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目的:选择2种神经毒物1-甲基-4-苯基吡啶离子(MPP+)和谷氨酸在体外诱导多巴胺能神经元退行性损伤,并检测士的宁对此细胞的药理毒理作用,及对其退行性损伤的协同作用或拮抗作用。方法:采用孕14 d小鼠,取出胎鼠中脑,分离出多巴胺能神经元,调整细胞密度7.5×105/mL,于5%CO2,37℃,100%湿度条件培养,于体外培养的第10天分别加入士的宁0.1,1,5,10μmol.L-1,MPP+10μmol.L-1,谷氨酸500μmol.L-1作用48 h,第12天染色。选取细胞数,神经元树突长度和数量作为观察指标,并做出分析。结果:士的宁对未损伤的多巴胺能神经元在48 h培养过程中无毒性影响。在10μmol.L-1士的宁作用下,多巴胺能神经元数量明显高于对照组(P<0.05);在5～10μmol.L-1士的宁作用下,树突数量与对照组相比也有明显提高(P<0.05)。士的宁(10μmol.L-1)和MPP+共同培养48 h后,细胞计数明显高于MPP+单独培养组(P<0.05);这种保护作用更为明显的表现在细胞形态学变化方面,在10μmol.L-1的士的宁剂量下,神经树突长度明显高于MPP+单独培养组(P<0.05);神经树突的数量在5μmol.L-1士的宁作用下与MPP+单独培养组相比有明显增加(P<0.05)。在对谷氨酸(500μmol.L-1)诱导的神经元损伤模型中,与谷氨酸组相比,10μmol.L-1的士的宁对所有的观察指标的数值都有递增影响(P<0.05)。结论:首次证实士的宁具有可在体外刺激中脑分离出的多巴胺能神经元的生长,并且对此神经元退行性损伤有保护作用。
AIM: To investigate the neurotoxic effect of strychnine on the degeneration of dopaminergic neurons induced by MPP + and glutamate in vitro. And its synergistic effect of degenerative injury or antagonism. Methods: The pregnant mice were taken out from the midbrain for 14 days. Dopaminergic neurons were isolated and the cell density was adjusted to 7.5 × 105 / mL. The cells were cultured in 5% CO2, 37 ℃ and 100% humidity conditions. 10 days were added strychnine 0.1, 1, 5, 10μmol.L-1, MPP + 10μmol.L-1, glutamate 500μmol.L-1 for 48 h, the first 12 days of staining. Select the number of cells, the length and number of neuronal dendrites as indicators of observation, and make an analysis. Results: Strychnine had no toxic effect on undamaged dopaminergic neurons during 48 h of culture. The number of dopaminergic neurons was significantly higher in the group of 10μmol.L-1 strychnine than that in the control group (P <0.05). Under the action of 5 ~ 10μmol.L-1 strychnine, the number of dendrites was Also significantly improved (P <0.05). The cell counts of strychnine (10μmol.L-1) and MPP + co-cultured for 48 h were significantly higher than those of MPP + alone group (P <0.05). This protective effect was more obvious in terms of cell morphological changes, In the dose of 10 μmol·L-1, the length of nerve dendrites was significantly higher than that of MPP + alone group (P <0.05). The number of neural dendrites in the group of MPP + alone cultured with 5 μmol·L-1 strychnine Compared with a significant increase (P <0.05). In the model of neuronal injury induced by glutamate (500μmol.L-1), all doses of 10μmol.L-1 strychnine had an increasing effect on all observed values compared with the glutamate group (P <0.05). Conclusion: It is the first time that strychnine can stimulate the growth of dopaminergic neurons isolated from midbrain in vitro and protect neurons from degeneration.