GENE REGULATION OF THROMBOPOIETIN EXPRESSION

造血的一个最不为人所知的方面是 巨核细胞发育。 血小板生成素（TPO）的克隆， 这一过程中的一种重要细胞因子调节剂， 维持生理上适当的分子基础 检测血小板水平 北方印迹显示 不同的组织表达TPO，但负责的细胞 TPO在体内的表达尚未确定。 两大主力车型 TPO血清水平的调节。 有人说，TPO 表达在肝脏和肾脏中是组成性的， 是通过蛋白质代谢介导的， 血小板量 第二种观点认为，在血小板显著减少的情况下， 在血小板聚集的可变性中，TPO mRNA水平可能与血小板质量成反比。 我们 提供支持组成型TPO表达的实验证据 在肝脏和肾脏中，以及在骨髓中基于mRNA的调节， 脾脏 我们计划研究TPO基因调控的分子基础 着眼于了解基础和诱导组织特异性 TPO基因的表达转化为生理上适当的 血清蛋白水平 为了实现这些目标，我们提出了一个研究计划， 三个具体目标：1。为了确定细胞和组织学部位 的基础和诱导型TPO的生产在小鼠模型 血小板减少症原位杂交，原代细胞RNA分析 组分和品系及RT-PCR; 2.为了完善体外模型， 组成型和诱导型组织特异性TPO基因表达， 确定转录增强的相对贡献， 细胞中的mRNA积累增加TPO mRNA水平， 对血小板减少血清; 3.要比较的职能组织 TPO基因通过DNAseI超敏位点定位，通过RNA酶保护 测定和5' RACE分析以表征在人肝细胞癌中hTPO 5' mRNA亚型。 血小板和巨核细胞质量的扰动，通过鉴定 TPO启动子的功能相关顺式作用元件， 报告基因分析，通过DNA酶I足迹来精炼这些序列 和迁移率变化分析，并确认功能贡献 这些序列的组成型和诱导型组织特异性TPO 通过定点突变表达并确定它们的功能 报告基因功能获得/功能丧失分析的作用 测定。 我们提供的数据表明，这些目标是可行的， 为今后的研究提供有用的数据。 了解 这种巨核细胞成熟的调节剂是通过 控制将提供洞察正常和失调 巨核细胞生成

One of the least understood aspects of hematopoiesis is the process of megakaryocyte development. The recent cloning of thrombopoietin (TPO), an essential cytokine regulator of this process, allows investigation of the molecular basis for maintenance of physiologically appropriate platelet levels to proceed. Northern blots have revealed several disparate tissues to express TPO, but the cells which are responsible for TPO expression in vivo have not been identified. Two main models of TPO serum level regulation have been proposed. One asserts that TPO expression is constitutive in liver and kidney, and that serum levels are mediated via protein metabolism by an expanding or contracting platelet mass. A second suggests that in states of significant platelet variability, TPO mRNA levels may vary inversely to platelet mass. We provide experimental evidence which supports constitutive TPO expression in the liver and kidney, and mRNA-based regulation in the marrow and spleen. We plan to study the molecular basis of TPO gene regulation with an eye to understanding how the basal and inducible tissue-specific expression of the TPO gene translates into physiologically appropriate serum protein levels. To achieve these ends, we propose a research plan of three specific aims: 1. To identify the cellular and histologic sites of basal and inducible TPO production in mouse models of thrombocytopenia by in situ hybridization, RNA analysis of primary cell fractions and lines and RT-PCR; 2. To refine in vitro models of constitutive and inducible tissue-specific TPO gene expression and identify the relative contribution of transcriptional enhancement and mRNA accumulation in cells which increase TPO mRNA levels in response to thrombocytopenic sera; 3. To compare the functional organization of the TPO gene by DNAseI hypersensitive site mapping, by RNAse protection assays and 5' RACE analysis to characterize hTPO 5' mRNA isoforms during perturbations in platelet and megakarcyocyte mass, by identification of functionally relevant cis-acting elements of the TPO promoter by reporter gene analysis, refining these sequences by DNAse I footprint and mobility shift assays, and confirming the functional contribution of these sequences to constitutive and inducible tissue-specific TPO expression by site directed mutagenesis and determining their functional role by gain of function/loss of function analysis in reporter gene assays. We provide data to suggest these aims are feasible and will result in useful data as a basis for future studies. Understanding the mechanisms by which this regulator of megakaryocyte maturation is controlled will provide insight into normal and dysregulated megakaryocytopoiesis.