HEMATOPOIETIC STEM CELL DIFFERENTIATION IN HUMAN BLOOD

本文提出的长期研究目标是确定生长因子刺激造血细胞增殖和分化的信号转导机制。 本研究的主要目的是了解红细胞生成素（Epo）在红细胞分化过程中调节离子通道的机制，并确定钙离子内流在红细胞生成中的功能作用。 将在CRC中对健康志愿者进行抽血和骨髓穿刺。 具体目标1：确定Epo调节钙通道的信号传导机制。 (A)在这里，我们将确定连接Jak 2和钙通道激活的Epo信号通路。STAT、Ras或IRS-2/PI 3激酶通路的参与将使用单个BFU-E衍生的成红细胞的显微注射和定量荧光显微镜结合数字视频成像来检查。(B)我们将确定促红细胞生成素调节钙通道开放所需的促红细胞生成素受体的结构域。 具体目的2：确定[Cai]在红细胞生成中调节转录因子活化的作用。 NF-kB和bHLH转录因子以及c-Jun N末端激酶（JNK）的功能受到钙的调节。 我们将确定红系细胞中Ca++反应的幅度或持续时间是否影响NF-κ B转录因子的激活，或者钙/钙调素水平是否影响bHLH蛋白的DNA结合，特别是SCL。 我们还将确定[Cai]对JNLK激活的影响，以及PI 3-激酶在该途径中的作用。

The long term goal of studies proposed here is to determine the signal transduction mechanisms through which growth factors stimulate hematopoietic proliferation and differentiation. The major goal of this project is to understand the mechanisms through which erythropoietin (Epo) regulates ion channels during erythroid differentiation and to determine the functional role of calcium influx in erythropoiesis. Blood will be drawn and bone marrow aspirates will be performed in the CRC on healthy volunteers. The following specific aims will be addressed: Specific Aim 1: Identification of the signaling mechanism through which Epo regulates calcium channels. (A) Here we will determine the Epo signaling pathways which link Jak2 to calcium channel activation. Involvement of STAT, Ras, or the IRS-2/PI 3 kinase pathways will be examined using microinjection of single BFU-E derived erythroblasts and quantitative fluorescence microscopy coupled digital video imaging. (B) We will determine the domains of erythropoietin receptor required for Epo modulation of calcium channel opening. Specific Aim 2:Determination of the role of [Cai] in regulation of transcription factor activation in erythropoiesis. The function of the NF-kB and bHLH transcription factors and the c-Jun N-terminal kinases (JNK) are modulated by calcium. We will determine if the amplitude or duration of the Ca++ response in erythroid cells affects NF-kB transcription factor activation, or if calcium/calmodulin levels influence DNA binding of bHLH proteins, particularly SCL. We will also determine the effect of [Cai] on JNLK activation on the role of PI 3-kinase in this pathway.