Heck S, Enz R, Richter-Landsberg C, Blohm D H
University of Bremen, Department of Biotechnology and Molecular Genetics, Germany.
Brain Res Dev Brain Res. 1997 Feb 20;98(2):211-20. doi: 10.1016/s0165-3806(96)00181-2.
The mouse EC cell line P19, differentiating in vitro into neural cell types under the influence of retinoic acid, represents a well established model system for neurogenesis. In this system the expression of the alpha (alpha 1-alpha 3) and beta subunits of the inhibitory glycine receptor (GlyR) and of gephyrin as well as their mRNA splice variants was analyzed by RT-PCR and by immunocytochemistry. In the course of neuronal differentiation of P19 cells mRNA of GlyR beta is constitutively expressed, GlyR alpha 1 and alpha 2 are induced and GlyR alpha 3 was not detected. From the three gephyrin transcripts known to be differently spliced in the C3/C4 cassette region, the C3 transcript was found at all stages while the C4 transcript was not detectable. The insert-free form was measurable in P19 cells only 3-4 days post induction by retinoic acid. In addition a GlyR beta splice variant and a fourth gephyrin transcript were detected. Primary glial cells do not contain significant amounts of GlyR alpha subunits while in primary neuronal cells transcripts of GlyR alpha 2 were found as well as the mRNA of the GlyR beta subunit and of gephyrin. PC12 cells do not express glycine receptor genes but do express gephyrin. Immunocytochemistry confirmed the constitutive expression of gephyrin at the protein level, whereas GlyR antigens could only be detected in islets of the 'P19 neurons'. In conclusion, P19 and primary neuronal cells but not PC12 cells express the transcripts of glycine receptor components, necessary to generate functional receptors.
小鼠胚胎癌细胞系P19在视黄酸的影响下可在体外分化为神经细胞类型，是一个成熟的神经发生模型系统。在该系统中，通过逆转录聚合酶链反应（RT-PCR）和免疫细胞化学分析了抑制性甘氨酸受体（GlyR）的α（α1-α3）和β亚基以及桥连蛋白的表达及其mRNA剪接变体。在P19细胞神经元分化过程中，GlyRβ的mRNA持续表达，GlyRα1和α2被诱导表达，未检测到GlyRα3。在已知在C3/C4盒区域存在不同剪接的三种桥连蛋白转录本中，C3转录本在所有阶段均被发现，而C4转录本未被检测到。无插入形式仅在视黄酸诱导后3-4天在P19细胞中可检测到。此外，还检测到一种GlyRβ剪接变体和第四种桥连蛋白转录本。原代神经胶质细胞不含大量的GlyRα亚基，而在原代神经元细胞中发现了GlyRα2的转录本以及GlyRβ亚基和桥连蛋白的mRNA。PC12细胞不表达甘氨酸受体基因，但表达桥连蛋白。免疫细胞化学证实了桥连蛋白在蛋白质水平的持续表达，而GlyR抗原仅在“P19神经元”的胰岛中可检测到。总之，P19细胞和原代神经元细胞而非PC12细胞表达产生功能性受体所需的甘氨酸受体成分的转录本。
