DNA甲基化转移酶调控候选基因表达在耐药性癫痫形成中的作用机制

DNA methylation is catalyzed by DNA methyltransferase (DNMT) and it can to regulate gene ranscription, it plays important roles in the central nervous system through regulating synaptic plasticity as well as neuronal network activity. However, it is still unclear whether DNA methylation regulate aberrant expression of candidate epileptogenesis genes expression.We have found that both DNMT1 and DNMT3a expression were significantly increased in brain tissue of patients with durg-refractory temporal lobe epliepsy and animal models of epilepsy,and DNMTs inhibitor decreased seizure susceptibility in responsse to convulasnt drug in rats. To prove the hyposies that aberrant expression of DNMTs associate with epileptogenesis,the surgical specimen of epileptic patients,cellular model of epilepsy’s disease in vitro and animal model of intractable epilepsy in vivo will be used to detect the relationship between the DNMTs and candidate genes GAD67,GluR2 and BDNF DNA methylation and mRNA as well as protien expression and to explicit the effect of candidate genes DNA methylation on imbalance excitability-inhibitory, synaptic Plasticity and mossy fiber sprouting by DNA bisulfitep pyro sequencing，molecular biology,morphology,electrophysiological technique, construction of recombinant lentiviral vector and expression technique in vitro. In this study we explore whether DNMTs inhibitor can improve the pathophysiological changes in the pathogenic process of intractable epilepsy by regulating candidate genes DNA methylation to reveal pathogenesis of pharmacoresistance in epilepsy.

DNA甲基化是经DNA甲基化转移酶（DNMTs）催化而影响基因转录水平的表达，在调控突触可塑性和神经元网络活性等神经生物过程中起重要作用，但DNA甲基化是否调控癫痫候选基因的表达尚不清楚。课题组前期发现DNMT1和DNMT3a在耐药性癫痫患者和动物模型脑组织中表达上调，经DNMTs抑制剂干预可使大鼠对致痫剂敏感性下降。本项申请拟采用耐药性癫痫患者手术标本、离体癫痫细胞和在体癫痫动物模型，在前期研究基础上用DNA甲基化测序、分子生物学、形态学、电生理技术、重组慢病毒载体构建及体外表达技术，进一步探索DNMTs与癫痫候选基因GAD-67、GluR2、BDNF启动子甲基化及其mRNA、蛋白表达的相关性，以及候选基因甲基化状态是否影响兴奋-抑制失衡、突触重塑及苔藓纤维芽生，探讨DNMTs抑制剂能否通过调控候选基因甲基化来改善耐药性癫痫形成过程中的病理生理改变，以揭示耐药性癫痫的发病机制。