锰对皮层神经元胞浆游离钙离子浓度的影响

Effect of Manganese on cytosolic free calcium concentration in cortical neurons

目的探讨皮层神经元胞浆游离钙离子浓度在锰(Mn)神经毒性作用中的变化。方法分离新生Wistar乳大鼠的皮层神经细胞进行体外培养,细胞生长至最佳状态时,予以分组处理。实验分染锰组和未处理组,染锰组分别与含低、中、高浓度氯化锰(MnCl_2·4H_2O)的培养液共培养,氯化锰的浓度分别为0．2、0．6、1．0mmol/L。未处理组则为正常培养的皮层神经细胞。各组处理24h后终止培养,钙离子荧光探针Fluo-3/AM标记后上激光扫描共聚焦显微镜检测荧光强度,以表示游离钙离子浓度,并用图像分析技术进行处理。结果氯化锰处理后引起皮层神经元胞浆游离钙离子不同程度的超载,中、高浓度锰组神经元的胞浆游离钙离子浓度明显高于低浓度锰组及未处理组,细胞钙离子荧光像素荧光强度增高且呈剂量依赖性,中、高浓度锰组分别为(131．1±16．7)、(183．2±22．9)；低浓度锰组为(84．6±10．2),差异有统计学意义(P＜0．05)。结论钙离子超载与锰的神经毒性有关,且钙离子超载与锰浓度呈剂量-反应关系。

Objective To investigate the change of free Ca^2＋ in cytoplasma in the neurotoxicity of the manganese（Mn）. Methods The cortical neurons were separated from the neonatal Wistar rats and cultured in vitre.The neurons were grouped as the Mn-treated groups and the untreated group. The neurons in the Mn-added groups were incubated in the culture media containing lower, medium and high dosage manganese chloride（ MnCl2·4H2O） with the concentration at 0.2,0.6, 1.0 mmol/L respectively. Meanwhile, neurons in control were cultured in the normalculture media. All treatments stopped 24 h later. Neurons were labeled Ca^2＋ sensitive prober, Fluo-3/ AM.The fluorescence intensity of Fluo-3 combined with Ca^2＋ was examined by LSCM（Laser scanning confocal microsope）and was treated by the picture analysis technique.The intensity was equal to the free Ca^2＋ concentrations in cytoplasma of neurons. Results MnCl2 can induce free Ca^2 ＋ overloaded in cytoplasma of neurons, but the increasing degree varied in MnCl2 dosage. Cytoplasma Ca^2 ＋ concentration in the mederate dosage The moderate dosage MnCl2 group and the high dosage MnCl2 group were significantly higher than that in the lower dosage MnCl2 group and the control group（ P 〈 0.05）. Conclusion The Ca^2＋ overload is involved in the neurotoxicity of manganese, and a dosage respone relationship is found between the manganese chloride dose and Ca^2 ＋ overload in cortical neurons.