Analysis of cell proliferation by GM-CSF

GM-CSF调节多种造血细胞的增殖和分化。我们对人GM-CSF受体的信号转导进行了分析，发现人GM-CSF受体有多种不同的信号通路。通过使用突变受体，我们发现我们可以激活或灭活某些信号通路。在这项研究中，我们试图使用突变的hGM-CSF受体来分析细胞增殖信号，该受体可以在不激活MAPK级联的情况下激活增殖。我们通过减法克隆了几个基因，但由于克隆方法是无功能的，我们发现很难揭示这些新基因的功能。为了用生化方法克隆和分析基因，我们尝试用分离细胞裂解物建立体外信号通路重构系统。我们成功地从造血细胞裂解液中制备了STAT5的体外激活系统，但未能将该系统应用于c-myc转录诱导。通过下拉分析克隆出hGM-CSSF受体结合蛋白，并利用STAT5体外系统分析克隆基因的功能。

GM-CSF regulates proliferation and differentiation of various hematopoietic cells. We have been analyzing signal transduction of human GM-CSF rcceptor and found that there are multiple distinct signaling pathways through human GM-CSF receptor. By using mutant receptors, we found that we can activate or inactivate certain signaling pathway. In this study, we tried to analyze signals for cell proliferation using the mutant hGM-CSF receptors which can activate proliferation without activation of MAPK cascade. We clones several genes by subtraction but we found difficulty to reveal function of these new genes since the cloning methods was non-functional. To clone and analyze genes by biochemical methods, we tried to establish in vitro reconstitution system of signaling pathway by fractionated cell lysates. We succeeded to make an in vitro system of STAT5 activation from hematopoietic cell lysate, but failed to apply this system to c-myc transcriptional induction. We cloned hGM-CSSF receptor binding proteins by pull-down analysis, and analyzed function of these cloned genes by STAT5 in vitro system.