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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 study the mechanism of action of caspase-3 in the apoptosis of hippocampal neurons in epileptic model. Methods TUNEL method was used to detect the apoptosis of hippocampal neurons in KA-induced epileptic rats. The expression of eIF2α was detected by Western blot. Take the rat hippocampal tissue to construct a cDNA library and construct the yeast three-hybrid bait vector of caspase-3 for screening experiments. Results The TUNEL-positive neurons increased at 12 h after seizure and reached the peak at 72 h. The fragment of eIF2 was digested by caspase-3 at 24 h after seizure and gradually increased to 72 h. A successful cDNA library was constructed, and a yeast three-hybrid bait vector containing caspase-3 was constructed. A new substrate of caspase-3, Miz1, was obtained from the library. Conclusion eIF2α is cleaved by caspase-3 in the hippocampal neuronal apoptosis in epileptic rats. It is feasible to find out the downstream substrate of caspase-3 by yeast three-hybrid system. Miz1, a substrate of caspase-3, was obtained from the hippocampus of epileptic rats.