| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 98 === Human embryonic stem cell (HES cells) is highly sensitive to dissociation, which cause HES cells death. So far, the usual way to passage HES cells is using type IV collagenase or accutase to prevent dissociation of HES cells. Generation of homogenous and single HESC is an obstacle, preventing the efficient gene transfer and clonal isolation. Previous study demonstrates Y-27632 permit survival of dissociated HES cells, but the mechanism is still unclear. In order to find out the mechanism that regulate dissociated HES cells survival, trypsin-resistant HES sublines were obtained to find out what happended in trypsin-resistant HES sublines can let it survival from trypsin-dissociation. To explore the differences in trypsin-resistant HES cells and normal HES cells, the gene expression patterns of both HES cells are analyzed at protein- and RNA-level. We have found out that cytoskeleton materials (beta-actin, alpha-tubulin and beta-tubulin) and genes that promoting cytoskeleton formation (ADD2, PAK3 and FGD1) are up-regulate in trypsin-resistant HES cells. We postulate that the polymerization and stabilization of actin-filaments and microtubules by activation of Rho/Rac/Cdc42 pathway may help trypsin-resistant HES cells overcome dissociation and survive. Moreover, we also find that the epidermal growth factor (EGF) which induces actin-filament formation also increases the survival of dissociated HES cells. However, the relationship between cytoskeletons and HES cell survival still need to be confirmed. We will further treat HES cells with microtubule regulator (Taxol and nocodazol) to confirm the relationship between microtubule and HES cell survival. Since we have find that actin-filaments and microtubules may improve survival of trypsin dissociated HES cells, the survival mechanism of dissociated HES cells may be solve. With the enhancement of cytoskeleton, it may be a simple and more efficient process to generate homogenous population of HES cells and improve gene transfer efficiency of HES cells in medical research and applications.
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