EXPRESSION & ROLE OF ACTIVIN A IN HEMATOPOIETIC TISSUES

拟议的研究将分析激活素A的表达及其 造血组织中的调控。我们还将调查 使用激活素A促进人红细胞生成的可能性 长期体外培养骨髓，并分析其分布情况。 造血细胞中的激活素A受体。具体目标如下 以下是： A.鉴定和鉴定骨髓中产生激活素A的细胞 基质细胞和/或克隆的基质细胞系。我们将使用免疫学 二聚体激活素A的原位检测技术 信使核糖核酸的杂交及生物活性和生物活性的检测 二聚体激活素A在骨髓基质细胞中的分布。除了骨髓 样本，我们将利用克隆的基质细胞系来进行建议的 学习。 B.分析激活素A基因5‘侧翼区。5英尺的一部分 对其侧翼区域进行了测序和分析。几种结构 这些特征耐人寻味，促使我们分析：(L) 转录起始点(S)和可能的附加内含子和外显子 在这个侧翼区域；(2)与GATA结合的能力 存在七个GATA基序的区域中的蛋白质；和(3)启动子 在这个5‘侧翼区的活动，可能控制表达 激活素A C.研究激活素A受体在人群中的分布 造血细胞。逆转录聚合酶链式反应，使用一对 简并的寡核苷酸引物，将使扩增 II型和各种形式的IIB型受体。这些研究将是 经激活素A受体免疫细胞化学分析证实 骨荧光激活细胞分选后的特定细胞谱系 骨髓或外周血单个核细胞使用谱系特异性 抗体。在另一个项目中，我们正在开发特定的抗体 抗激活素A受体作为GST融合蛋白，将用于 当这些研究可用时。 D.分析激活素A对小鼠红细胞生成的促进作用 LTMC。我们将调查剂量效应和谱系特异性 外源添加激活素A和激活素A/EPO的各种组合 其他重组造血因子，以及动力学和 这些培养物中不同造血系的增殖状态。 我们还将把激活素A基因插入基质细胞群体中，并 调查当地生产的激活素A是否在微环境中 与对照组相比，LTMC患者维持红细胞生成的时间更长。这些研究将 有助于确定激活素A或其与其他因素的组合 有潜力作为红细胞成分治疗的新药物 未来。

The proposed studies will analyze the expression of activin A and its regulatory control in hematopoietic tissues. We will also probe the possibility of using activin A to enhance the erythropoiesis in the human long-term marrow cultures in vitro, and analyze the distribution of activin A receptors in hematopoietic cells. The specific aims are as follows: A. To identify and characterize activin A producing cells in marrow stromal cells and/or cloned stromal cell lines. We will use immunological techniques to detect the production of dimeric activin A, in situ hybridization for mRNA expression and bioassay/ELISA for bioactive and dimeric activin A among marrow stromal cells. In addition to marrow samples, we will utilize cloned stromal cell lines for the proposed studies. B. To analyze the 5' flanking region of activin A gene. Part of the 5' flanking region has been sequenced and analyzed. Several structural features are intriguing and have prompted us to analyze: (l) the transcription initiation site(s) and possible additional introns and exons in this flanking region; (2) the capacity of interacting with GATA binding proteins in a region where seven GATA motifs are present; and (3) promoter activities in this 5' flanking region, which may control the expression of activin A. C. To investigate the distribution of activin A receptors among hematopoietic cells. The reverse transcription PCR, using a pair of degenerate oligonucleotide primers, will make it possible to amplify the type II and various forms of type IIB receptors. These studies will be confirmed with immunocytochemical analysis of activin A receptors among specific cell lineages after fluorescence-activated cell sorting of bone marrow or peripheral blood mononuclear cells using lineage specific antibodies. In another project, we are developing specific antibodies against activin A receptor as GST fusion protein, which will be used in these studies when they are available. D. To analyze the potentiating effects of activin A on erythropoiesis in LTMC. We will investigate the dosage effect and lineage specificity of exogenously added activin A and various combinations of activin A /Epo and other recombinant hematopoietic factors, and the kinetics and proliferative states of various hematopoietic lineages in these cultures. We will also insert activin A cDNA into stromal cell populations and investigate whether the locally produced activin A in microenvironment sustains erythropoiesis longer than controls in LTMC. These studies will help to determine whether activin A or its combination with other factors have the potential as novel agent for red cell component therapy in the future.