Watanabe E, Hosokawa H, Kobayashi H, Murakami F
Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Japan.
Eur J Neurosci. 1994 Aug 1;6(8):1354-61. doi: 10.1111/j.1460-9568.1994.tb00326.x.
Accumulating evidence suggests that an inhibitory influence of the environment on growth cones plays a crucial role in development and regeneration of neuronal projections. Oligodendrocyte-associated neurite growth inhibiting substance is one of the most extensively studied molecules. Molecular biological studies, however, remain slow in progress. Although finding clonal cells that express such factors would facilitate the analysis of inhibitory influences on neurite growth, few cell lines have been reported to express neurite growth inhibitor. We therefore investigated the possibility of a clonal glial cell line to differentiate and express inhibitory or non-permissive features for neurite outgrowth in culture. We chose the C6 glioblastoma cell line and examined neurite extension from chick dorsal root ganglion (DRG) explants. Neurites from embryonic day 9 DRG extensively grew on C6 cells that were cultured at low cell density, while they failed to grow on C6 cells cultured at high density, even in the presence of nerve growth factor in high concentrations. Membrane extract from high density C6 cells, when used as culture substratum, was less permissive for neurite outgrowth compared to extract from low density cells. Treatment of the membrane extract derived from high density C6 cells with trypsin made it less non-permissive for neurite growth. These results suggest that C6 cells are induced to express a non-permissive property for neurite outgrowth by culturing them at high density.
越来越多的证据表明，环境对生长锥的抑制作用在神经元突起的发育和再生中起着关键作用。少突胶质细胞相关神经突生长抑制物质是研究最为广泛的分子之一。然而，分子生物学研究进展仍然缓慢。虽然找到表达此类因子的克隆细胞将有助于分析对神经突生长的抑制作用，但据报道很少有细胞系能表达神经突生长抑制剂。因此，我们研究了一种克隆神经胶质细胞系在培养中分化并表达对神经突生长的抑制或非允许特性的可能性。我们选择了C6胶质母细胞瘤细胞系，并检测了鸡背根神经节（DRG）外植体的神经突延伸情况。来自胚胎第9天DRG的神经突在低密度培养的C6细胞上广泛生长，而在高密度培养的C6细胞上则无法生长，即使存在高浓度的神经生长因子也是如此。与低密度细胞提取物相比，高密度C6细胞的膜提取物作为培养底物时，对神经突生长的允许性较低。用胰蛋白酶处理高密度C6细胞的膜提取物后，其对神经突生长的非允许性降低。这些结果表明，通过高密度培养C6细胞可诱导其表达对神经突生长的非允许特性。
