caspase-3在颞叶癫痫大鼠海马神经凋亡中作用的实验研究

Experimental Study on the Role of Caspase-3 in the Neuronal Apoptosis of Hippocampus in Epileptic Model Rat

目的构建癫痫模型大鼠海马凋亡神经元的cDNA文库,采用酵母三杂交技术从中寻找caspase-3潜在的酶切底物。方法制作海人藻酸大鼠癫痫模型,取其海马组织制作酵母三杂交cDNA文库,克隆获得caspase-3的P12、P17两亚基,然后分别定向插入同一pBridge质粒,构建caspase-3的酵母三杂交诱饵载体并进行验证,然后对所构建文库进行筛库。结果成功构建癫痫模型大鼠海马组织cDNA文库,构建了caspase-3酵母三杂交诱饵载体,并通过caspase-3与eIF2α之间的相互作用验证,经筛库获得caspase-3的底物MIZ1。结论酵母三杂交技术可用于寻找caspase-3下游底物,从癫痫模型大鼠海马的cDNA文库中筛库获得caspase-3的底物MIZ1,为进一步研究caspase-3在癫痫发作引起的海马神经元损伤中的作用奠定了基础。

Objective To construct the yeast three-hybrid cDNA library with hippocampi of epileptic model rats and to screen the library with the yeast three-hybrid system for latent substrates of caspase-3. Methods The yeast three-hybrid cDNA library was constructed with hippocampi of epileptic model rats. P12 and P17 subunits of caspase-3 were obtained by PCR and subcloned into the Multiclone Sites （MCS） of a pBridge for screening caspase-3 interacted substrates using yeast three-hybrid system. Feasibility of the bait vector was verified with eIF2 α and the library was screened with the bait vector for this novel substrates of caspase-3. Results Construction of the yeast three-hybrid cDNA library with hippocampal neurons of epileptic model rats was accomplished. The bait vector in yeast three-hybrid system for caspase-3 was constructed. And a novel substrate of caspase-3 was harvested. Conclusions The bait vector is feasible. With the bait we are able to verify that caspase-3 can interact with eIF2α. MIZ1 was cloned as a novel substrate of caspase-3 in the hippocampus of epileptic model rat, therefor it may play a role in hippocampal neuronal apoptosis after status epilepsy.