Establishment And Biological Characterization Of Recombinant Cell Lines Stably Express Wild Or Mutated Type Human C-Kit Receptor

Stem cell factor (SCF) is a growth factor critical for hematopoiesis as well as for the regulation of survival, proliferation and differentiation of mast cells, melanocytes and germ cells. SCF was able to enhance the formation of granulocyte-macrophage colonies, erythroid bursts and colonies of multipotential progenitors. The human proto-oncogene c-kit encodes a transmembrane protein known as the receptor for SCF with a molecular weight of 145 kD. It is a member of type III receptor tyrosine kinase subfamily which is characterized by the presence of five Ig-like repeats in the extracellular domain and an insert that splits the cytoplasmic kinase domain into an adenosine triphosphate (ATP)-binding region and the phosphotransferase domain. The interaction between SCF and c-Kit rapidly induces dimerization of the receptors and increasing in autophosphorylation activity. Downstream of c-Kit, multiple signal transduction components are activated. SCF has the remarkable species-specific difference, because the encoding sequences of the extracellular domain of c-Kit are different between human and murine.The bioassay of recombinant human SCF is some difficulty, which has to be resolved in its pharmaceutical development. At present, the human megakaryoblastic leukemia cell line Mo7e is generally used to detect the proliferation induced by rhSCF. However, the growth of Mo7e relies on some growth factors, such as GM-CSF, IL-3, Epo and insulin and is weak in remarkable and specific induced-proliferation to rhSCF. It is an optimal approach to resolve the problem that establish an artificial cell line which proliferates dependently on rhSCF by means of genetic engineering techniques. The cell lines expressing human c-Kit receptor or its mutant stably should be a useful tool for the bioassay of rhSCF and the research of its regulation mechanisms and the roles which plays in the signal transduction activated by SCF/c-Kit in hematopoietic progenitor cell. This thesis comprises the following three parts:1. The full-length cDNA sequence encoding wild-type human c-Kit receptor was cloned and the first 3 Ig-like domain (hc-KitIg1-3) was expressed in E.coli and preliminarily purified. Four overlapping fragments F1-F4 of hc-kit cDNA, which share lengths of 716 bp, 600 bp, 901 bp and 798 bp, were obtained from human megakaryoblastic leukemia cell...