海马神经元无镁致痫性损伤对CREB磷酸化水平的影响

Effects of magnesium-free epileptiform damage on the CREB phosphorylation in hippocampal neurons

摘要:

目的：观察原代培养的海马神经元经无镁人工脑脊液诱发癫痫样放电后，cAMP反应元件结合蛋白（cAMP response-element binding protein，CREB）磷酸化表达水平的变化。方法：将所培养的神经元随机分成癫痫组和对照组，癫痫组中的神经元培养至第10天时，经无镁人工脑脊液（artificial cerebrospinal fluid，ACSF）处理3 h建立癫痫样放电模型，对照组中的神经元培养至第10天时，使用正常人工脑脊液处理3 h。使用Western blot检测p-CREB和β-actin的表达，免疫荧光双重标记磷酸化CREB（p-CREB）和神经元核抗原（neuronal nuclei，NeuN）的表达。结果：Western blot显示p-CREB水平在癫痫样放电后2 h即较对照组增强且在6 h达到高峰，在12 h和24 h都维持在增强水平，差异皆有统计学意义（P＜0.01）。取癫痫样放电后6 h的神经元行免疫荧光检测，其荧光强度绝对值较对照组明显增强，差异有统计学意义（P＜0.01）。结论：p-CREB水平在海马神经元无镁致痫性损伤后增强，可能在癫痫样放电中起到重要作用。

Abstract:

Objective：To observe the expression of phosphorylated cAMP response-element binding protein（p-CREB） after epilepti－form discharge of hippocampal neurons. Methods：All cultured neurons were divided into two groups：epilepsy group：on the 10th d after the incubation，maintenance medium was replaced with magnesium-free artificial cerebrospinal fluid for 3 h；control group：on the 10th d after the incubation，maintenance medium was replaced with normal artificial cerebrospinal fluid for 3 h. Changes in ex－pressions of p-CREB and β-actin were observed by Western blot. Changes in p-CREB and neuronal nuclei（NeuN） expression was investigated by double labeled immunofluorescence. Results：Results of Western blot demonstrated that p-CREB expression was in－creased at 2 h after epileptiform discharges in epilepsy group than in control group，peaked at 6 h and maintained increased at 12 h and 24 h. Compared with that in control group，expression of p-CREB was significantly increased in epilepsy group at each time point（P＜0.01）. Immunofluorescence intensity of p-CREB was also increased significantly in epilepsy group than in control group（P＜0.01）. Conclusions：p-CREB expression in epileptiform damage neurons is significantly enhanced，which probably closely relate with epilep－tiform discharge.