THROMBOPOIETIN: IMMUNOASSAY AND CHARACTERIZATION

血小板生成刺激因子（TSF或血小板生成素）， 现在已知它是血小板生成的主要控制因素。 确定化学特性所需的研究， 生产地点、作用机制、感觉机制 尚未执行TSF释放和行动需求，因为 缺乏合适的分析技术和可用的 稳定的纯化TSF来源。 本文中描述的研究 提案将提供新的分析程序和适当的供应 TSF的进一步研究。 这项建议的目的是 收件人： 1)继续生产TSF，并建立其化学和 生物学性质， 2)确定氨基酸序列并克隆TSF基因， 3)表征来自人胚肾（HEK）细胞的mRNA， 4)测量TSF在骨髓抑制动物中的功效， 5)开发TSF的放射免疫测定法， 6)测量TSF后动物中巨核细胞的倍性 治疗， 7)在巨核细胞上建立TSF结合位点， 8)阐明缺氧对小鼠巨核细胞生成的影响， 9)从产生TSF的肿瘤中建立细胞系， 10)血小板聚集患者体液中TSF的连续检测 生产障碍，以及 11)开始对TSF遗传转移的新研究。 这些目标将利用各种物理 化学和免疫学方法： 1)血小板减少动物血清和HEK培养基 细胞培养物将用于TSF生产; 2)抗血清将通过使用常规的和 单克隆抗体技术; 3)将通过以下方法从各种来源纯化TSF： HPLC和免疫学技术; 4)蛋白水解切割将用于产生 用于序列测定的TSF; 5)TSF的氨基酸序列将用于产生DNA探针 为了鉴定目前在人cDNA文库中产生TSF的克隆， 可在Abbott Laboratories获得; 6)mRNA将从HEK细胞中提取并与其他细胞分离。 寡聚-dT纤维素亲和层析法测定核酸 列; 7)将mRNA注射到青蛙卵母细胞中并产生TSF 测量; 8)将小鼠暴露于缺氧和/或89 Sr以确定 脾和骨髓的相对作用 巨核细胞生成; 9)小鼠和狗将注射TSF及其血小板 为确定TSF的功效而测量的产量;以及 10)在动物模型中逆转录病毒介导的基因转移将 企图。 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。 实现本提案中列出的目标将使 可用于治疗血小板减少患者的重组TSF 生产障碍，在加速血小板恢复， 接受放疗或化疗后的血小板减少症患者 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.