EXPRESSION & ROLE OF ACTIVIN A IN HEMATOPOIETIC TISSUES

拟开展的研究将分析激活素A的表达及其与细胞凋亡的关系。 造血组织中的调节控制。我们还将探讨 使用激活素A增强人红细胞生成的可能性 体外长期骨髓培养，分析 造血细胞中的激活素A受体。具体目标如下： 如下所示： 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 用于mRNA表达杂交和用于生物活性和 骨髓基质细胞中的二聚体激活素A。除了骨髓 样本，我们将利用克隆基质细胞系的建议， 问题研究 B。分析激活素A基因5'端侧翼序列。部分5' 侧翼区进行了测序和分析。几个结构 特征是耐人寻味的，并促使我们分析：（l） 转录起始位点和可能的额外内含子和外显子 （2）与加塔结合相互作用的能力 存在七个加塔基序的区域中的蛋白质;和（3）启动子 在这个5'侧翼区的活性，这可能控制的表达， 活化素a C.为了研究激活素A受体的分布， 造血细胞逆转录PCR，使用一对 简并寡核苷酸引物，将使得有可能扩增 II型和各种形式的IIB型受体。 这些研究报告将 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异性细胞谱系 骨髓或外周血单核细胞， 抗体的在另一个项目中，我们正在开发特异性抗体 针对激活素A受体的GST融合蛋白，其将用于 这些研究如果有。 D.分析激活素A对红细胞生成的增强作用， LTMC。 我们将研究的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， 其他重组造血因子，以及动力学和 这些培养物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， 研究微环境中局部产生的激活素A是否 在LTMC中维持红细胞生成的时间长于对照。这些研究将 有助于确定激活素A或其与其他因子的组合是否 具有作为红细胞成分治疗的新试剂的潜力， 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.