ROLE OF HEMATOPOIETIC GROWTH FACTORS AND THEIR RECEPTORS DURING HEMATOPOIESIS

造血生长因子及其受体在造血过程中的作用

• 批准号: 6236732
• 负责人: DAVID G. NATHAN
• 金额: $ 14.35万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-10 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6236732
• 关键词: animal tissue; blood cell count; bone marrow transplantation; cell differentiation; cell growth regulation; cell population study; colony stimulating factor; embryonic stem cell; flow cytometry; gene mutation; gene therapy; genetic manipulation; genetic transduction; genetically modified animals; growth factor receptors; hematopoiesis; hematopoietic growth factor; hematopoietic stem cells; human tissue; interleukin 3; interleukin 6; laboratory mouse; tissue /cell culture

The long-term objectives of this proposal are to develop the means whereby human hematopoietic stem cells (HSC) can be manipulated in culture to provide a cellular source for bone marrow transplantation and target cells for gene transfer. The working hypothesis is that specific hematopoietic growth factors (HGFs) can be identified that are crucial to the regulation of stem cell self-renewal and differentiation. This hypothesis will be tested in two ways: first, (Aim 1) murine, simian, and human stem and progenitor cells will be assessed for their self- renewal and differentiation capacity after incubation in selected HGF combinations; and second (Aim 2), the genes for candidate HGF receptors will be disrupted in embryo-derived stem cells (ES) to provide a critical measure of their importance in vivo. In Aim 1, species-appropriate methods will be used to purify or enrich for hematopoietic stem cells and separate them from committed progenitors. These cells will be incubated in a combination of HGFs for 6-7 days and then evaluated for their stem and progenitor cell and T lymphocyte content, both in vitro and in vivo in murine and simian species, and in vitro in the human studies. In addition, the effect of combined HGFs on the efficiency of gene transfer into stem and progenitor cells and the number of clones that contribute to hematopoiesis will be determined. In Aim 2, genes for the interleukin-3 receptor will be disrupted in ES cells by homologous recombination and evaluated for function in vitro and in vivo. Both the alpha and beta subunits of the IL-3 R will be disrupted using a vector that allows positive-negative selection. ES cells with heterozygous mutations of IL-3 Ralpha or beta will be injected into blastocysts and transferred to foster mothers. Mice with germ line integration will be bred to homozygosity and interbred to yield IL-3 R alpha, beta, and alphabeta "null" animals. In addition, the heterozygous mutant ES cells will be "homozygosed" in vitro and then evaluated directly in chimeric animals by glucose phosphate isomerase analysis. Last, we plan to use IL-3 R "null" murine ES cells for structure function analysis of the human IL-3 R/GM-CSF R components. In these ways, we hope to identify methods to expand the population of pluripotent HSC for eventual clinical use in bone marrow transplantation, either unmodified or, ultimately, modified by gene insertion. In addition, we hope to establish gene mutation methods that would provide a powerful means to investigate the physiological importance of IL-3 and other HGFs in hematopoiesis.

这项建议的长期目标是发展手段， 由此可以在体外操作人造血干细胞（HSC）， 培养以提供用于骨髓移植的细胞来源， 用于基因转移的靶细胞。 我们的假设是 造血生长因子（HGFs）可以被鉴定为至关重要的 干细胞自我更新和分化的调节。 这 假设将以两种方式进行检验：第一，（目标1）鼠，猿， 人类干细胞和祖细胞将被评估其自身， 在选择的HGF中孵育后的更新和分化能力 组合;第二（目标2），候选HGF受体的基因 将在胚胎来源的干细胞（ES）中被破坏，以提供关键的 在体内的重要性。 在目标1中，适合物种 方法将用于纯化或富集造血干细胞， 将它们与承诺的祖先分开。 这些细胞将被培养 在HGF的组合中培养6-7天， 以及祖细胞和T淋巴细胞含量，包括体外和体内 在鼠和猿物种中，以及在人体研究中的体外。 在 此外，还研究了组合的HGF对基因转移效率的影响。 分化成干细胞和祖细胞， 造血功能将被确定。 在目标2中， 白细胞介素-3受体将在ES细胞中被同源 重组并在体外和体内评价功能。 两者 IL-3 R的α和β亚基将使用载体 允许正负选择。 ES细胞杂合子 将IL-3R α或β的突变注射到胚泡中， 转移到养母身上 具有生殖系整合的小鼠将 繁殖至纯合性并杂交以产生IL-3 R α、β和 alphabeta“null”动物。 此外，杂合突变ES细胞 将在体外“纯合”，然后在嵌合体中直接评估。 动物通过葡萄糖磷酸异构酶分析。 最后，我们计划使用 IL-3 R“null”小鼠ES细胞用于IL-3受体的结构功能分析 人IL-3 R/GM-CSF R成分。 我们希望通过这些方式， 扩增多能HSC群体用于最终临床应用的方法 在骨髓移植中的应用，无论是未经修饰的，还是最终， 通过基因插入进行修饰。 此外，我们希望建立基因 突变方法，这将提供一个强大的手段来调查 IL-3和其他HGF在造血中的生理重要性。