EXPRESSION & ROLE OF ACTIVIN A IN HEMATOPOIETIC TISSUES

表达

• 批准号: 2141226
• 负责人: JOHN C YU
• 金额: $ 20.76万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-02-15 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2141226
• 关键词: DNA binding protein; RNA splicing; bioassay; bone marrow; complementary DNA; dimer; enzyme linked immunosorbent assay; erythropoiesis; erythropoietin; flow cytometry; gene expression; genetic promoter element; growth factor receptors; hematopoietic growth factor; hematopoietic stem cells; hormone regulation /control mechanism; human subject; immunocytochemistry; in situ hybridization; inhibin; messenger RNA; oligonucleotides; polymerase chain reaction; structural genes; tissue /cell culture

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.

拟议的研究将分析激活素 A 的表达及其 造血组织的调节控制。我们还将探究 使用激活素 A 增强人类红细胞生成的可能性 长期体外培养骨髓，并分析其分布 造血细胞中的激活素 A 受体。具体目标如下 如下： A. 鉴定和表征骨髓中产生激活素 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 原位检测二聚体激活素 A 产生的技术 mRNA 表达杂交和生物活性和生物测定/ELISA 骨髓基质细胞中的二聚体激活素 A。除了骨髓 样品，我们将利用克隆的基质细胞系来进行拟议的 研究。 B.分析激活素A基因的5'侧翼区域。 5' 的一部分 侧翼区域已被测序和分析。几种结构 这些特征很有趣，促使我们分析：(l) 转录起始位点和可能的附加内含子和外显子 在这个侧翼区域； (2)与GATA结合的相互作用能力 存在七个 GATA 基序的区域中的蛋白质； (3)发起人 该 5' 侧翼区域的活动，可能控制 激活素A。 C. 研究激活素 A 受体在 造血细胞。逆转录PCR，使用一对 简并寡核苷酸引物，将使扩增成为可能 II型和各种形式的IIB型受体。 这些研究将 通过激活素 A 受体的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 使用谱系特异性的骨髓或外周血单核细胞 抗体。在另一个项目中，我们正在开发特异性抗体 对抗激活素 A 受体的 GST 融合蛋白，将用于 当这些研究可用时。 D. 分析激活素 A 对红细胞生成的增强作用 LTMC。 我们将研究剂量效应和谱系特异性 外源添加的激活素A和激活素A/Epo的各种组合和 其他重组造血因子，以及动力学和 这些文化中各种造血谱系的增殖状态。 我们还将激活素 A cDNA 插入基质细胞群中，并 研究微环境中本地产生的激活素A是否 LTMC 中红细胞生成的维持时间比对照更长。这些研究将 有助于确定激活素A或其与其他因素的组合 具有作为红细胞成分治疗新药的潜力 未来。