GENES CONTROLLING NEURTROPHIL DEVELOPMENT FROM STEM CELL

在这项建议中，一种新的小鼠淋巴造血干细胞系MER 它的衍生品将用于分离控制中性粒细胞的基因 承诺和差异化。MERE细胞系依赖于干细胞 细胞因子(SCF；c-kit配体)和HAS-淋系和造血系 (“髓系”)电位。从这个干细胞系我们建立了一种 中性粒细胞衍生物(Mer-Pro)阻断在早幼粒细胞阶段。这 用高浓度的维甲酸可以克服阻滞。这 互补的一对细胞系代表“冷冻”的造血细胞 可逆地处于两个发展阶段，特别适合作为 干细胞承诺和谱系受限研究的模型系统 差异化。这项建议的具体目的是：(一)隔离和 中性粒细胞祖细胞特异性基因的特性，包括 参与了血统承诺。差分技术 用杂交法筛选mer-Pro的cdna文库 (中性早幼粒细胞)使用从 Mer-Pro和Mer细胞系；(Ii)分离激活或 维甲酸在细胞终末分化过程中的失活 嗜中性的早幼粒细胞。要么是cDNA文库差异 以聚合酶链式反应为基础的杂交技术 可以使用mRNAs的“差异显示”。这些基因比 在特定目标I的下游，但在后者中很重要 中性粒细胞分化阶段；(Iii)体内特征 淋巴造血干细胞系MER及人淋巴造血干细胞系的建立 相当于MER细胞系。MER细胞系的能力 作为干细胞的功能将在受辐射的同基因小鼠中进行检验。 将尝试从人骨中创建类似的细胞系 骨髓。该细胞系可能是第一个依赖干细胞因子的细胞。 符合淋巴和造血细胞的潜能，应该大大 促进干细胞研究。从这个独特的故事中获得的知识 实验系统可能有助于我们理解 人类淋巴造血干细胞生物学。 这项提议的长期目标是直接识别分子 介导干细胞的自我更新、承诺和分化 以及这些分子是如何被荷尔蒙和 细胞外基质。希望这一新知识将使我们能够 控制干细胞的行为以达到治疗目的，如基因 未来的治疗。

In this proposal, a novel murine lymphohematopoietic stem cell line MER and its derivative will be used to isolate genes controlling neutrophil commitment and differentiation. The MER cell line is dependent on stem cell factor (SCF; c-kit ligand) and has-both lymphoid and hematopoietic ("myeloid") potentials. From this stem cell line we established a neutrophilic derivative (MER-PRO) blocked at the promyelocyte stage. This block can be overcome with high concentrations of retinoic acid. This complementary pair of cell lines represent hematopoietic cells "frozen" reversibly at two developmental stages and are particularly suitable as a model system for studying stem cell commitment and lineage-restricted differentiation. The specific aims of this proposal are: (I) Isolation and characterization of neutrophil progenitor-specific genes, including those involved in lineage commitment. The technique of differential hybridization will be used to screen the cDNA library of MER-PRO (neutrophilic promyelocytes) using subtracted cDNA probes prepared from the MER-PRO and MER cell lines; (II) Isolation of genes activated or inactivated by retinoic acid during the terminal differentiation of neutrophilic promyelocytes. Either the cDNA library differential hybridization technique or the polymerase chain reaction-based "differential display" of mRNAs can be used. These genes are more downstream to those in Specific Aim I but are important in the later stages of neutrophil differentiation; (III) In vivo characterization of the lymphohematopoietic stem cell line MER and establishment of a human equivalent of the MER cell line. The capacity of the MER cell line to function as stem cells will be examined in irradiated syngeneic mice. Attempts will be made to create a similar cell line from human bone marrow. The MER cell line may be the first stem cell factor-dependent cell line with both lymphoid and hematopoietic potentials and should greatly facilitate stem cell research. The knowledge gained from this unique experimental system is likely to contribute to our understanding of the biology of human lymphohematopoietic stem cells. The long-term objective of this proposal is to identify molecules directly mediating the self-renewal, commitment and differentiation of stem cells and how these molecules are in turn modulated by hormones and extracellular matrix. It is hoped that this new knowledge will enable us to control stem cell behavior for therapeutic purposes such as gene therapy in the future.