【作者】 刘旭；

【导师】 白虹；

【作者基本信息】 天津医科大学 ， 免疫学， 2008， 硕士

【摘要】 目的脑缺血后再灌注虽可挽救濒临死亡的细胞,但也可加重缺血细胞的损伤,甚至导致细胞的死亡,此为缺血再灌注损伤。以往的研究结果表明,脑缺血再灌注后脑组织局部过度的炎症反应是造成再灌注损伤的主要原因之一,级联的炎症反应是导致细胞损伤的重要病理环节,能引起继发性神经元损伤。本文通过研究大鼠局灶性脑缺血再灌注(I/R)后白细胞髓过氧化物酶(MPO)活性的动态变化、白细胞浸润及脑病理形态学改变与相关粘附分子ICAM-1、P-选择素(P-selection)、E-选择素(E-selection)mRNA及蛋白表达动态变化的相互关系,探讨粘附分子介导的脑缺血再灌注炎症损伤的免疫学机制。方法(1)用线栓法制作大鼠大脑中动脉再灌注(MCAO)模型,分为再灌注后3h、6h、12h、24h和48h共5个时间点,每个时间点又分为假手术组和模型组。(2)通过TTC染色、HE染色和电镜检测进行脑缺血再灌注病理形态学动态观察。(3)用酶检测技术检测缺血侧皮层和纹状体新鲜组织白细胞髓过氧化物酶(MPO)活性的动态变化。(4)采用原位杂交法检测PMN浸润相关粘附分子ICAM-1、P-selection、E-selectionmRNA的表达;应用免疫组织化学技术检测脑缺血区粘附分子ICAM-1、P-selection、E-selection蛋白的表达。(5)应用计算机图像分析系统进行脑缺血再灌注图像分析。结果(1)TTC染色观察显示:MCAO模型的梗死部位主要局限于额顶叶皮层和纹状体等区域,再灌注24h梗死面积达到峰值,48h梗死灶开始软化坏死。(2)HE染色镜下观察显示:缺血再灌注6h开始,缺血侧皮层和纹状体缺血半暗区出现白细胞的粘附及浸润,并逐渐加剧,同时坏死灶中心区逐渐扩大。(3)白细胞活性检测:再灌注6h皮层和纹状体MPO活性均开始增加,24h进一步增加,48h达到较高值。I/R后6h、24h、48h模型组皮层和纹状体MPO活性均显著升高,与假手术组相比有显著性差异(P＜0.01)。(4)粘附分子ICAM-1、P-selection、E-selection mRNA在脑缺血3h后在缺血侧皮层和纹状体开始表达增加,6-12h达到高峰;免疫组化证实ICAM-1、P-selection、E-selection蛋白表达在毛细血管与mRNA表达区域基本一致,不同的是其表达高峰后移,于脑缺血再灌注24h达到高峰。结论脑缺血再灌注后缺血区ICAM-1、P-selection、E-selection mRNA和蛋白表达增加,从而促进了粘附分子介导的内皮细胞与中性粒细胞的粘附浸润所引起的“炎症级联反应”。更多还原

【Abstract】 ObjectiveThe cerebral ischemia-reperfusion may save cells from the borders of death, but may aggravate the cell damage even the cell death caused by the ischemia-reperfusion. The previous studies have shown that the excessive inflammatory reaction in the ischemic reperfusion of the brain is the main mechanisms of cerebral ischemic reperfusion injury. The inflammation response is the important pathological process to result in the cell injury and the secondary nerve cell destroy. In order to study the immunological mechanism of inflamatory injury mediated by the cell-adhesion molecules in cerebral ischemic reperfusion, we examined the relationship between inflammatory response and the expression of mRNA and protein of cell-adhesion molecules, ICAM-1、P-selection and E-selection.Methods(1) The animal models of middle cerebral artery occlusion (MCAO) were established with nelon threads in Wistar rats. The model group of rats were randomly divided into the five time points: 3h, 6h, 12h, 24h, 48h after reperfusion respectively and each time point include sham-operation group and model group.(2) Observe the pathology and morphology of ischemia-reperfusion injury by TTC staining, Hematoxylin-Eosin staining and electron microscope.(3) Observe the dynamic changes of the myelperoxidase activity of PMN by Enzyme assay technic.(4) mRNA and protein expression of cell-adhesion molecules ICAM-1, P-selection and E-selection during cerebral ischemia-reperfusion were examined by insitu hybridization and imunohistochemistry techniques respectively.(5) Image analysis was processed by computer picture analysis system.Results(1) The injury areas in MCAO were located in frontal parietal lobe cortex and striatum observed by TTC staining. The infarct area was peaked at 24h reperfusion and the infarct focus was already softened and necrosed at 48h time point of reperfusion.(2) The adhension and infiltration of neutrophils were found to begin at ischemia-6h reperfusion in cerebral cortex and striatum by HE staining, and became more and more serious with the process. At the same time, the necrosis center in inflammatory area magnified gradually.(3) MPO activity began to increas in the striatum and cortex 6h reperfusion and peaked at 48h post reperfusion.(4) mRNA expression of ICAM-1, P-selection and E-selection began to be detected at capillary endothelia cells in cerebral ischemic cortex and corpus striatumn 3h reperfusion and peaked at 6-12h of reperfusion. The protein expression of ICAM-1, P-selection and E-selection were coincident with that of mRNA expression in ischemic area by imunohistochemistry assay. However, the peak point was extended and reached at 24h reperfusion.ConclusionsBoth of the mRNA and protein expression of ICAM-1, P-selection and E-selection were upregulated after cerebral ischemia-reperfusion, which boosted the inflammatory cascade response that mediated by the cell-adhesive molecules.更多还原