G-CSF RECEPTOR AND PROGENITOR MOBILIZATION

这项研究的长期目标是描述涉及造血祖细胞动员的分子机制。在清髓治疗后，使用HPC重建造血系统已显著改善了多种恶性肿瘤患者的临床预后。最近，动员的外周血HPC被用来代替骨髓来源的HPC，因为它们减少了植入时间，相对容易收集，并可能降低移植物抗宿主病的风险。目前，从骨髓到血液的HPC动员耐受性良好，但并不是普遍有效，而且往往与肿瘤细胞的共动员有关。更好地理解管理HPC动员的机制可能会导致设计新的动员战略来克服这些问题。我们最近发现，在粒细胞集落刺激因子受体(G-CSFR)缺陷小鼠中，对环磷酰胺(CY)或白介素8(IL-8)反应的HPC的动员明显受损，而对Fit-3配体的反应不明显。这些令人惊讶的结果表明，造血细胞或骨髓基质细胞中的G-CSFR信号在HPC迁移中扮演着以前意想不到的重要角色。这项建议旨在描述这些依赖G-CSFR的HPC动员机制。提出了以下具体目标。1.我们将详细研究G-CSFR缺陷小鼠对CY、白介素12(IL-12)或干细胞因子(SCF)的动员反应。将分析G-CSFR缺陷小鼠对SCF或IL-12的反应中的HPC动员。为了探讨G-CSFR缺陷小鼠动员缺陷的机制，我们将比较CY治疗后野生型和G-CSFR缺陷小鼠骨髓中造血细胞的表型，特别是造血细胞的表型。2.我们将确定负责G-CSFR依赖动员的细胞类型。对G-CSFR缺乏辐射嵌合体的HPC动员的初步研究表明，CY诱导的动员需要成熟造血细胞上的G-CSFR，而不是HPC或基质细胞上的G-CSFR。这一具体目标的第一个目标是确认这些令人惊讶的结果，并确定原始高性能混凝土是否以类似的方式动员起来。这一特定目的的第二个目标是表征G-CSF在这些辐射嵌合体中诱导的动员。3.我们将明确中性粒细胞在G-CSFR依赖动员中的作用。通过使用小鼠组织蛋白酶G调控序列在髓系细胞中驱动减毒白喉毒素A亚单位的表达，将产生一个中性粒细胞减少的小鼠系。CY-和G-CSF诱导的HPC动员将是这些小鼠的特征。4.我们将确定动员HPC所需的G-CSFR区域。CY和G-CSF诱导的动员的特征是最近产生的G-CSFR的两个靶向“敲入”突变。将研究STAT-3在G-CSFR产生HPC动员信号中的作用。

The long range objective of this research is to characterize the molecular mechanisms involved in hematopoietic progenitor cell (HPC) mobilization. The use of HPC to reconstitute hematopoiesis following myeloablative therapy has significantly improved the clinical outcome in patients with a variety of malignancies. Recently, mobilized peripheral blood HPC instead of bone marrow-derived HPC have been used because of their reduce engraftment times, relative ease of collection, and possibly reduced risk of graft-versus-host disease. Currently to mobilize HPC from the bone marrow to blood are well tolerate but not universally effective and are often associated with co-mobilization of neoplastic cells. A better understanding of the mechanisms that regulate HPC mobilization may lead to the design of novel mobilization strategies that overcome these problems. We recently showed that mobilization of HPC in response to cyclophosphamide (CY) or interleukin-8 but not fit-3 ligand is markedly impaired in granulocyte colony-stimulating factor receptor (G-CSFR) deficient mice. These surprising results suggested that G-CSFR signals in hematopoietic- or bone marrow stromal-cells play an important and previously unexpected role in HPC migration. This proposal is designed to characterize these these G- CSFR-dependent mechanisms of HPC mobilization. The following specific aims are proposed. 1. We will characterize in detail the mobilization response in G-CSFR deficient mice to CY, interleukin-12 (IL-12), or stem cell factor (SCF).. HPC mobilization in G-CSFR deficient mice in response to SCF or IL-12 will be analyzed. To explore mechanisms for the mobilization defect in G- CSFR deficient mice, the phenotype of hematopoietic cells, in particular HPC , in the bone marrow of wild-type versus G-CSFR deficient mice after CY treatment will be compared. 2. We will identify the cell type responsible for G-CSFR dependent mobilization. Preliminary studies of HPC mobilization in G-CSFR deficient radiation chimeras suggest that a functional G-CSFR on mature hematopoietic cells but not on HPC or stromal cells is required for CY- induced mobilization. The first objective of this specific aim is to confirm these surprising results and to determine whether primitive HPC are mobilized in a similar fashion. The second objective of this specific aim is to characterize G-CSF induced mobilization in these radiation chimeras. 3. We will define the role of neutrophils in G-CSFR dependent mobilization. A neutropenic mouse line will be generated by driving expression of the attenuated diphtheria toxin A subunit in myeloid cells using murine cathepsin G regulatory sequences. CY- and G-CSF induced HPC mobilization will be characterized in these mice. 4. We will define the regions of the G-CSFR that are required for HPC mobilization. CY- and G-CSF-induced mobilization will be characterized in two recently generated targeted "knock-in" mutations of the G-CSFR. The role of STAT-3 in the generation of the HPC mobilization signal by the G- CSFR will be examined.