突触囊泡蛋白2A在耐药性颞叶癫痫大鼠海马组织苔藓纤维发芽中的作用

The pharmacoresistance is a great challenge in the treatment of epilepsy.The mechanism of the pharmacoresistance is unclear.Studies have demonstrated that the mossy fiber sprouting plays an important role in the epileptogenesis of pharmacoresistant temporal epilepsy.Synaptic vesicle 2A(SV2A) decreased and laminins increased in the hippocampal tissues in patients with pharmacoresistant temporal epilepsy, and the location of the SV2A decreace is consistant with the spot of mossy fiber sprouting.The SV2A and the laminins formed complex on the surface of the electic organ synaptosomes in experimental animals.Hence,we postulate that the decrease of SV2A leads to the increase of the laminins, finally results in the mossy fiber sprouting. The aim of the present study is to observe the changes of SV2A and laminins and their correlation, so as to explore whether the SV2A result in the mossy fiber sprouting by the increase of the laminins. In the present study,we will prepare firstly the pharmacoresistant epileptic model and then upregulate and downregulate the SV2A expression. Secondly,we use an immunohistochemical method,Western blot to observe the exprssion of the SV2A and the laminins, as well as the condition of the mossy fiber sprouting by microscopic Timm`s staning technique. An co-immunoprecipitation method,slow virus over-expression and RNA interference technique will be used to observe the correlation between the SV2A and the laminins. The present studies are expected to provide the theoretical evidences for the mechanism of mossy fiber sprouting in the hippocampal tissues of the pharmacoresistant epileptic rats,and probably discover a potential new target of antiepileptic drugs for the treatment of pharmacoresistant epilepsy.

药物耐受是癫痫治疗中的重大挑战，但耐药的机制并不清楚，苔藓纤维发芽在颞叶癫痫的耐药机制中占有重要地位。耐药性癫痫患者海马组织中突触囊泡蛋白2（SV2A）减少、层粘连蛋白明显增加，SV2A降低的部位就在苔藓纤维发芽的位置，而且SV2和层粘连蛋白在生物体电器官的突触体上形成复合物，因此推测SV2A减少引起层粘连蛋白增加，最终导致苔藓纤维出芽。本研究目的是观察耐药性癫痫大鼠海马组织中SV2A、层粘连蛋白的表达变化及两者间的关系，从而探讨SV2A是否通过层粘连蛋白促进苔藓纤维的发芽。本研究通过制作杏仁核点燃耐药性颞叶癫痫模型，用免疫组化、蛋白印迹、电镜及光镜下的Timm`s染色技术来观察SV2A、层粘连蛋白及苔藓纤维发芽的变化，用免疫共沉淀、慢病毒过表达及RNA干扰技术观察SV2A与层粘连蛋白的关系，有望对耐药性颞叶癫痫大鼠海马组织苔藓纤维发芽机制提供理论依据，同时可能发现新的药物治治疗靶点。

本研究发现突触囊泡蛋白2（SV2A）在颞叶癫痫的耐药机制中占有重要地位。本课题建立多药耐药性颞叶癫痫模型，以癫痫发作、杏仁核电活动、层粘连蛋白表达、苔藓纤维发芽，突触结合蛋白、整合蛋白等的表达。抑郁、焦虑和认知功能障碍的实验为观察指标，观察突触囊泡蛋白2A在耐药性癫痫发生机制中的作用。主要结果如下：①耐药性癫痫模型SV2A及层粘连蛋白α5表达降低，层粘连蛋白β1表达升高，苔藓纤维出芽明显；SV2A过表达明显增加海马组织层粘连蛋白α5的表达及抑制癫痫发生、改善癫痫性脑电活动；SV2A干扰明显降低层粘连蛋白α5和β1表达、增加苔藓纤维发芽，增加癫痫发作。②耐药性癫痫大鼠的焦虑、抑郁、认知障碍程度较药物敏感大鼠明显加重，而SV2A过表达可改善癫痫大鼠焦虑、抑郁及认知障碍；下调SV2A的表达可能加重癫痫大鼠焦虑、抑郁和认知障碍。③下调SV2A可增加突触结合蛋白(syt)-1、syt-4、整合蛋白(integrin-alpha)3以及integrin-beta1的表达，加重癫痫发作；上调V2A可减少syt-1、syt-4、integrin-alpha3以及integrin-beta1的表达，减轻癫痫发作。本课题明确了SV2A在耐药性癫痫中发挥重要的作用，可以影响焦虑，抑郁，认知功能障碍，而且通过调节层粘连蛋白，突触结合蛋白，整合素等的表达来拮抗苔藓纤维发芽，对耐药性癫痫起着保护的作用。本项目共发表论文16篇，其中第一标注的SCI论文4篇，中华神经科1篇，北图核心期刊2篇，科技核心期刊3篇。第二标注的SCI论文3篇，科技核心3篇。荣获2017年贵州省医学会颁发的“贵州医学科技奖一等奖”1项，2018年贵州省人民政府颁发的“贵州省自然科学三等奖”1项。