Qu Y, Vadivelu S, Choi L, Liu S, Lu A, Lewis B, Girgis R, Lee C S, Snider B J, Gottlieb D I, McDonald J W
Department of Neurology, Washington University School of Medicine, Box 8111, St, Louis, MO 63110, USA.
Exp Neurol. 2003 Nov;184(1):326-36. doi: 10.1016/j.expneurol.2003.07.001.
We determined whether embryonic stem (ES) cells could provide a model system for examining neuronal death mediated by glutamate receptors. Although limited evidence indicates that normal neurons can be derived from mouse ES cells, there have been no studies examining pathophysiological responses in mouse ES cell systems. Mouse ES cells, induced down a neural lineage by retinoic acid (RA), were found to have enhanced long-term survival when plated onto a layer of cultured mouse cortical glial cells. In these conditions, the ES cells differentiated into neural cells that appeared normal morphologically and displayed normal features of immunoreactivity when tested for neuron-specific elements. Varying the culture medium generated cultures of mixed neuronal/glial cells or enriched in oligodendrocytes. These cultures were viable for at least four weeks. Real-time PCR analysis of N-methyl-D-aspartate (NMDA) receptor subunits revealed an appropriate age-in-vitro dependent pattern of expression. Neurons derived from ES cells were vulnerable to death induced by a 24-h exposure to the selective glutamate receptor agonists NMDA, kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). This vulnerability to agonist-induced death increased with age in vitro, and related closely to expression of receptor subunits, as it does in cultured primary neurons. Experiments with selective receptor antagonists showed that glutamate receptors mediated the NMDA- and kainate-induced death. Neuronal differentiated ES cells therefore exhibited an excitotoxic response resembling that displayed by central nervous system (CNS) neurons. Thus, ES cells, which are very amenable to genetic manipulation, provide a valid system for studying glutamate receptor-mediated toxicity at the molecular level.
我们确定胚胎干细胞（ES细胞）是否能为研究谷氨酸受体介导的神经元死亡提供一个模型系统。尽管有限的证据表明正常神经元可从小鼠ES细胞分化而来，但尚无关于小鼠ES细胞系统病理生理反应的研究。发现经视黄酸（RA）诱导向神经谱系分化的小鼠ES细胞，接种到一层培养的小鼠皮质神经胶质细胞上时，其长期存活率有所提高。在这些条件下，ES细胞分化为形态上看似正常的神经细胞，在检测神经元特异性成分时显示出正常的免疫反应特征。改变培养基可生成混合神经元/神经胶质细胞培养物或富含少突胶质细胞的培养物。这些培养物至少存活了四周。对N-甲基-D-天冬氨酸（NMDA）受体亚基的实时PCR分析显示出合适的体外年龄依赖性表达模式。源自ES细胞的神经元易受24小时暴露于选择性谷氨酸受体激动剂NMDA、海人藻酸和α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）诱导的死亡影响。这种对激动剂诱导死亡的易感性随体外年龄增加，且与受体亚基的表达密切相关，如同在培养的原代神经元中一样。用选择性受体拮抗剂进行的实验表明，谷氨酸受体介导了NMDA和海人藻酸诱导的死亡。因此，神经元分化的ES细胞表现出类似于中枢神经系统（CNS）神经元的兴奋性毒性反应。所以，非常适合进行基因操作的ES细胞为在分子水平研究谷氨酸受体介导的毒性提供了一个有效的系统。
