Genomic approach to investigating ex vivo hematopoiesis

研究离体造血的基因组方法

• 批准号: 6579514
• 负责人: Eleftherios T Papoutsakis
• 金额: $ 26.02万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6579514
• 关键词: CD34 molecule; acidity /alkalinity; apoptosis; cell differentiation; cell growth regulation; cell population study; clinical research; cytokine; functional /structural genomics; genetic transcription; granulocyte; hematopoiesis; hematopoietic stem cells; human tissue; megakaryocytes; microarray technology; oxygen tension; polymerase chain reaction; tissue /cell culture

DESCRIPTION (provided by applicant): High-dose chemotherapy with autologous or allogeneic stem-cell rescue results in prolonged pancytopenia that is accompanied by infectious and bleeding complications requiring antibiotic and transfusion therapy and, at times, prolonged hospitalization. Infusion of large numbers of ex vivo expanded hematopoietic cells -as a supplement to the conventional auto- or allograft - has the potential to close the window of neutropenia and/or thrombocytopenia. Furthermore, the recently discovered plasticity of hematopoietic stem cells suggests that these readily available stem cells may be used for generating autologous or allogeneic cells and tissues for non-hematopoietic cell and gene therapies. Success of such therapies depends on the ability to generate large numbers of cells with the desired, therapy-dependent state of cell differentiation. This remains an elusive task despite the great progress in basic and applied biology. Culture conditions, such as cytokine combinations and presentation, oxygen tension (pC2) and pH, alter stem- and progenitor-cell differentiation and proliferation with substantial patient-to-patient variability. Little is known about the underlying molecular biology of these effects, and specifically, about the large-scale transcriptional program during differentiation. Such knowledge has large predictive and diagnostic potential for both ex vivo and in vivo outcomes. Thus, a comprehensive examination of the transcriptional program of ex vivo expanded human primary myeloid cells - initiated with CD34+ cells - will be examined using 8,300-gene DNA microarrays, and key findings further explored using standard molecular-biology tools. Studies include examination of the temporal and differential transcriptional program of G and Mk cells cultured either under high or low pC2 and/or pH, and with different cytokine combinations. Specific issues to be examined include the extent to which apoptosis is linked to Mk differentiation; if Mk apoptosis employs a machinery similar to that of general apoptosis; and why, in contrast to Mk cultures, there is such a low level of apoptosis in G cultures. Furthermore, gone-clustering and regulatory-network techniques applied to DNA-microarray data may lead to the discovery of unknown Mk- and G-differentiation genes. These experiments will provide the basis for future studies in which clinical specimens could be examined in the context of clinical stem cell transplantation.

描述（由申请人提供）：高剂量化疗联合自体或同种异体干细胞拯救会导致长期全血细胞减少症，并伴有感染和出血并发症，需要抗生素和输血治疗，有时还需要延长住院时间。输注大量离体扩增的造血细胞——作为传统自体或同种异体移植的补充——有可能关闭中性粒细胞减少症和/或血小板减少症的窗口。此外，最近发现的造血干细胞的可塑性表明这些容易获得的干细胞可用于生成用于非造血细胞和基因治疗的自体或同种异体细胞和组织。此类疗法的成功取决于产生大量具有所需的、依赖于疗法的细胞分化状态的细胞的能力。尽管基础生物学和应用生物学取得了巨大进步，但这仍然是一项难以捉摸的任务。细胞因子组合和表达、氧张力 (pC2) 和 pH 等培养条件会改变干细胞和祖细胞的分化和增殖，并且患者之间存在很大差异。对于这些效应的潜在分子生物学，特别是分化过程中的大规模转录程序，人们知之甚少。这些知识对于离体和体内结果都具有巨大的预测和诊断潜力。因此，将使用 8,300 个基因 DNA 微阵列对离体扩增的人原发性骨髓细胞的转录程序（以 CD34+ 细胞启动）进行全面检查，并使用标准分子生物学工具进一步探索关键发现。研究包括检查在高或低 pC2 和/或 pH 下以及使用不同细胞因子组合培养的 G 和 Mk 细胞的时间和差异转录程序。需要研究的具体问题包括细胞凋亡与 Mk 分化的关联程度； Mk细胞凋亡是否采用与一般细胞凋亡类似的机制；以及为什么与 Mk 培养物相比，G 培养物中的细胞凋亡水平如此之低。此外，应用于 DNA 微阵列数据的消失聚类和调控网络技术可能会导致未知 Mk 和 G 分化基因的发现。这些实验将为未来的研究奠定基础，在未来的研究中可以在临床干细胞移植的背景下检查临床标本。