幹細胞、ニューロンとグリアの分化 Stem Cells, Neuronal and Glial Production/Differentiation
P1-1-54 成体脊髄における前駆細胞の人為的細胞分化調節の試み Attempts of cell fate intervention of potential progenitor cells in the adult rodent spinal cord ○北田容章1, 鈴木潤一1, 出澤真理1 ○Masaaki Kitada1, Junichi Suzuki1, Mari Dezawa1 東北大学大学院 医学系研究科 細胞組織学1 Dept Stem Cell Biol and Histol, Tohoku Univ Grad Sch Med, Sendai1 Anatomical reconstruction and functional recovery after spinal cord injury (SCI) in the adult mammals are known to be considerably limited. On the other hand, even after severe injury of the spinal cord in fishes and amphibians complete anatomical repair and functional recovery are known to be spontaneously achieved, in which the ependymal cells play the remarkable roles such as cell proliferation, epithelial-mesodermal transition, cell migration, and neurogenesis to primarily contribute to functional recovery from SCI. In this study, we tried to manipulate the activity of the ependymal cells in the adult rodent spinal cord by virus-mediated gene transfer to make them mimicking the reactions that are observed in the ependymal cells of fishes and amphibians after SCI. We introduced the gene, which is known to regulate the cell fate of neural stem cells and is normally expressed in the ependymal cells. We found that the rate of the proliferation in the ependymal cells was significantly raised. In this case, the differentiation of the ependymal cells into astrocytes was also observed. Transfer of another neural stem cell-related gene led to cell proliferation without differentiation into astrocytes. These findings indicate that the cell fate of the ependymal cells in the adult rodent spinal cord can be modulated by intervention. Future studies will clarify whether cell fate intervention in the ependymal cells can contribute to anatomical reconstruction and functional recovery in the adult mammalian damaged spinal cord.	P1-1-55 Tet3はNeuro2A細胞のニュローン分化を制御する Role of Tet3 protein in neuronal differentiation ○黒田貴雄1, 中澤孝哉2, 川端一範2, 水谷健一1,3 ○Takao Kuroda1, Takaya Nakazawa2, Kazunori Kawabata2, Ken-ichi Mizutani1,3 同志社大院・脳科学1, 同志社大・生命医・医生命システム2, 科学技術振興機構・さきがけ3 Grad Sch Brain Sci, Doshisha Univ1, Dept Med Life System, Fac Life Med Sci, Doshisha Univ.2, PRESTO, JST3 DNA methylation is crucial for gene silencing, cell differentiation, and development. Although the mechanisms of DNA methylation have been well studied, those of DNA demethylation have been elusive. It has been reported that DNA demethylaion of the Hes5 and GFAP promoters occurs during early neurogenesis and gliogenesis, respectively. Therefore, it is thought that DNA demethylation is an important event for neural development. Recent studies indicates that Tet family proteins (Tet1, Tet2, and Tet3) are able to catalyze the conversion of 5-methylcytosine of DNA to 5-hydroxymethylcytosine, suggesting a potential role for Tet proteins in DNA demethylation. We predicted that Tet family proteins could be involved in neural development. To examine this possibility, we investigated the role of Tet family proteins in neuronal differentiation of neuroblastoma Neuro2A cells. In Neuro2A cells, Tet3 transcripts were present at high copy numbers, Tet2 transcripts were about 4-fold less abundant than Tet3 but still well expressed, and Tet1 transcript levels were very low. When neuronal differentiation was induced by retinoic acid treatment, Tet3 but not Tet1 or Tet2 mRNA levels increased more than 2.5-fold after 5 days culture. We investigated the effects of Tet3 knockdown on neuronal differentiation and found that the induction of NeuN, neuronal marker gene, mRNA was significantly impaired in Tet3 knockdown cells. These results indicate that Tet3 plays an important role for neuronal differentiation.
P1-1-56 PC12の神経細胞分化に伴うカスパーゼ―8とカスパーゼ―9の活性化について Activation of Caspase-8 and Caspase-9 are required for PC12 cells differentiation ○茂木眞希雄1, 森田あや美1 ○Makio Mogi1, Ayami Morita1 愛知学院大学・薬学部・生体機能化学1 Dept Medicinal Biochem, Sch of Pharmacy, Aichi-Gakuin Univ, Nagoya, JAPAN1 Background: Although caspase activation is indispensable for the proper execution of apoptosis, little is known about other possible physiologic functions for this class of protease in addition to their well-defined role in apoptosis. Since nerve growth factor (NGF) is important regulators of rat pheochromoctyoma PC12 differentiation with neurite outgrowth, we sought to determine if NGF-induced differentiation of PC12 cells depends on the activity of the key apoptotic proteases, i.e., the caspase-8 and caspase-9 as initiator proteinase. Materials & method: PC12 cells were grown in DMEM supplemented with 10% horse serum and 10% fetal bovine serum. Assay for caspase-1, caspase-3, caspase-8, and caspase-9 utilized fluorescence (MCA)-substrate and/or Western blot using anti-caspase antibody. Neurite outgrowth was evaluated by IN cell analyzer. Cytochrome c (Cyt.c) released from mitochondria was determined by commercially available ELISA.Result: In addition to previous report on the activation of caspasse-1 and caspase-3 as effector proteinase, NGF induced potent activation of caspase-8, and caspase-9 in PC12 cells. Interestingly, although it is known that caspae-9 activation was associated with apoptosis and the potent release of Cyt.c, neither apoptosis nor release of Cyt.c could be detected in NGF-treated cells. Since peptide inhibition of caspase activity led to a potent reduction in neurite outgrowth in NGF-treated cells, we demonstrated the unique caspase cascade, caspase-1 to caspase-9, and caspase-3 to caspase-9 to occur, in this order. Conclusion: these results confirm a unique role for the caspase-8 and caspase-9 mediated signal cascade in the differentiation of PC12 cells.

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