Role of C/EBPepsilon in myeloid differentiation

C/EBPepsilon 在骨髓分化中的作用

• 批准号: 7574465
• 负责人: Stephanie Halene
• 金额: $ 13万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7574465
• 关键词: Affect; Appearance; Back; Biological Assay; Bone Marrow; Bone Marrow Transplantation; Candidate Disease Gene; Cell Line; Cell Nucleus; Cells; Characteristics; Chemotaxis; Cytoplasmic Granules; Defect; Estrogen Receptors; Event; Failure; Gelatinase A; Gene Expression; Gene Proteins; Genes; Hematopoietic stem cells; Infection; Injection of therapeutic agent; Knockout Mice; Lactoferrin; Marrow; Microarray Analysis; Modeling; Mus; Myelocyte; Myelodysplastic/Myeloproliferative Disease; Myelogenous; Myeloproliferation; NOD/SCID mouse; Neutrophil Collagenase; Patients; Phagocytosis; Phenotype; Play; Process; Progranulocytes; Proteins; Reporting; Respiratory Burst; Retroviral Vector; Role; Series; Site; Staging; Transcobalamin (I); Transcriptional Regulation; Transducers; Transplantation; Up-Regulation; bactericide; established cell line; granulocyte; in vivo; insight; leukemia; neutrophil; progenitor; programs; protein expression; retroviral transduction; transcription factor

Mature neutrophils arise from the hematopoietic stem cell via a series of commitment steps. The appearance of the secondary granule proteins (SGP) lactoferrin (LF), transcobalamin I (TCI), neutrophil collagenase (NC) and neutrophil gelatinase (NG) marks the commitment to terminal neutrophil differentiation. C/EBPepsilon (C/EBPe) plays a critical role in the coordinate upregulation of SGP genes. Disruption of the C/EBPe gene in mice leads to morphologic and functional defects in neutrophil maturation with a defectivetransition from the promyelocyte to the myelocyte stage. The neutrophils have bilobed nuclei, abnormal respiratory burst activity, and impaired chemotaxis and bactericidal activity. They lack specific granules and fail to express mRNAs encoding for secondary and tertiary granule content proteins. The mice die within 3-5 months of infection or from complications of "myeloproliferation". Phenotypic and functional defectsof the C/EBPe -/- mice closely parallel those in patients with secondary granule deficiency. We have made 2 different cell lines from the bone marrow of the C/EBPe -/- mice and corresponding wildtype littermates that mimic the morphologic and functional defects in the knockout mice. Using these cell lines and primary marrow cells, we propose to further characterize the transcriptional regulation of terminal neutrophil differentiation and specifically the role of C/EBPe in the neutrophil maturation program. Our specific aims are:1) To complete the characterization of the newly generated cell lines and establish them as a faithful model of the C/EBPE -/- phenotye: 2) To identify downstream targets of C/EBPepsilonthrough microarray analysis of the cell lines and primary C/EBPe +/+ and -/- bonemarrow; and 3) To rescue the C/EBPepsilon -/- phenotype by retroviral transduction with candidate genes identified in specif aim 2 . Genes will be transferred first into the C/EBPe-/- cell line to determine reversalof phenotype. Verified important targets will be transduced into C/EBPe-/- marrow progenitors and transplanted back into C/EBPe-/- mice to assess reversal of phenotype.

成熟的中性粒细胞通过一系列的定型步骤由造血干细胞产生。这 次级颗粒蛋白 (SGP)、乳铁蛋白 (LF)、转钴胺素 I (TCI)、中性粒细胞的出现 胶原酶（NC）和中性粒细胞明胶酶（NG）标志着对终末中性粒细胞的承诺 差异化。 C/EBPepsilon (C/EBPe) 在 SGP 基因的协调上调中发挥着关键作用。 小鼠 C/EBPe 基因的破坏导致中性粒细胞成熟的形态和功能缺陷 从早幼粒细胞到粒细胞阶段的转变有缺陷。中性粒细胞有双叶 细胞核、呼吸爆发活动异常以及趋化性和杀菌活性受损。他们缺乏 特定颗粒，并且无法表达编码二级和三级颗粒内容蛋白的 mRNA。 小鼠在感染后 3-5 个月内死亡或死于“骨髓增殖”并发症。表型和 C/EBPe -/- 小鼠的功能缺陷与二次颗粒患者的功能缺陷非常相似 不足。我们从 C/EBPe -/- 小鼠的骨髓中制备了 2 种不同的细胞系， 相应的野生型同窝小鼠模仿了基因敲除小鼠的形态和功能缺陷。 使用这些细胞系和原代骨髓细胞，我们建议进一步表征转录 终末中性粒细胞分化的调节，特别是 C/EBPe 在中性粒细胞中的作用 成熟计划。我们的具体目标是：1）完成新生成细胞的表征 线并将其建立为 C/EBPE -/- 表型的忠实模型：2) 确定下游目标 通过细胞系和原代 C/EBPe +/+ 和 -/- 骨髓的微阵列分析来分析 C/EBPepsilon； 3) 通过逆转录病毒转导并鉴定出候选基因来拯救 C/EBPepsilon -/- 表型 在具体目标2中。基因将首先转移到 C/EBPe-/- 细胞系中以确定逆转 表型。经过验证的重要靶点将被转入 C/EBPe-/- 骨髓祖细胞中 移植回 C/EBPe-/- 小鼠以评估表型的逆转。