培养大鼠海马脑片模拟痫性发作后pCREB在细胞凋亡中的作用的初步研究

Effect of pCREB on the apotosis of neurons in cultured hippocampal slices after seizure like activity

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5 μg/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。

Abstract:

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(P<0.05).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(P<0.05).(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 (P<0.05).The expressions of pCREB were （172.60±17.68） remarkably up-regulated compared with those in the control group (P<0.05). 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 phosphorylation to cultured hippocampal slice,and blocking CREB phosphorylation may lead to neuronal cell apoptosis accentuation.