Turetsky D M, Huettner J E, Gottlieb D I, Goldberg M P, Choi D W
Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Neurobiol. 1993 Sep;24(9):1157-69. doi: 10.1002/neu.480240904.
While primary neuronal cell cultures have been used to investigate excitotoxicity, development of cell lines exhibiting glutamate receptor-mediated death is desirable. P19 mouse embryonal carcinoma cells, exposed to retinoic acid and plated onto a layer of cultured mouse cortical glial cells, differentiated into neuron-like elements immunoreactive for neurofilaments and neuron-specific enolase. Whole-cell recordings revealed inward currents in response to extracellular application of either NMDA or kainate. The NMDA-induced currents exhibited a voltage-dependent blockade by magnesium, required glycine for maximal activation, and were blocked by the NMDA antagonist dizocilpine. Kainate-induced currents were blocked by the AMPA/kainate receptor antagonist CNQX. Exposure to 500 microM NMDA for 24 h destroyed most P19 cells (EC50 approximately 70 microM); death was prevented by dizocilpine or D-APV. Exposure to 500 microM kainate also resulted in widespread death reduced by CNQX. Thus differentiated P19 cells exhibited both excitatory amino acid responses and vulnerability to excitotoxicity, characteristic of CNS neurons. These cells may provide a genetically open system useful for studying glutamate receptor-mediated phenomena at a molecular level.
虽然原代神经元细胞培养已被用于研究兴奋性毒性，但开发表现出谷氨酸受体介导死亡的细胞系是很有必要的。将P19小鼠胚胎癌细胞暴露于视黄酸，然后接种到一层培养的小鼠皮质神经胶质细胞上，这些细胞分化为对神经丝和神经元特异性烯醇化酶有免疫反应的神经元样细胞。全细胞记录显示，细胞外施加NMDA或海人酸后会出现内向电流。NMDA诱导的电流表现出镁离子对其电压依赖性的阻断作用，需要甘氨酸才能实现最大激活，并且被NMDA拮抗剂地卓西平阻断。海人酸诱导的电流被AMPA/海人酸受体拮抗剂CNQX阻断。将细胞暴露于500μM NMDA 24小时会破坏大多数P19细胞（半数有效浓度约为70μM）；地卓西平或D-APV可防止细胞死亡。暴露于500μM海人酸也会导致广泛的细胞死亡，而CNQX可使其减少。因此，分化的P19细胞表现出兴奋性氨基酸反应以及对兴奋性毒性的易感性，这是中枢神经系统神经元的特征。这些细胞可能提供一个遗传开放的系统，有助于在分子水平上研究谷氨酸受体介导的现象。
