ORP3促进血管内皮细胞外泌体释放在脑缺血损伤中的作用及机制研究

脑缺血损伤引起炎症反应，破坏血脑屏障。脑微血管内皮细胞(BMVEC)是血脑屏障的重要组成部分。已发现血管内皮细胞在不利刺激下会分泌外泌体等多种信号物质吸引炎症细胞，但这一现象的调控机制尚不明确。我们前期发现膜脂锚定蛋白ORP3在人BMVEC中促进外泌体分泌，且这些外泌体可能参与炎症反应。而免疫共沉淀初步证实ORP3可能与外泌体转运蛋白RAB27以及微管驱动相关蛋白CRMP2结合。.由于这三个分子在脑缺血损伤中的功能尚不明确。本项目将在前期工作基础上，首先通过调控BMVEC中ORP3表达，确认该分子对外泌体分泌和炎症的影响。再使用免疫共沉淀和荧光共振能量转移技术确认ORP3、RAB27和CRMP2直接结合。最后通过调控三个分子的表达，确认它们在脑缺血损伤中对外泌体分泌和炎症等的影响。从而证实ORP3作为新的外泌体运输调节蛋白，结合RAB27和CRMP2共同促进外泌体释放，加重脑缺血炎症反应。

Cerebral ischemic injury causes inflammation and destroys the blood-brain barrier (BBB). The brain microvascular endothelial cell (BMVEC) is an important part of the BBB. It has been found that vascular endothelial cells increase exosome secretion with adverse stimulation, but the underlying mechanisms are still unclear. We preliminarily found that ORP3 as a membrane anchor protein promoted exosome release from human BMVECs, and these exosomes might participate in the inflammatory response. Moreover, co-immunoprecipitation confirmed that ORP3 might bind to exosome transporter RAB27 and kinesin related CRMP2..The role of ORP3, RAB27 and CRMP2 in cerebral ischemia is still unknown. Based on the above work, firstly, we will regulate ORP3 expression in BMVECs to explore its role in exosome secretion and inflammatory response. Next, co-immunoprecipitation and fluorescence resonance energy transfer will be used to confirm the interactions of ORP3, RAB27 and CRMP2. Finally, the role of ORP3/RAB27/CRMP2 complex in exosome transport and inflammation will be determined by regulating their expression in BMVECs. Thus, this project will aim to reveal that ORP3 as a new exosome transporter regulate inflammatory response in BMVECs after cerebral ischemic injury. And it will provide a new perspective and experimental evidence for the treatment of ischemic stroke.