耐药性颞叶癫痫大鼠海马组织环磷酸腺苷反应原件结合蛋白及磷酸化环磷酸腺苷反应原件结合蛋白表达量的变化

Expressions of cAMP response element binding protein and phosphorylated cAMP response element binding protein in the hippocampus of drug resistant temporal lobe epileptic rats

目的 建立杏仁核点燃耐药性颞叶癫痫大鼠模型，观察环磷酸腺苷反应原件结合蛋白（CREB）及磷酸化环磷酸腺苷反应原件结合蛋白（p-CREB）在大鼠海马组织中的表达变化，探讨其在耐药性颞叶癫痫形成中的作用。方法 将80只成年雄性SD大鼠按照完全随机数字表法分为对照组（n＝10）和模型制作组（n＝70）。模型制作组70只大鼠用于制作慢性杏仁核点燃模型，模型制作成功后用苯妥英钠及苯巴比妥进行耐药性癫痫模型的筛选，根据癫痫大鼠对苯妥英钠的反应性，筛选出耐药性癫痫大鼠及药物敏感癫痫大鼠，然后根据完全随机数字表法随机选择药物敏感组（n＝10）和耐药模型组（n＝10）进行实验，通过行为学、电生理学及病理学HE染色方法观察癫痫模型的特征，用免疫组织化学染色方法和蛋白质印迹法检测CREB及p-CREB的变化，观察正常对照组、药物敏感组及耐药模型组之间的不同。结果 正常对照组大鼠杏仁核脑电图频率为（8.700±1.494）Hz，药物敏感组为（14.700±1.159）Hz，耐药模型组为（19.800±1.686）Hz，3组间差异有统计学意义（F＝144.202，P＝0.000）。耐药模型组经免疫组织化学染色可见大量CREB、p-CREB阳性细胞的表达。与对照组蛋白表达水平相比（CREB 0.197±0.058、p-CREB 0.260±0.176），敏感组的蛋白表达水平（CREB 0.361±0.151、p-CREB 0.656±0.234）及耐药组的蛋白表达水平（CREB 0.591±0.150、p-CREB 1.077±0.400）升高，差异有统计学意义（F＝24.206，P＝0.000；F＝20.376，P＝0.000）。结论 耐药性癫痫大鼠海马中CREB及p-CREB表达均升高，提示其可能在耐药性癫痫发生发展机制中起重要作用。

Objective To establish drug resistant models of temporal lobe epilepsy induced by amygdala kindling, and to investigate the changes of cAMP response element binding protein （ CREB ） and phosphorylated cAMP response element binding protein （p-CREB） expression in the hippocampus tissues in order to explore their roles in drug resistant epileptogenesis. Methods Eighty adult male SD rats were randomly divided into control group （n=10） and model group （n =70）. The 70 rats were used to prepare the amygdaloid kindled model of epilepsy by chronic stimulation of amygaloid basal lateral nucleus. The successful kindled models were randomly selected as drug resistant epileptic group （ n = 10 ） and drug sensitive epileptic group （ n = 10 ） according to their response to the phenytoin and phenobarbital. On the basis of behavioral observation, electrophysiology, pathological HE staining, CREB and p-CREB expression changes, we verified the reliability of the models and explored the differences among the three groups above. The changes of CREB and p-CREB expression were detected by immunohistochemical method and Western blotting assay. Results In control group, the electroencephalogram （EEG） frequency was （8. 700± 1. 494） Hz; in drug sensitive epileptic group, the EEG frequency was （14. 700 ± 1. 159） Hz; in drug resistant epileptic group, the EEG frequency was （19. 800 ± 1. 686） Hz. The frequency differences among the three groups were statistically significant （ F = 144. 202, P = 0. 000）. By immunohistochemical staining, a large number of CREB and p-CREB positive cells were observed in drug resistant epileptic group. As compared with the control group （CREB 0. 197 ± 0. 058, p-CREB 0. 260 ±0. 176）, the expression levels of CREB and p-CREB were increased in drug sensitive epileptic group （CREB 0. 361 ±0. 151, p-CREB 0. 656±0. 234） and in drug resistant epileptic group （ CREB 0. 591± 0. 150, p-CREB l. 077±0. 400）. The difference among the three groups had statistical significance （ F = 24. 206, 20. 376, both P 〈 0. 01 ） . Conclusions The expressions of CREB and p-CREB were significantly increased in drug resistant epileptic rats. These findings indicate that the expressions of CREB and p-CREB may play certain roles in the drug- resistant epileptogenesis.