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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钙通道Ca v1.2和钙调蛋白激酶Ⅱ的表达变化。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12d,无镁细胞外液处理一部分神经元3h后,应用全细胞膜片钳技术记录神经元的放电情况以及免疫印迹法检测电压门控性钙通道Ca v 1.2和钙调蛋白激酶Ⅱ的蛋白表达。无镁细胞外液处理另一部分细胞12h后检测Ca v1.2和钙调蛋白激酶Ⅱ的蛋白表达。结果在无镁细胞外液处理3h后,神经元存在自发的“癫痫样”放电,而神经元Ca v1.2和磷酸化钙调蛋白激酶Ⅱ表达不变;无镁诱导12h后,神经元电压门控性钙通道Ca v1.2表达下调,而磷酸化钙调蛋白激酶Ⅱ表达上调。结论电压门控性钙通道Ca v1.2和钙调蛋白激酶Ⅱ的表达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 changes of voltage-gated calcium channel Ca v1.2 and calmodulin-1 in primary cultured rat hippocampal neurons induced by 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 12 days, some neurons were treated with magnesium-free extracellular fluid for 3 hours. Whole-cell patch-clamp technique was used to record the discharge of neurons and Western blotting was used to detect the expression of voltage-gated Ca v 1.2 and calmodulin-Ⅱ Protein. The other part of the cells were treated with magnesium-free extracellular solution for 12h and then detected the protein expression of Ca v1.2 and calmodulin-2. Results After 3h treatment with magnesium-free extracellular fluid, spontaneous epileptiform discharges were found in neurons, whereas expression of Ca v1.2 and phosphorylated calmodulin-Ⅱ did not change. After 12h of magnesium-free induction, The voltage-gated calcium channel Ca v1.2 expression was down-regulated, while phosphorylated calmodulin-Ⅱ was up-regulated. Conclusions The changes of voltage-gated calcium channel Ca v1.2 and calmodulin-1 may be related to the basic pathological mechanism of spontaneous abnormal discharge of hippocampal neurons induced by magnesium in vitro.