论文部分内容阅读
背景:一氧化氮合酶(nitric oxide synthase,NOS)是一氧化氮(nitric oxide,NO)合成的关键因素,由于NO在体内易与氧和血红蛋白等物质结合而迅速失活,不易准确定量测定。因此,测定NOS活性是深入研究NO在脑缺血再灌注损伤发病机制的重要环节。目的:研究脑内不同类型NOS在脑缺血再灌注损伤过程中的作用。设计:随机对照的动物实验。单位:青岛大学医学院附属医院脑血管病研究所。材料:实验于2005-05/12在山东省脑病防治重点实验室完成。选择成年健康雄性Wistar大鼠28只,清洁级,体质量220~260g,由山东大学实验动物中心提供。按随机数字表法分为假手术组和脑缺血组,假手术组4只,脑缺血组24只。脑缺血组又分为缺血1h再灌注6h,12h,1d,3d,7d,14d6个时间点,其中每个时间点4只。方法:应用线栓法经左侧颈外-内动脉插线建立大鼠大脑中动脉闭塞再灌注模型。应用免疫组织化学技术检测脑缺血再灌注后不同时间点脑内不同类型NOS的表达。主要观察指标:①甲苯胺蓝染色的两组神经细胞;②脑缺血组大鼠脑内神经元型NOS(neuronalNOS,nNOS)、内皮型NOS(endothelial NOS,eNOS)和诱导型NOS(inducible NOS,iNOS)在不同时间点的表达和分布。结果:①脑缺血组损伤区神经细胞出现核固缩、细胞碎片等,各时间点间细胞差异无显著性。②再灌注后6h脑内神经细胞即出现nNOS,eNOS和iNOS表达,随着再灌注时间的延长逐渐增强,脑组织神经元细胞不同类型NOS表达的区域一致,主要在皮质和纹状体区。脑内nNOS和iNOS于再灌注12h~7d保持较高的表达水平,而eNOS于再灌注6h~3d保持较高水平,持续时间短,升高和降低时间均早于nNOS和iNOS;但3种NOS均于再灌注1d到达表达高峰。3种NOS在皮质区和纹状体区的表达变化趋势基本一致。结论:脑缺血再灌注损伤后eNOS高表达时间较早,持续时间短,而nNOS和iNOS高表达时间稍迟,持续时间长。
BACKGROUND: Nitric oxide synthase (NOS) is a key factor in the synthesis of nitric oxide (NO). Because NO is rapidly inactivated by the combination of oxygen and hemoglobin in the body, NO can not be accurately quantified . Therefore, the determination of NOS activity is an in-depth study of NO in the pathogenesis of cerebral ischemia-reperfusion injury an important part. Objective: To study the role of different types of NOS in cerebral ischemia-reperfusion injury. Design: Randomized controlled animal experiments. Unit: Institute of Cerebrovascular Diseases, Affiliated Hospital of Qingdao University Medical College. MATERIALS: Experiments were performed at Key Laboratory of Encephalopathy Prevention and Control of Shandong Province between May 2005 and December 12. 28 adult healthy male Wistar rats were selected and their cleaning grade and body weight were 220-260g, which were provided by Experimental Animal Center of Shandong University. According to the random number table, the rats were divided into sham operation group and cerebral ischemia group, 4 sham operation group and 24 cerebral ischemia group. Cerebral ischemia group was divided into ischemia 1h reperfusion 6h, 12h, 1d, 3d, 7d, 14d6 time points, of which 4 at each time point. Methods: The model of middle cerebral artery occlusion and reperfusion in rats was established via the left external carotid artery and internal carotid artery. Immunohistochemistry was used to detect the expression of different types of NOS in the brain at different time points after cerebral ischemia-reperfusion. MAIN OUTCOME MEASURES: ① Toluidine blue staining of two groups of nerve cells; ② Cerebral ischemia in rats with neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS , iNOS) at different time points of expression and distribution. Results: ①Nuclear pyknosis and cell debris were observed in the neurons in the ischemic region of the cerebral ischemia group. There was no significant difference in the cells between the time points. At 6h after reperfusion, the expression of nNOS, eNOS and iNOS in brain neurons increased gradually with the reperfusion time prolonging. The expression of NOS in different types of neurons in brain tissue were consistent, mainly in cortex and striatum. The levels of nNOS and iNOS in brain remained high at 12h ~ 7d after reperfusion, while eNOS maintained high level at 6h ~ 3d after reperfusion. The duration of eNOS was earlier than that of nNOS and iNOS. However, NOS reached the peak of expression at 1 day after reperfusion. The expressions of three kinds of NOS in cortex and striatum were basically the same. Conclusion: The eNOS overexpression time is earlier and the duration is shorter after cerebral ischemia-reperfusion injury, but the nNOS and iNOS expression time is slightly later, and the duration is longer.