气体信号分子H2S对脑缺血后神经血管单元的影响及其机制研究

The co-ordination and symbiosis involved in the neuron - astrocyte -blood vessels (neurovascular unit, NVU) is very important for the neuroprotection after cerebral infarction(CI). New multi-targets protective agents are hoped to be developed for the treatment of CI. Hydrogen sulfide is an endogenously produced gas and has been touted as the body's third gaseous signaling molecule. It mainly generated by astrocytes in the CNS. However, the function of H2S in NVU after CI is rarity described. This project is mainly observed: ①the changes of endogenous H2S and its related enzymes in the vitro NVU model of cerebral ischemia; ②the effect and relative molecular mechanism of H2S generated from astrocytes regulated by optogenetical technology in NVU under injury of OGD; ③Based on the optogenetical technology to regulate astrocytes in a specific site of mice' brain, observe the histopathologic features of micro-cerebral infarction in mice which modeling by photochemical method. ④ Intraluminal suture occlusion and ligation of the distal branch of internal carotid artery of rat after injection of H2S donor-NaHS to make rats CI model, and then assess the neurological deficit symptoms, the infart size and the histopathologic features. The above study was designed to deepen the understanding of the pathophysiology of cerebral ischemia and provide a new theoretic proof and technical means for the treatment.

脑梗死后神经功能保护依赖于神经元-星形胶质细胞-血管（神经血管单元，NVU）之间的协调、共生，这就呼唤着多靶点的保护剂出现。研究表明，气体信使硫化氢(H2S)参与哺乳动物多系统/细胞的调节。H2S在脑中主要由星形胶质细胞生成，但目前仍未见H2S在NVU的缺血性损伤中的详细报道。本项目主要观察：①内源性H2S及其相关酶学在NVU体外模型脑缺血损伤中的表达变化；②光感基因调控星形胶质细胞H2S生成量对神经血管单元缺血性损伤的影响及相关的分子机制；③对小鼠特定部位星型胶质细胞进行光感基因调控以增加H2S的生成量，在此基础上观察光化学法所致小鼠脑微梗死局部NVU的病理学特点；④对大鼠进行H2S供体- - NaHS体外注射，经线栓法致脑梗死模型，评估不同NaHS浓度组及空白组大鼠神经功能缺损症状，并比较梗死灶面积及病理学特点。以上研究旨在深化对脑缺血病理生理的认识，为疾病的治疗提供新的理论依据和技术手段

脑梗死后神经功能保护依赖于神经元-星形胶质细胞-血管（神经血管单元，NVU）之间的协调、共生，这就呼唤着多靶点的保护剂出现。本研究探讨气体信使硫化氢(H2S)对哺乳动物多系统/细胞的调节。本研究共分体外和体内实验2个部分，体外实验是在利用transwell细胞共培养板构建体外神经血管单元（neurovascular, NVU）模型。通过SAM（S-腺苷-甲硫氨酸）激活内源性H2S生成限速酶CBS（胱硫醚-β合成酶）观察OGD/R（糖氧剥夺再灌注）损伤后体外NVU体系中星形胶质细胞内H2S产生数量。证实了内源性H2S对OGD/R损伤后NVU共培养体系中神经元和BMECs（脑微血管内皮细胞）的保护作用。提出其保护作用可能通过星形胶质细胞中内源性H2S能降低体外NVU体系中神经元线粒体介导的凋亡和坏死，同时降低体外NVU体系中BMECs的坏死率，且降低神经元和BMECs中ROS（活性氧簇）水平和增加GSH（谷胱甘肽）生成的抗氧化应激途径来发挥保护作用。同时在大鼠缺血再灌注模型中证实了H2S对NVU的保护作用，通过行为学评分、梗死面积、Q-PCR和WB的结果证实H2S的合成代谢对维持血脑屏障的稳定，保护神经血管单元具有一定的作用。本试验还采用微流控芯片构建新型的神经血管单元模型，探索了神经元、脑微血管内皮细胞以星形胶质细胞为桥梁微流控内肩并肩生长的接种顺序、接种时间、接种细胞密度以及接种方式，并测定了共培养状态下三种要素细胞的细胞活力。本研究按计划完成研究任务，课题经费使用合理，在SCI期刊发表论文3篇，国内期刊发表论文2篇，正在投稿（SCI期刊）2篇，申请国家发明专利1项。培养硕士研究生5名，博士研究生1名。