REGULATORS OF MEGAKARYOCYTOPOIESIS IN HUMAN URINE

该项目的目标是分离、逐步纯化和 表征源自人尿的因子，其可导致 单个巨核细胞的数量、成熟度和大小增加， 小鼠骨髓细胞血浆凝块培养物中的巨核细胞集落。 涉及尺寸选择性超滤的纯化技术， 常规和高压液体分级沉淀 将使用色谱法。 据推测， 巨核细胞生成依赖于至少两种 调节因素;一个因素，主要是增加数量 细胞能够培养成可识别的巨核细胞， 这是导致巨核细胞成熟所必需的。 我们发现 巨核细胞集落刺激因子（MEG-CSF）不同于 促红细胞生成素（EPO）存在于人尿中。 它将试图 辨别第二个“增强”因素，类似于所描述的 在条件培养基中，也存在于尿液中。 剂量依赖性和 通过实验研究了这些因素的作用机理 包括小鼠骨髓靶细胞预孵育的设计 在具有一种因子或多种因子组合悬浮培养中， 随后用适当凝血因子攻击这些细胞 文化 将确定是否增加了 巨核细胞集落形成单位（CFU-MEG）所带来的这些因素 是基于细胞复制或细胞活化（致敏）。 的 因子的靶细胞的性质（直接作用与间接作用， 单能对多能祖细胞）进行研究。 的 血小板计数异常患者的尿MEG-CSF含量 不同的原因和正常人将被测量，以检测 可能与其他血液学参数相关。 的测定 检测和定量促进巨核细胞形成的因子 在培养物中基于对小鼠中5-羟色胺摄取的刺激 将开发骨髓细胞悬浮培养物以补充 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.