基于周细胞介导AQP4极性分布的癫痫病理机制及防治策略研究

As a common clinical neurological disorder, epilepsy is still difficult to be controlled in 25% patients with regular drug treatment. Risk factors for vasogenic injury are one of the main pathological mechanisms of epilepsy. AQP4 accumulation occurs at higher densities in endfoot membrane domains facing pericytes than endothelial cells, and the pericytic vascular coverage of epileptic brain is decreased. Therefore, it is speculated that the pericyte-mediated AQP4 polarity distribution is closely related to the pathological process of epileptogenesis. In our previous work, a mouse model of spontaneous epilepsy was constructed by specific knockout the Cdk5 gene in the pericyte and it has reduced pericytic vascular coverage and decreased AQP4 polarity. This project intends to carry out the following research: (1) Resolve which cue molecule in the pericyte regulates the polar distribution of AQP4 and its associated biochemical processes by molecular biology methods. (2) Investigate the mechanisms through which pericyte regulates AQP4 polar distribution during the pathological process of epilepsy, using aforementioned transgenic or chemical-induced animal models with spontaneous epilepsy. (3) Explore a new strategy to control epilepsy through maintaining the AQP4 polar distribution by pharmacological means to stabilize pericyte around the vessels or regulate the cue molecular. The in-depth study of this project will provide an experimental basis for the involvement of angiogenic causes in the pathological process of epileptogenesis, as well as possible novel drug targets and research focus for the prevention and treatment of epilepsy.

癫痫为临床常见神经疾患，约有25%患者用药物难以控制。血管源性损伤危险因素是癫痫的主要病理机制之一。星形胶质细胞AQP4主要分布于血管腔面的周细胞区域，而癫痫脑内周细胞血管覆盖率降低，由此推测周细胞介导的AQP4极性分布与癫痫发生病理过程密切关联。申请人前期研究发现小鼠周细胞特异性敲除Cdk5基因后出现自发性癫痫表型，其周细胞血管覆盖率减少，AQP4极性降低。本项目拟开展如下研究：1）利用分子生物学手段分析周细胞介导AQP4极性的诱导分子，该诱导分子及与其相关的生物化学过程调控AQP4极性的分子机制；2）采用转基因自发性癫痫小鼠等模型研究周细胞介导癫痫进程中AQP4极性改变的机制；3）探索通过药理学手段稳定周细胞于血管周围、调控周细胞源诱导分子维持AQP4极性对癫痫的防治作用。本项目的深入研究，将为血管源性病因参与癫痫病理过程提供实验依据，为防治癫痫发现新的药物作用靶标和研究思路。

癫痫为临床常见神经疾患，约有25%患者用药物难以控制。血管源性损伤危险因素是癫痫的主要病理机制之一。星形胶质细胞AQP4主要分布于血管腔面的周细胞区域，而癫痫脑内周细胞血管覆盖率降低，由此推测周细胞介导的AQP4极性分布与癫痫发生病理过程密切关联。本项目发现周细胞特异性敲除Cdk5基因小鼠出现自发性癫痫表型并对其癫痫发作规律进行研究。利用该模型和经典的癫痫动物模型研究癫痫病理进程中周细胞血管覆盖率、AQP4极性分布的经时变化，以证实周细胞血管覆盖率与AQP4极性分布的相关性。利用Transwell实验和转录组学对周细胞介导AQP4极性分布的分子进行研究。观察调节AQP4极性分布对癫痫发作及其病理改变的作用。研究结果表明周细胞敲除Cdk5转基因小鼠为自发性癫痫小鼠，我们提供了一血管源性癫痫动物模型。癫痫发生发展过程中周细胞血管覆盖率、AQP4极性分布先增加后降低，脑脊液和血清中的PDGF-BB在急性期保持正常水平，但慢性期降低。三者时序变化的一致性证实了周细胞血管分布与AQP4极性分布的相关性。Transwell实验中经周细胞培养的星胶细胞AQP4总量不变，但细胞膜AQP4相较于内皮细胞增多，转录组学结果表明周细胞介导的AQP4极性分布不是由星形胶质细胞DDC复合物的改变，而主要由周细胞分泌的层黏连蛋白(如LAMA1等)细胞外基质所引起。AQP4极性抑制剂三氟拉嗪能够减少PTZ诱导的癫痫持续时间，延长癫痫潜伏期，抑制癫痫后脑水肿。调节AQP4极性分布抑制癫痫、脑实质出血等导致的细胞毒性水肿。以AQP4极性分布为支撑的类淋巴系统参与神经损伤和脑水肿。癫痫病理进程中AQP4的极性改变与周细胞血管覆盖率改变，周细胞层黏连蛋白等细胞外基质改变相关。调节周细胞在血管周围的分布，调节AQP4极性分布有助于缓解癫痫、脑水肿等病理进展。本项目的实施为癫痫防治提供了新的研究视角和药物作用靶标。