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目的探讨在谷氨酸毒性作用时星形胶质细胞是否释放作为三羧酸循环中重要介质的丙酮酸来保护神经元,以及可能的机理。方法本实验第一步采用离体培养星形胶质细胞,加入浓度达到神经元毒性作用的谷氨酸,用分光光度计测量培养液中的丙酮酸含量。设立两组,一组为相同浓度谷氨酸作用下,不同时间段的丙酮酸浓度测定。另一组为在梯度浓度的谷氨酸作用下,同一时间段的丙酮酸浓度测定。以不加谷氨酸的培养液作为对照。第二步离体培养神经元细胞,加入具有神经元毒性的谷氨酸,并同时加入丙酮酸,观察神经元的生存情况。第三步在神经元培养液中加入谷胱苷肽合成抑制剂BSO,观察神经元存活情况。结果①加入谷氨酸后星形胶质细胞培养液中的丙酮酸浓度增加大于对照组,且丙酮酸浓度与谷氨酸的浓度有关。②丙酮酸加入已加有谷氨酸的神经元培液,神经元死亡数显著减少。③单独用低浓度BSO及单独用10mM丙酮酸作用5小时都没有使神经元死亡量增加,但当两者同时加入培养液中,神经元的死亡量显著上升。结论①体外实验中谷氨酸可促进星形胶质细胞释放丙酮酸,这可能与其保护神经元有关。②丙酮酸能抵制谷氨酸的神经毒性作用。③丙酮酸对谷氨酸神经毒性的保护作用需要谷胱甘肽(GSH)的参加,因而认为丙酮酸保护神经元的机理与增加细胞能量代谢有关。
OBJECTIVE: To investigate whether astrocytes release pyruvate, an important mediator of tricarboxylic acid cycle, during neurotoxicity of glutamate to protect neurons and its possible mechanism. Methods The first step of this experiment using astrocytes cultured in vitro, adding concentration of glutamic acid to achieve neurotoxic effects, using a spectrophotometer to measure the content of pyruvate in the culture medium. Two groups were set up, one set of pyruvate concentration for different time periods under the same concentration of glutamic acid. In the other group, the concentration of pyruvate in the same period of time was measured under the action of glutamic acid with a gradient concentration. With glutamate-free medium as a control. The second step in vitro cultured neuronal cells, adding neurotoxicity of glutamate, and while adding pyruvate to observe the survival of neurons. The third step in the neuron culture medium adding glutathione synthesis inhibitor BSO, observe the survival of neurons. Results ① The concentration of pyruvate in astrocyte culture medium increased more than that of control group after adding glutamate, and the concentration of pyruvate was related to the concentration of glutamate. ② pyruvate added glutamate-added neuron culture, neuronal death significantly reduced. (3) Neuronal deaths were not increased by low concentrations of BSO alone and 5 hours of treatment with 10mM pyruvate alone. However, when both of them were added to the culture medium at the same time, the death rate of neurons increased significantly. Conclusion ① Glutamic acid can promote pyruvate release from astrocytes in vitro, which may be related to the protection of neurons. ② pyruvate can resist the neurotoxic effect of glutamate. (3) Pyruvate needs glutathione (GSH) to protect against glutamate neurotoxicity. Therefore, the mechanism by which pyruvate protects neurons is related to increased cellular energy metabolism.