Caspase-3在癫痫模型大鼠海马神经细胞凋亡中作用的研究

Role of caspase-3 in the course of the neuronal apoptosis of hippocampus of epileptic model rat

目的对癫痫模型海马神经细胞凋亡中caspase-3的作用机制进行实验研究。方法以TUNEL法检测KA致痫大鼠海马神经细胞凋亡情况,以Western blot法检测其中eIF2α的表达变化。取模型鼠海马组织构建cDNA文库,构建caspase-3的酵母三杂交诱饵载体,进行筛库实验。结果在癫痫发作后12 h TUNEL阳性神经元增加,72 h达高峰;发作后24 h出现eIF2α被caspase-3酶切的片段,逐渐增加至72 h。制作成功cDNA文库,构建了caspase-3的酵母三杂交诱饵载体,筛库获得caspase-3的新底物Miz1。结论在癫痫大鼠海马神经细胞凋亡中eIF2α被caspase-3酶切。酵母三杂交寻找caspase-3下游底物有可行性,从癫痫大鼠海马中获得caspase-3的底物Miz1。

Objective To explore the role of caspase-3 in hippocampal neuronal apoptosis of epileptic model. Methods TUNEL staining was used to detect the apoptosis of hippocampal neurons of the KA-induced epileptic rats and Western blot to detect the change of eIF2α expression. The yeast hybrid cDNA library was constructed using hippocampi of epileptic rats and the bait vector of yeast three-hybrid system for caspase-3 was constructed with PCR. The library was screened with the bait. Results The TUNEL positive neurons start to grow at 12 h after epileptic onset and the apoptotic rate of neurons reached a peak at 72 h after epileptic onset. The cleavage of eIF2α by caspase-3 was detected in hippocampal neurons of epileptic rat 24 h after onset and escalated until 72 h after onset. The yeast hybrid cDNA library was successfully constructed and the yeast three-hybrid system for caspase-3 was accomplished as well. As a result, a novel substrate of caspase-3 was obtained. Conclusion The cleavage of eIF2α by caspase-3 occurs in the SE-induced hippocampal neuronal apoptosis. The bait vector is available by way of yeast three-hybrid system for caspase-3. Miz1, as a novel substrate of caspase-3 obtained from hippocampal neurons of epileptic rat, may play a certain role in neuronal apoptosis at epileptic onset.