Role of C/EBPepsilon in myeloid differentiation

成熟的中性粒细胞通过一系列的承诺步骤从造血干细胞分化而来。这个 次级颗粒蛋白(SGP)、乳铁蛋白(LF)、转钴胺I(TCI)、中性粒细胞 胶原酶(NC)和中性粒细胞明胶酶(NG)标志着对终末期中性粒细胞的承诺 差异化。C/EBPepsilon(C/EBPE)在Sgp基因协同上调中起重要作用。 C/EBPE基因突变导致中性粒细胞成熟过程中的形态和功能缺陷 伴随着从早幼粒细胞到粒细胞的排泄转变。中性粒细胞已经形成双叶 核异常，呼吸爆发活动异常，趋化性和杀菌活性受损。他们缺乏 特定颗粒，不表达编码第二级和第三级颗粒内容蛋白的mRNAs。 这些小鼠在感染后3-5个月内死亡，或死于“骨髓增殖”并发症。表型和 C/EBPE-/-小鼠的功能缺陷与次级颗粒患者非常相似 缺乏症。我们从C/EBPE-/-小鼠的骨髓中制备了两种不同的细胞系 相应的野生型窝产仔，模仿基因敲除小鼠的形态和功能缺陷。 利用这些细胞系和原代骨髓细胞，我们建议进一步表征转录 中性粒细胞终末分化的调控及C/EBPE在中性粒细胞中的作用 成熟程序。我们的具体目标是：1)完成新生成细胞的表征 并将它们建立为C/EBPE-/-Phonotye的忠实模型：2)以确定下游目标 通过对细胞系和原代C/EBPE/和-/-骨髓进行微阵列分析，获得C/EBPE/和-/-骨髓； 3)通过逆转录病毒转导来挽救C/EBPepsilon/-表型，并确定候选基因 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。经验证的重要靶点将被转导入C/EBPE-/-骨髓前体细胞和 移植回C/EBPE-/-小鼠体内以评估表型逆转。

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.