基于缺血再灌注所致神经系统自噬发生的机制研究

Ischemia/hypoxia cause nervous system damageneuronal apoptosis and therefore is the important reason for the pathogenesis of vascular dementia. Autophagy is a substance involved in the lysosomal catabolism primarily cytosolic degradation of various components, including the unfolded protein, aging organelle damage and intracellular pathogens. Autophagy occurs in the hours after cerebral ischemia. However, whether autophagy play a protective or damage roles in the process of ischemia hypoxiaare still unclear. Endogenous hydrogen sulfide, the third type of gas signal molecule in central nervous system (CNS), has an important regulation and protective effects in CNS. However, its protection has not been fully elucidated. The present research project is to investigate the possible regulation mechanism of CBS/H2S in the pathogenesis of vascular dementia in the cerebral ischemia reperfusion rats model, the low sugar oxygen nerve cell lines as the research object by using siRNA, spectrophotometry and immunofluorescence, Real time PCR, Western blotting as well as gene transfection. The mechanism of autophagy and its signaling pathways in vascular dementia ; The possible role of hydrogen sulfide on the regulation of autophagy and related signaling pathways. This study will provide useful clues in the diagnosis, treatment and prevention of vascular dementia.

缺血/低氧导致神经系统损伤、神经元凋亡是血管性痴呆发病的重要原因。自噬是一种溶酶体参与的物质分解代谢过程，主要降解胞浆内的多种组分，包括未折叠蛋白、衰老损伤的细胞器、胞内病原体。自噬在脑缺血后数小时即发生，然而，自噬在缺血缺氧过程中到底是对神经元起保护还是加重损伤作用目前还不清楚。内源性H2S作为第三类气体信号分子对中枢神经系统功能有着重要的调节和保护作用。本研究以全脑缺血再灌注大鼠模型、缺氧低糖神经细胞株为研究对象，应用siRNA、分光光度法、免疫荧光、Real-time PCR、Western-blotting、基因转染等方法探讨CBS/H2S体系在VaD发病机制中的作用；VaD发病过程中自噬的发生机制、信号通路；H2S对自噬发生的调控作用及相关的信号通路。本研究将为协助VaD的诊断，治疗及预防提供有意义的研究基础。

对氧糖剥夺再复氧对神经细胞损伤、内质网应激与自噬的关系、干扰内源性H2S关键酶CBS基因对氧糖剥夺再复氧对神经细胞的影响、外源性NaHS对缺血再灌注损伤大鼠神经元的保护作用进行了研究。结果发现氧糖剥夺再复氧可导致神经细胞线粒体损伤、内质网应激、自噬增加从而导致细胞凋亡，引起自噬可能与mTOR/Akt通路有关； OGD/R后引起PC12细胞及原代海马神经元发生内质网应激及自噬，甚至细胞的凋亡，并且呈动态变化，内质网应激通路PERK-eIF2α-ATF4介导了自噬的发生，PERK抑制剂GSK2604146的作用同干扰PERK基因的作用一致，自噬抑制剂CQ增加了内质网应激通路PERK-eIF2α-ATF4的蛋白表达并抑制了自噬流，自噬流受阻将导致自噬小体包裹的错误蛋白无法降解，内质网应激加重，细胞凋亡增多，表明OGD/R损伤期间神经元细胞发生的自噬反馈调节内质网应激信号通路PERK-eIF2α-ATF4的发生；CBS基因干扰对细胞自噬和凋亡均无明显影响；缺血再灌注能引起大鼠脑组织海马区域NF-κBp65、COX-2及总Caspase-3表达的变化，导致神经细胞线粒体功能受损，且引发线粒体自噬，低剂量H2S能够调节大鼠海马组织NF-κBp65、COX-2及Caspase-3表达，减轻VaD大鼠海马神经元线粒体自噬程度并且可以保护线粒体功能，从而改善VaD大鼠的学习记忆功能水平，减轻神经元损伤。