一氧化氮和一氧化氮合酶在大鼠局灶性脑缺血中的表达特点  

To investigate the expression of NOS and NO in the cerebral ischemic tissue. after ischemic injury

作　　者：杨卫忠[1] 陈春美[1] 王春华[1] 石松生[1] 雷军荣[1] 张永亮[1] 

机构地区：[1]福建医科大学附属协和医院神经外科福建省神经外科研究所,福州350001

出　　处：《中国神经精神疾病杂志》2007年第6期335-339,共5页Chinese Journal of Nervous and Mental Diseases

基　　金：福建省教育厅科技计划项目资助(编号:JA04200)

摘　　要：目的建立脑缺血SD大鼠模型,探讨一氧化氮(NO)和一氧化氮合酶(NOS)在脑缺血模型中的表达特点。方法应用线栓法制作大脑中动脉阻塞(MCAO)局灶性脑缺血模型,根据缺血不同时间分为8组,设立假手术组和正常对照组,每组各有6只大鼠。应用硝酸还原酶法测定脑组织NO的含量,流式细胞术(FCM)定量检测硝基酪氨酸(NT)表达,化学比色法测定脑组织NOS的活性,免疫组织化学方法定位检测eNOS、nNOS和iNOS表达位置,逆转录反应系统(RT-PCR)半定量分析eNOS、nNOS和iNOS的 mRNA在脑缺血区域的表达。结果神经功能缺失评分发现缺血时间越长,神经功能缺失越明显;脑组织中NO含量与缺血时间正相关;缺血1h后NT阳性细胞百分比开始明显升高(9.50%);缺血0.5h时NOS的活性开始升高,缺血3d达到高峰[0.94nmol/(g.min)];免疫组织化学提示eNOS在神经细胞和血管内皮细胞胞浆均有表达,nNOS和iNOS抗体主要在神经细胞胞浆中表达;RT-PCR半定量分析在缺血早期(0.5h^6h),随着缺血时间延长,eNOS和nNOS表达增加,iNOS未见表达或低表达;缺血中晚期(6h^5d),iNOS高表达,并与缺血时间呈正相关,eNOS和nNOS表达明显减少。结论脑缺血时间延长,NOS的活性升高,NO在体内特异性代谢产物NT量增加,神经功能缺失越明显;NOS在缺血早期以eNOS和nNOS为主,在缺血晚期以iNOS为主。Objective TO establish the focal cerebral ischemia model and investigate the expression of NOS and NO in the cerebral ischemic tissue. Methods Focal cerebral ischemia model was established via MCAO with the intraluminal filament method. Rats were randomized into normal group, sham operation group and the ischemia group, which were divided into 8 group according to different ischemia time. There were 6 rats every group. NO and NOS were evaluated respectively by nitrate reductase （NR） and spectrophotometry. The expression of nitrotyrosine （NT） was tested by flow cytometry （FCM）. eNOS, nNOS and iNOS proteins were semi-quantitively analyzed by RT-PCR and locationally detected by immunochemistry as well. Results According to neurological grading system, the longer ischemic insult lasted, the more severe neurofunction reduced. The deficit peaked at day 3 and 5 with 3.67 and 3.83 points respectively. NO increased with ischemic time and NT positive cell percentage ascended markedly after 1 hour occlusion （9. 50% ,P 〈 0.05）. NOS increased as early as 0. 5 hour and peaked at day 3 with 0. 94 nmol/（ g · min）. Immunohistochemical findings indicated that eNOS was located in the cytoplasm of both neurons and vascular endothelial cells while nNOS and iNOS mainly in the cytoplasm of neurons. Analysis of RT-PCR showed upregulation of eNOS and nNOS occurred at early ischemic injury （0.5 h-6 h） while that of iNOS at mid-late injury （6 h -5 d）. Conclusions Neural function deteriorated with cerebral ischemia time which was accompanied by the elevation of NOS activity and NT production, eNOS and nNOS predominated in the early phase of the ischemia whereas iNOS in the late phase.

关 键 词：脑缺血 一氧化氮 内皮型一氧化氮合酶 神经元型一氧化氮合酶 诱导型一氧化氮合酶 

分 类 号：R743.3[医药卫生—神经病学与精神病学]