CONTROL OF ERYTHROPOIESIS BY GROWTH PROMOTING PEPTIDES

许多多肽生长因子（GF）表达红细胞生成素（EPO）， 在培养物中以及可能在体内的活性。 近期 哺乳动物细胞化学成分确定培养基的可用性 培养允许评价激素的作用和调节 在细胞和亚细胞水平的机制。 我们将雇用一名 用于培养人红系细胞的“无血清”系统， 我们的发展是为了： 1.确定定义的GF的细胞效应。 我们将 确定所选肽的剂量-反应关系 在血清剥夺下的单层和双层培养中。 我们将扩展我们的发现，一个GF，L-三碘甲状腺原氨酸， 不仅刺激蛋白质释放，而且刺激旁分泌（爆发 促进活性，BPA）从培养的 淋巴细胞 累积蛋白质和BPA释放， 放射性标记的GF的释放和亚细胞定位将 被定义。 特定的细胞内加工途径将 被研究。 新合成的类BPA 分子到未诱导的和促分裂原驱动的BPA将是 在生化和免疫学上定义的。 2.分离和纯化未定义的GF。 可溶性BPA释放， 在没有诱导剂的情况下， 介质 柱层析，凝集素亲和力 高效液相色谱法 将使用色谱法。 此外，抗BPA IgG将 用于免疫亲和层析纯化。 3.表征GF靶细胞并检查GF结合。 我们 将利用杂交瘤抗体选择性地“淘选” 红系祖细胞 与抗T- 和B细胞群，它们将被用来识别骨髓 对GF效应敏感或具有抗性的亚群。 胰岛素和胰岛素样生长因子与panned 还将测定骨髓细胞。 4.关联体内和组织培养反应。 我们将 获得低增殖性贫血患者，以确定 体外雄激素敏感性试验的预测价值。 骨髓细胞和血浆细胞的研究 过度增殖性骨髓恶液质将探索潜在的 涉及血小板衍生生长的体液异常 因子（PDGF）。 这些调查集中在我们的长期 评价组织生理相关性的目标 健康和疾病的文化发现。

Many polypeptide growth factors (GFs) express erythropoietic activity in culture and possibly in vivo as well. The recent availability of chemically defined media for mammalian cell culture permits evaluation of hormone effects and regulatory mechanisms at cellular and subcellular levels. We will employ a serum "free" system for the culture of human erythroid cells that we have developed in order to: 1. Determine cellular effects of defined GFs. We will determine dose-response relationships for selected peptides in single and double layer cultures under serum deprivation. We will extend our finding that one GF, L-triiodothyronine, stimulates not only protein release but also paracrine (burst promoting activity, BPA) release from cultured lymphocytes. Cumulative protein and BPA release, rates of release and subcellular localization of radiolabeled GF will be defined. Specific intracellular processing pathways will be studied. The relationship of newly synthesized BPA-like molecules to uninduced and mitogen-driven BPA will be defined biochemically and immunologically. 2. Isolate and purify undefined GFs. Soluble BPA released with and without inducting agents will be purified from liquid medium. Column chronmatograpy, lectin affinity chromatograpy and reverse-phase high-performance liquid- chromatography will be used. In addition, anti-BPA IgG will be used for purification by immunoaffinity chromatography. 3. Characterize GF target cells and examine GF binding. We will utilize hybridoma antibodies to selectively "pan" for erythroid progenitors. Together with monoclonal IgG to T- and B-cell populations, they will be used to identify marrow subpopulations that are sensitive or resistant to GF effects. Insulin and insulin-like growth factor binding to panned marrow cells will also be determined. 4. Correlate in vivo and tissue culture responses. We will accure patients with hypoproliferative anemia to determine the predictive value of in vitro androgen sensitivity testing. Studies of plasma and marrow cells from patients with hyperproliferative marrow dyscrasias will explore potential humoral abnormalities involving platelet-derived growth factor (PDGF). These investigations focus on our long term goal of evaluating the physiological relevance of tissue culture findings in health and disease.