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在急性、慢性神经退行性疾病和炎症引发的神经系统疾病的发病机制中,兴奋性毒性可能是造成后期神经元死亡的共同途径.小脑颗粒神经元谷氨酸兴奋性毒性模型是研究上述过程的重要实验手段,该模型的稳定性和可重复性是开展相关研究的重要基础.然而,文献报道的建模方法条件各异,说法不一,很难适从.本工作针对小脑颗粒神经元谷氨酸兴奋性毒性模型建立的关键环节,包括小脑颗粒神经元的培养、兴奋性毒性刺激条件的确定,毒性标志性指标的表征,分别进行了比较和优化,从培养皿的包被、神经元消化、兴奋性刺激的溶液介质选择、神经元刺激的最佳时间及谷氨酸的最佳刺激浓度等方面分别给出了优化条件.通过特征性钙离子曲线、NMDA受体特异性抑制剂MK-801的干预作用以及c-fos基因转录水平的动力学变化等指标,确认了毒性模型的成功建立.本工作不仅对建立小脑颗粒神经元谷氨酸兴奋性毒性模型的实验室具有重要参考意义,而且,其针对不同条件分析比较的结果及优化原则,对其他神经毒性模型的建立也具有普遍参考意义.
Excitotoxicity may be the common pathway leading to late neuronal death in the pathogenesis of acute, chronic neurodegenerative diseases and inflammatory-induced neurological diseases.Glutamate excitotoxicity model of cerebellar granule neurons is important to study the above process Experimental methods, the stability and repeatability of the model is an important basis for the relevant research.However, the modeling methods reported in the literature have different conditions, different arguments, it is difficult to adapt to.This work aimed at cerebellar granule neurons glutamic acid Excitotoxicity models were established, including the culture of cerebellar granule neurons, the determination of excitotoxicity stimuli, the characterization of toxicity markers, and the comparison and optimization of the excitotoxicity models respectively. From the petri dish coating, neuron digestion, Excitatory stimulus solution medium selection, the best time for neuron stimulation and glutamate optimum stimulation concentration were given optimized conditions.Through the characteristic calcium curve, NMDA receptor specific inhibitor MK-801 And the dynamic changes of c-fos gene transcription level, confirming the successful establishment of toxicity model.This work not only The establishment of cerebellar granule glutamate excitotoxicity model laboratory has an important reference value, and its analysis and comparison of results and optimization principles for different conditions, also has a universal reference for the establishment of other neurotoxic model.