Analysis of gene related with the epileptogenesis of inherited epileptic model

在本研究中，我们试图通过随机引物聚合酶链式反应的方法，在人类区域相关性癫痫模型--EL小鼠中寻找癫痫发生过程中出现的基因。我们在100日龄的失控刺激和惊厥经历过的EL小鼠(EL[S])脑中发现了20个存在于癫痫发生过程中的基因，而在惊厥未经历过的EL[ns]脑中不存在。将第二次扩增产物亚克隆到pBSK II载体上，测定部分序列，并与已知基因进行同源性比较。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。有9个克隆与癫痫或癫痫有关，但功能未知或尚未清除，尽管它们已经是已知的基因。我们试图通过Norten blotting分析15个克隆，但只有两个克隆与两个EL小鼠脑中的mRNA杂交，没有意义。经Norten印迹杂交和RNA保护实验，其余13个克隆均未与两只小鼠的mRNA杂交。用定量RT-PCR方法检测到12个克隆在EL[S]和EL[ns]小鼠之间没有显著差异。只有一个克隆与琵琶蛋白同源性超过98%，显然在EL[S]小鼠脑中的含量比EL[ns]小鼠丰富。由于Pinin是一种细胞连接相关蛋白，对其在脑内的分布及其与癫痫或癫痫发作的关系知之甚少，我们正在进行组织化学研究，并与两种EL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.