THROMBOPOIETIN: IMMUNOASSAY AND CHARACTERIZATION

一种血小板生成刺激因子(TSF或血小板生成素) 现在已知是血小板生成的主要控制因素。 确定化学特性所需的研究， 生产现场，作用机制(S)，机制(S)那意义 尚未执行TSF放行和行动的需要，因为 缺乏合适的检测技术和可获得性 一种稳定的TSF纯化源。这篇文章中描述的研究 提案将提供新的化验程序和适当的供应 以供进一步研究。这项提议的目标是 致： 1)继续生产TSF，并建立其化学和 生物本性， 2)测定TSF的氨基酸序列，克隆TSF基因， 3)鉴定人胚胎肾(HEK)细胞中的mRNA， 4)测定TSF对骨髓抑制动物的疗效； 5)建立TSF的放射免疫分析方法。 6)TSF术后动物巨核细胞倍性测定 治疗， 7)在巨核细胞上建立TSF结合部位， 8)阐明低氧对小鼠异核细胞生成的影响， 9)建立产生TSF的肿瘤细胞系， 10)继续检测血小板患者体液中的TSF 生产障碍，以及 11)开展TSF基因转移的新研究。 这些目标将利用各种物理手段来实现 化学和免疫学方法： 1)血小板减少性动物血清和HEK培养液 细胞培养将用于TSF的生产； 2)抗血清将在动物体内用常规和 单抗技术； 3)从各种来源提纯TSF将由 高效液相色谱和免疫学技术； 4)将使用蛋白水解性切割来生成片段 TSF用于序列测定； 5)TSF的氨基酸序列将用于生成DNA探针 鉴定目前人cDNA文库中产生TSF的克隆 可在雅培实验室购买； 6)从HEK细胞中提取信使核糖核酸并与其他 核酸在寡聚DT纤维素上的亲和层析 栏目； 7)将信使核糖核酸注入青蛙卵母细胞并产生TSF 被测量的； 8)小鼠将暴露在低氧和/或89sr中，以确定 脾与骨髓在骨质疏松症中的相对作用 巨核细胞增多症； 9)给小鼠和狗注射TSF和它们的血小板 为确定三氟化硫的效力而测量的产量；以及 10)在动物模型中逆转录病毒介导的基因转移将是 已尝试。 这些研究将提供必要的信息来预测 TSF的作用方式及其作用机制。 完成本提案中列出的目标将使 可用于治疗血小板的重组TSF 生产障碍，在加速血小板恢复中 放化疗后的血小板减少患者 骨髓抑制、骨髓移植和艾滋病。

A thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) is now known to be a major controlling factor of platelet production. The studies necessary to determine the chemical characteristics, site of production, mechanism(s) of action, mechanism(s) that sense the need for TSF release and action have not been performed because of the lack of suitable assay techniques and the availability of a stable purified source of TSF. Research described in this proposal will provide new assay procedures and a suitable supply of TSF for further studies. The objectives of this proposal are to: 1) continue production of TSF and establish its chemical and biological nature, 2) determine the amino acid sequence and clone the gene for TSF, 3) characterize mRNA from human embryonic kidney (HEK) cells, 4) measure the efficacy of TSF in bone marrow suppressed animals, 5) develop a radioimmunoassay for TSF, 6) measure ploidy of megakaryocytes in animals after TSF treatment, 7) establish TSF combining sites on megakaryocytes, 8) clarify effects of hypoxia on murine metakaryocytopoiesis, 9) establish cell-lines from tumors that produce TSF, 10) continue TSF testing in body fluids of patients with platelet production disorders, and 11) begin new studies on genetic transfer of TSF. These objectives will be pursued utilizing a variety of physical chemical and immunological methods: 1) sera from thrombocytopenic animals and culture media from HEK cell cultures will be utilized for TSF production; 2) antisera will be raised in animals by use of conventional and monoclonal antibody technology; 3) purification of TSF from a variety of sources will be made by HPLC and immunological techniques; 4) proteolytic cleavage will be employed to generate fragments of TSF for sequence determination; 5) amino acid sequences of TSF will be used to generate DNA probes to identify TSF producing clones in a human cDNA library currently available at Abbott Laboratories; 6) mRNA will be extracted from HEK cells and separated from other nucleic acids by affinity chromatography on oligo-dT cellulose columns; 7) mRNA will be injected into frog oocytes and TSF production measured; 8) mice will be exposed to hypoxia and/or 89Sr to determine the relative roles of the spleen and bone marrow in megakaryocytipoiesis; 9) mice and dogs will be injected with TSF and their platelet production measured in order to determine the efficacy of TSF; and 10) retroviral-mediated gene transfer in an animal model will be attempted. These studies will provide the necessary information to predict the mode of action of TSF and it mechanisms of action. Accomplishment of the goals listed in this proposal will make available recombinant TSF for treating patients with platelet production disorders, in accelerating platelet recovery in thrombocytopenic patients following radio- or chemotherapy-induced bone marrow depression, bone marrow transplantation, and AIDS.