演題番号 : P3-d02
米山 雅紀 / Masanori Yoneyama:1 芝 達雄 / Tatsuo Shiba:1 荻田 喜代一 / Kiyokazu Ogita:1
1:摂南大学薬学部 薬理学 / Department of Pharmacology, Faculty of Pharmaceutical Sciences
Various neurological injuries are widely recognized as promoting endogenous neurogenesis in hippocampal dentate gyrus of adulthood. Our previous studies demonstrated that the granule cells in the hippocampal dentate gyrus are injured and disappeared by treatment with trimethyltin chloride (TMT), with being regenerated in the dentate granule cell layer after neuronal loss. Using TMT-treated mouse as an in vivo model, we established the efficient culture system of adult mouse dentate gyrus-derived neural stem/progenitor cell after injury. Hippocampal dentate gyrus was isolated from 5-week-old Std-ddY male mice day 3 after injection of TMT, and then cell suspensions were maintained in DMEM/F-12 supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). The obtained cells were fixed for immunostaining of nestin and GFAP. TMT led to a significant increase in the nestin-positive cells, without affecting that of GFAP-positive cells. Cells cultured floatingly in DMEM/F-12 medium containing EGF/bFGF resulted in the formation of round spheres immunoreactive to nestin in 27 days in vitro (DIV). The number of neurospheres was significantly increased compared with those derived from naive animals. Cells were seeded on dishes that had been previously coated with poly-L-ornithine and then cultured in DMEM/F12 medium containing bFGF/EGF. Under these conditions, cells grew at monolayer without forming neurospheres. Most cells were immunoreactive with both nestin and GFAP. ELISA for 5'-bromo-2'-deoxyuridine revealed that a marked increase in the proliferative activity was seen by treatment with TMT at 28 DIV. Our data suggest that hippocampal neural stem/progenitor cells can be abundantly prepared from the dentate gyrus of mice treated with TMT. Thus, TMT-treated mice are an attractive model for achieving the neural stem/progenitor cell from hippocampal dentate gyrus.