制）的独特基因 从Hoechst长期繁殖中产生的子细胞 33342/罗丹明123-选择性HSC暴露于特异性生长因子，+ /- 增殖抑制剂和/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.