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目的利用无镁细胞外液诱导原代培养大鼠海马神经元癫痫放电模型来检测电压门控性钠通道Nav1.1和钙调蛋白的表达与定位。方法采用新生24h内Wistar大鼠,取海马进行神经元原代培养。体外培养至10d,无镁细胞外液处理(实验组)或正常细胞外液(对照组)处理神经元3h后,一部分细胞应用全细胞膜片钳技术记录神经元的放电情况并应用免疫印迹法检测电压门控性钠通道Nav1.1和钙调蛋白的蛋白表达;另一部分细胞用正常培养液继续培养12h后分别通过免疫印迹法和免疫荧光双标法检测Nav1.1和钙调蛋白的蛋白表达与共定位。结果无镁细胞外液处理3h后的神经元存在自发的“癫痫样”放电,而与正常细胞外液处理组相比,Nav1.1和钙调蛋白表达不变;无镁处理3h正常培养液再继续培养12h后,与正常细胞外液处理组相比,神经元Nav1.1表达上调,而钙调蛋白表达不变。另外,与正常细胞外液处理组相比,Nav1.1和钙调蛋白共定位的阳性细胞数增加。结论 Nav1.1和钙调蛋白的表达与定位变化可能与无镁诱导体外培养大鼠海马神经元自发异常放电的基础病理机制相关。
Objective To detect the expression and localization of voltage-gated sodium channel Nav1.1 and calmodulin by inducing epileptiform discharge in primary cultured rat hippocampal neurons by using magnesium-free extracellular fluid. Methods Newborn Wistar rats were used for 24 hours. Hippocampal neurons were cultured in primary culture. After cultured in vitro for 10 days, the neurons were treated with magnesium-free extracellular fluid (experimental group) or normal extracellular fluid (control group) for 3 h. Part of the cells were subjected to whole-cell patch clamp recording of neuronal discharge and Western blotting Voltage-gated sodium channels Nav1.1 and calmodulin protein expression; the other part of the cells cultured with normal medium for 12h after incubation by Western blot and immunofluorescence double-labeled method to detect Nav1.1 and calmodulin protein expression With co-location. Results Neurons in spontaneous “epileptiform” after 3h treatment without magnesium extracellular fluid had no change in expression of Nav1.1 and calmodulin compared with those in normal extracellular fluid group. Normal magnesium-free treatment for 3h After cultured for 12 hours, the expression of Nav1.1 in neurons was up-regulated but the expression of calmodulin was unchanged in normal extracellular fluid-treated group. In addition, co-localization of Nav1.1 and calmodulin increased the number of positive cells compared to the normal extracellular fluid-treated group. Conclusion The changes of expression and localization of Nav1.1 and calmodulin may be related to the basic pathological mechanism of spontaneous abnormal discharge of hippocampal neurons induced by magnesium in vitro.