白细胞介素1β转化酶在脑缺血再灌注损伤中的表达及对细胞凋亡的调节作用(英文)

Expression of interleukin-1-beta converting enzyme and its effect on cell apoptosis in cerebral ischemia-reperfusion injury

背景:细胞凋亡是缺血再灌注损害的重要形式之一,白细胞介素1β转化酶是细胞凋亡关键调节因子,其激活可导致蛋白降解引起细胞凋亡。目的:观察在脑缺血再灌注过程中白细胞介素1β转化酶基因的表达及其与细胞凋亡的关系,探讨其在调控脑缺血后细胞凋亡中的作用。设计:随机对照试验。材料:实验于1996-03/2000-12在解放军第三军医大学神经科学中心完成。选择成年W istar大鼠64只,随机分为2组,脑缺血再灌注组:采用四血管阻塞全脑缺血模型,缺血30m in 后再灌注,根据处死时间分为再灌注3,6,12,24,48,72h 和7d 共7个时间点,每个时间点8只。假手术组8只,仅分离,未夹闭双侧颈总动脉。方法:假手术组和脑缺血再灌注组再灌注后每个时间点随机选取4只大鼠采用免疫组化和原位杂交检测。其余4只用于原位末端标记检测。主要观察指标:①各组大鼠脑组织白细胞介素1β转化酶蛋白和m RNA的表达。②各组大鼠脑组织神经细胞凋亡情况。结果:64只大鼠全部进入结果分析。①各组大鼠脑组织白细胞介素1β转化酶m RNA 和蛋白的表达:假手术组脑组织多数脑区白细胞介素1β转化酶mRNA 和蛋白有少量表达,脑缺血组白细胞介素1β转化酶m RNA 和蛋白在神经元内及小胶质细胞均有表达,分布在大脑皮质、小脑蒲肯野细胞、海马及皮质下白质。在缺血再灌注12h 后白细胞介素1β转化酶基因表达增加,48～72h 为表达高峰,第7天表达下降。②各组大鼠脑组织神经细胞凋亡情况:细胞凋亡在缺血再灌注12h 出现犤(49.4±6.8)个/切片犦,高峰时间在72h犤(228.6±29.8)个/切片犦,且白细胞介素1β转化酶蛋白和m RNA 的表达在分布上与神经细胞凋亡的发生有显著相关性(r=0.89,0.68,P <0.05)。结论:①白细胞介素1β转化酶蛋白和m RNA 的表达增加,与缺血后细胞凋亡有显著相关性,从时间上支持白细胞介素1β转化酶表达是导致细胞凋亡的重要因素。②在脑缺血再灌注过程中大脑皮质、海马及基底节区是凋亡细胞的多发部位,而这些部位也是白细胞介素1β转化酶表达增高的区域,进一步证明缺血后白细胞介素1β转化酶的表达参与神经细胞凋亡的调节。

BACKGROUND： Cell apoptosis is one of the important pathological changes in ischemic-reperfusion （IR） injury. As the key factor involved in cell apoptosis regulation, interleukin （IL）-1β converting enzyme, when activated, leads to cell apoptosis via protein degradation. OBJECTIVE： To investigate the relationship between the expression of IL-1β converting enzyme and cell apoptosis in cerebral IR injury and explore the role of this enzyme in post-ischemia cell apoptosis. DESIGN： Randomized controlled experiment. MATERIALS： The experiment was performed at the Center of Neuroscience of the Third Military Medical University between March 1996 and December 2000. Totally 64 adult healthy Wistar rats were randomized into two groups, namely IR group （n=56） and sham operation group （n=8）. In IR group, the rats were subjected to four vessel occlusion to mimic whole brain IR injury, and reperfusion was carried out after 30 minutes of ischemia for 3, 6, 12, 24, 48, 72 hours and 7 days, respectively （8 rats at each time point）. Only separation but not occlusion of the bilateral common carotid artery was performed in sham operation group. METHODS： Four rats were randomly selected from IR group at each time point and 4 from the sham operation group for immunohistochemical study and in situ hybridization, with the other 4 rats for in situ end-labeling assay. MAIN OUTCOME MEASURES： Protein and mRNA expression of IL-1β converting enzyme and neural cell apoptosis in the brain. RESULTS： Totally 64 rats were used in this study and all data were statistically analyzed. In the sham operation group, IL-1β converting enzyme protein and mRNA were expressed in small amount in most of the normal brain tissues, and their expressions were also detected in the neurons and small glial cells in IR group localized mainly in the cerebral cortex, cerebellum Purkinje＇s cells, hippocampal and subcortical white matters. The expression of IL-1β converting enzyme began to increase at IR 12 hours, reaching the peak level at 48-72 hours followed by declination since 7 days after the operation. Cell apoptosis occurred 12 hours after IR （49.4±6.8） /section and peaked at 72 hours （228.6±29.8）/section, showing significant correlation with the temporal expression of IL-1β converting enzyme protein and mRNA （r=0.89, 0.68, P 〈 0.05）. CONCLUSIONS： Expressions of IL-1β converting enzyme protein and mRNA increased after IR in close correlation with post-ischemia cell apoptosis, and their temporal expression pattern supports the presumption that 1L-1β converting enzyme is an important factor in cell apoptosis. Apoptosis is mostly likely to occur in the cerebral cortex, hippocampus and basal ganglion in IR injury, where IL-1β converting enzyme is highly expressed, further demonstrating that post-ischemia expression of IL-1β converting enzyme might be involved in cell apoptosis regulation.