【摘要】 目的阐明癫痫模型大鼠海马神经元凋亡中caspase-3的作用机制。方法以TUNEL法检测KA致痫大鼠海马神经元凋亡情况,以WesternBlot方法检测其海马神经元中eIF2α的表达变化。取模型鼠海马组织制备cDNA文库,以caspase-3的大小亚单位构建酵母三杂交诱饵载体,并进行酵母三杂交筛库实验。结果在癫痫发作后12h海马出现凋亡神经元,72h达高峰;发作后24h海马神经元出现eIF2α被caspase-3酶切的片段,逐渐增加至72h。成功制备癫痫模型大鼠海马组织cDNA文库,构建了caspase3酵母三杂交诱饵载体,验证了caspase3与eIF2α之间的相互作用,并经筛库获得caspase3的新底物PIAS1。结论在癫痫模型大鼠海马凋亡神经元中eIF2α被caspase3酶切。酵母三杂交技术可用于筛库寻找caspase3下游底物,从癫痫大鼠海马中获得caspase3的新底物PIAS1,为进一步研究在癫痫发作致神经元损伤中caspase3的作用建立了基础。更多还原

【Abstract】 Objective To explore the role of caspase-3 in hippocampal neuronal apoptosis of epileptic model rat. Methods TUNEL staining and Western Blot were used to detect the apoptotic neurons and the changes of eIF2α expression respectively in hippocampi of the KA-induced epileptic model rats. Construction of the yeast hybrid cDNA library with hippocampi of epileptic model rats was performed.The bait vector in yeast three-hybrid system for caspase-3 was constructed and used to screen the library. Results The apoptotic rate of neurons increased since 12 h and reached the peak at 72 h after epileptic discharge. The cleavage of eIF2α by caspase-3 was detected in hippocampal neurons of epileptic rat. Construction of the bait vector for caspase-3 and the cDNA library was accomplished . PIAS1 was obtained as a novel substrate of caspase-3 through screening the library. Conclussions The cleavage of eIF2α by caspase-3 occured in the epileptic hippocampal neuronal apoptosis. The yeast three-hybrid systom is available for screening the substrates of caspase-3. PIAS1 may play certain role in neuronal apoptosis after epileptic discharge.更多还原