Analysis of gene related with the epileptogenesis of inherited epileptic model

本研究试图用RNA任意引物PCR的方法，对人类区域性癫痫模型EL小鼠癫痫发生过程中出现的基因进行鉴定。我们得到20个PCR产物存在于失稳刺激和有癫痫发作经历的EL小鼠（EL[s]）脑中，而不存在于100日龄无癫痫发作经历的EL小鼠（EL[ns]）脑中。将二次PCR产物亚克隆到pBSK II载体上，确定部分序列，并与已知基因进行同源性研究。6个克隆为未知基因，3个克隆为线粒体dna， 2个克隆为基因组dna。9个克隆体的功能未知，或与癫痫或癫痫发作的关系尚不清楚，尽管它们已经是已知的基因。我们试图用Northen blotting分析15个克隆，但只有两个克隆与两个El小鼠大脑的mRNA杂交，但没有显著性。其余13个克隆通过Northen印迹或RNA保护实验未与两种小鼠的mRNA杂交。通过定量RT-PCR检测，EL [s]与EL [ns]小鼠间有12个克隆无显著性差异。只有一个克隆与pipin蛋白同源性超过98%，其在EL [s]小鼠脑中的丰度明显高于EL [ns]。pinin是一种细胞连接相关蛋白，目前对其在脑内的分布及其与癫痫发作的关系尚不清楚，我们正在进行组织化学研究，并与两种EL小鼠脑进行比较。

In present study we tried to identify the gene appeared on the process of the epileptogenesis in the EL mouse that is a model for human regional related epilepsy by RNA arbitrarily primed PCR method.We got twenty PCR products existing in loss-up stimulated and seizure experienced EL mouse (EL [s]) brain but not existing in seizure unexperienced EL mouse (EL[ns]) brain at 100 days of age. After second PCR products were subcloned to the pBSK II vector, we determined partial sequence, followed by researched the homology with already known genes. Six clones were unknown genes and three clones were mitochondrial DNAs and two clones were genomic DNAs. Nine clones were unknown of function or have not cleared relating with epilepsy or seizure, although they were already known genes. We tried to analyze fifteen clones by Northen blotting, but only two clones hybridized with mRNA from both El mice brains without significance. Remaining thirteen clones did not hybridize with mRNA from both mice by Northen blotting or RNA protection assay. Using quantitative RT-PCR, twelve clones were not significant different between EL [s] and EL [ns] mice. Only one clone, that was homologous with pipin protein over 98%, was clearly more abundant in EL [s] mice brain that EL [ns]. Since pinin is a cell junction-associated protein with a little understanding about its distribution in brain and relationship with epilepsy or seizure, we are investigating in histochemistory and comparing with both EL mouse brains.