REGULATORS OF MEGAKARYOCYTOPOIESIS IN HUMAN URINE

该项目的目标是分离、逐步提纯和 表征从人类尿液中提取的因子，这可以带来 单个巨核细胞的数量、成熟度和大小增加 小鼠骨髓细胞浆凝块培养中的巨核细胞集落形成。 包括尺寸选择性超滤在内的净化技术， 分步沉淀法常规高压液体 将采用层析法。人们一直假设，增加 巨核细胞生成依赖于至少两种类型的 监管因素；一个主要增加数量的因素 能够诱变为可识别的巨核细胞的细胞和一个因素 这是导致巨核细胞成熟所必需的。我们发现了 巨核细胞集落刺激因子(MEG-CSF)与 促红细胞生成素(EPO)存在于人体尿液中。它将被尝试 辨别是否有第二个“增强”因素，类似于所述 在条件培养液中，也存在于尿液中。剂量依赖关系和 这些因素的作用机理将在实验中进行研究。 将包括预先培养小鼠骨髓靶细胞的设计 在悬浮培养中使用一种因素或多种因素和组合 用适当的因子(S)在凝块中对这些细胞的后续挑战 文化。将确定是否增加的数量 这些因素带来的巨核细胞集落形成单位(CFU-MEG) 是基于细胞复制或细胞激活(敏化)。这个 因子的目标细胞的性质(直接作用与间接作用， 单能和多能祖细胞)将被研究。这个 慢性阻塞性肺疾病血小板计数异常患者尿液中的MEG-CSF含量 不同的病因和来自正常人的测量将被检测到 可能与其他血液学参数相关。一种化验 巨核细胞形成促进因子的检测与定量 基于刺激小鼠摄取5-羟色胺的培养 将开发骨髓细胞悬浮培养，以补充 血浆凝块测定。

The objective of this project is to isolate, progressively purify and characterize facators derived from human urine, which can bring about increases in the number, maturity and size of single megakaryocytes and of megakaryocyte colonies in mouse marrow cell plasma clot cultures. Purification techniques involving size selective ultrafiltration, fractional precipitation conventional and high pressure liquid chromatography will be employed. It has been hypothesized that an increase in megakaryocytopoiesis is dependent on the action of at least two types of regulatory factors; one factor which primarily increases the number of cells capable of muturing into recognizable megakaryocytes and one factor which is needed to bring about megakaryocyte maturation. We have found that megakaryocyte colony stimulating factor (MEG-CSF) different from erythropoietin (EPO) is present in human urine. It will be attempted to discern whether a second "potentiating" factor, similar to that described in conditioned media, is also present in urine. The dose dependence and machanism of action of these factors will be studied in experimental designs which will include preincubation of the mouse marrow target cells in suspension culture with a factor or with a combination of factors and subsequent challenge of these cells with appropriate factor(s) in clot culture. It will be determined whether an increase in numbers of megakaryocyte colony forming units (CFU-MEG) brought about by these factors is based on cell replication or on cell activation (sensitization). The nature of the target cells for the factors (direct versus indirect action, unipotent versus multipotent progenitor cells) will be investigated. The MEG-CSF content of urine from patients with abnormal platelet counts due to different causes and from normal individuals will be measured to detect possible correlations with other hematological parameters. An assay for the detection and quantitation of factors promoting megakaryocyte formation in culture based on the stimulation of the uptake of serotonin in mouse bone marrow cell suspension cultures will be developed to complement the plasma clot assay.