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背景:核因子κB是一种重要的转录因子,激活后能促进许多靶基因转录。目的:研究核因子κB在局部脑缺血及再灌注中的表达及N-乙酰半胱氨酸预处理的影响。设计:随机分组设计、动物实验。单位:哈尔滨医科大学第二临床学院神经内科。材料:实验于2004-01/05在哈尔滨医科大学动物实验中心及病理实验室完成。选择健康雄性Wister大鼠99只,随机分3组,假手术组11只,生理盐水对照组44只,N-乙酰半胱氨酸组44只。方法:3组大鼠采用Longa等改良线栓法制备大鼠局灶性脑缺血模型。将头端加热成0.26mm直径的光滑圆球的尼龙线经颈总动脉近分叉处切口插入,扎紧颈总动脉备线,打开颈内动脉上的微动脉夹,尼龙线进入颈内动脉,N-乙酰半胱氨酸组和生理盐水对照组插入长度由颈内动脉和颈外动脉分叉部计约(18.5±0.5)mm,阻断大脑中动脉血供,假手术组插入深度<15mm,大脑中动脉血供正常。N-乙酰半胱氨酸组于缺血前30min腹腔注射N-乙酰半胱氨酸(150mg/kg),生理盐水对照组于缺血前30min注射等体积生理盐水。生理盐水对照组和N-乙酰半胱氨酸组于缺血6,24h,缺血6,24h再灌注1h时间点将大鼠断颈处死,每次11只。应用免疫组织化学法观察脑组织核因子κB的表达情况,红四氯氮唑染色测定各组大鼠脑梗死体积百分比,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法检测脑组织细胞凋亡。主要观察指标:各组大鼠脑梗死体积百分比,核因子κBp65结合活性,凋亡细胞。结果:纳入动物99只,均进入结果分析。①缺血6h及24h再灌注1hN-乙酰半胱氨酸组梗死体积百分比分别为(8.39±2.54)%,(24.54±6.02)%,相应生理盐水对照组为(15.50±4.18)%,(32.22±3.99)%。缺血24h各组较缺血6h各组梗死灶增大,使用N-乙酰半胱氨酸组较生理盐水对照组梗死体积明显缩小(P<0.01)。②缺血及再灌注后核因子κBp65明显从胞质转移到胞核。缺血6h及24h再灌注N-乙酰半胱氨酸组p65阳性细胞率分别为(0.462±0.022)%,(0.452±0.015)%,与相应生理盐水对照组[(0.563±0.028)%,(0.554±0.013)%]比较表达减少(P<0.01)。③N-乙酰半胱氨酸预处理较生理盐水预处理凋亡细胞减少。结论:局灶脑缺血及再灌注能使核因子κBp65活化,参与脑缺血及再灌注损伤。N-乙酰半胱氨酸可抑制p65表达,减轻神经损伤,具有脑保护作用。
Background: Nuclear factor κB is an important transcription factor that activates transcription of many target genes. Objective: To investigate the expression of nuclear factor kappa B in focal cerebral ischemia and reperfusion and the effect of N-acetylcysteine pretreatment. Design: randomized block design, animal experiments. SETTING: Department of Neurology, Second Clinical College, Harbin Medical University. MATERIALS: The experiment was performed at Animal Experimental Center and Pathology Laboratory, Harbin Medical University from January to May 2004. A total of 99 healthy male Wister rats were randomly divided into 3 groups: sham operation group (n = 11), saline control group (n = 44) and N-acetylcysteine group (n = 44). Methods: Three groups of rats were subjected to focal cerebral ischemia with Longa et al. The head heated to a 0.26mm diameter smooth ball of nylon line through the common carotid artery incision near the incision, tie the carotid artery preparation line, open the internal carotid artery on the micro-artery clip, the nylon line into the internal carotid artery , N-acetyl cysteine group and saline control group (18.5 ± 0.5) mm from the bifurcation of the internal carotid artery and external carotid artery to block the blood supply to the middle cerebral artery. The insertion depth of the sham operation group was < 15mm, middle cerebral artery for normal. The N-acetyl cysteine group was intraperitoneally injected with N-acetylcysteine (150mg / kg) 30 minutes before ischemia, and the normal saline control group was injected with equal volume of normal saline 30min before ischemia. Rats in normal saline control group and N-acetylcysteine group were sacrificed at 6,24 hours after ischemia, 6,24 hours after reperfusion and 1 hour after reperfusion. Immunohistochemistry was used to observe the expression of nuclear factor kappa B in brain tissue. The percentage of infarct volume of rats in each group was determined by red tetrazolium dye staining. The expression of nuclear factor kappa B in brain tissue was detected by nick-end labeling Apoptosis. MAIN OUTCOME MEASURES: Volume percentage of cerebral infarction, nuclear factor κBp65 binding activity, apoptotic cells in each group. Results: 99 animals were included in the analysis of the results. ① The percentage of infarction volume in N-acetylcysteine group at 6h and 24h after reperfusion was (8.39 ± 2.54)% and (24.54 ± 6.02)% respectively at 1h and 6h after reperfusion, and (15.50 ± 4.18)% and ± 3.99)%. The ischemic infarct size increased in all groups at 6h after ischemia, and the volume of infarction in N-acetylcysteine group was significantly smaller than that of saline control group (P <0.01). ② After ischemia and reperfusion, NF-κBp65 obviously migrated from the cytoplasm to the nucleus. The rates of p65 positive cells were (0.462 ± 0.022)% and (0.452 ± 0.015)%, respectively, compared with the corresponding normal saline control group [(0.563 ± 0.028)%, 0.554 ± 0.013)%] compared with the decreased expression (P <0.01). ③N-acetyl cysteine pretreatment compared with saline pretreatment decreased apoptotic cells. Conclusion: Focal cerebral ischemia and reperfusion can activate nuclear factor κBp65 and participate in cerebral ischemia and reperfusion injury. N-acetyl cysteine can inhibit the expression of p65, reduce nerve damage, with brain protection.