摘要
目的:观察痫性发作对体外培养海马脑片的损伤作用,研究细胞磷酸化cAMP反应元件结合蛋白(Phosphorylated cAMP response element-binding protein,pCREB)对损伤海马神经元的作用。方法:以培养11 d的海马脑片为研究对象,随机分为3组:(1)正常对照组:正常海马脑片培养液培养至14 d。(2)痫性发作模型组:正常海马脑片培养液培养至14 d,更换为含匹罗卡品(0.5 mmol/L)的完全培养液作用1 h后终止培养。(3)抗pCREB抗体干预组:用含pCREB抗体的海马脑片培养液(pCREB抗体终浓度为5μg/ml)预培养3 d,换为含匹罗卡品(0.5 mmol/L)的完全培养液作用1 h后终止培养。采用TUNEL原位末端标记法检测各组培养海马脑片凋亡细胞;免疫组化染色观察海马脑片神经元内pCREB蛋白的表达变化。结果:(1)痫性发作模型组:海马脑片CA1区发生凋亡的细胞(95.44±14.30)显著多于对照组(41.30±9.30)(P<0.05);pCREB表达(201.36±19.31)较对照组(75.12±13.71)显著升高(P<0.05)。(2)抗pCREB抗体干预组:海马脑片CA1区发生凋亡的细胞(140.88±18.32)较痫性发作模型组显著增多(P<0.05);pCREB表达(172.60±17.68)较对照组显著升高、较模型组显著降低(P<0.05)。结论:(1)痫性发作可以增加体外培养海马脑片神经细胞凋亡发生,加重神经元损伤。(2)痫性发作后海马脑片神经细胞CREB的磷酸化增强,阻断CREB的磷酸化可导致神经细胞凋亡加重。
Objective：To study the effect of seizure-like activity and pCREB on the neurons in cultured hippocampal slices in vitro.Methods： Hippocampal slice cultured for 11 days were randomly divided into 3 groups：（1）control group：cultured hippocampal slices were exposed to regular media to 14 d.（2）seizure-like activity model group：cultured hippocampal slices were exposed to regular media to 14 d,then returned to regular media with pilocarpine（final concentration：5 mmol/L） for another 1 h.（3）anti-pCREB antibody treated group：cultured hippocampal slices were pre-cultured with regular media with anti-pCREB antibody（final concentration：5 μg/ml） to 14 d,then returned to regular media with pilocarpine（final concentration：5 mmol/L） for another 1 h.The apoptosis neurons of cultured hippocampal slices was detected by terminal deoxynucleotidyl dUTP nick end-labeling（TUNEL）staining in situ.Expression of pCREB protein in the neurons of cultured hippocampal slices was detected by immunocytochemistry.Results：（1）Seizure-like activity model group：the number of apoptotic neurons in CA1 of cultured hippocampal slices was（95.44±14.30） significantly increased compared with that of（41.30±9.30） in the control group（P0.05）.The expressions of pCREB in neurons in CA1 of cultured hippocampal slices were（201.36±19.31）,remarkably up-regulated compared with those of（75.12±13.71） in the control group（P0.05）.（2）Anti-pCREB antibody treated group：the number with of apoptotic of neurons in CA1 of cultured hippocampal slices was（140.88±18.32） significantly increased compared with that in the seizure-like activity model group（P0.05）.The expressions of pCREB were（172.60±17.68） remarkably up-regulated compared with those in the control group（P0.05）.Conclusion：（1）Seizure-like activity can induce neuronal apoptosis and aggravate neuronal injury in cultured hippocampal slices.（2）Seizure-like activity can up-regulate the level of CREB phosphorylati
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2012年第4期289-293,共5页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:30672217)
关键词
PCREB
海马脑片
痫性发作
凋亡
pCREB
hippocampal slice
seizure-like activity
apoptosis
