人尿激肽原酶对大鼠脑缺血再灌注后作用机制的研究
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摘要
第一部分人尿激肽原酶对大鼠脑缺血再灌注后神经细胞凋亡的影响
目的：观察人尿激肽原酶(HUK)对大鼠脑缺血再灌注(I/R)后大脑缺血侧皮质缺血灶周围区Caspase-3.凋亡诱导因子(AIF)和Survivin的时相表达及其对神经细胞凋亡的影响。
方法：将Wistar大鼠随机分为假手术组、模型组及HUK干预组,应用线栓法建立大鼠大脑中动脉阻塞(MCAO)再灌注模型。假手术组于术后,模型组、HUK干预组于缺血2h再灌注6h、24h、48h、72h,应用免疫组化技术和原位末端标记法(TUNEL)检测不同时相各组缺血灶周围脑区Caspase-3. AIF. Survivin和TUNEL阳性细胞的表达。
结果：大鼠I/R后在缺血灶周围区各个时间点均可以见到Caspase-3.AIF.Survivin和TUNEL阳性细胞的表达,HUK组与模型组相比较,两组Caspase-3.AIF.Survivin和TUNEL阳性细胞的表达趋势基本一致,Caspase-3.AIF和TUNEL的表达高峰在I/R后24h,Survivin的表达高峰在I/R后48h。HUK组各时间点Caspase-3、AIF和TUNEL阳性细胞的光密度值较模型组明显降低,Survivin的光密度值较模型组明显升高(均为P<0.05).
结论：HUK对大鼠脑缺血再灌注损伤有保护作用,其作用机制可能与HUK抑制Caspase-3.AIF的表达,促进Survivin的表达,减轻迟发性神经元的凋亡有关。关键词：人尿激肽原酶；细胞凋亡；Caspase-3；AIF；Survivin；脑缺血再灌注
第二部分人尿激肽原酶对大鼠脑缺血再灌注后空间学习记忆功能及皮质巢蛋白表达的影响
目的：探讨人尿激肽原酶(HUK)对大鼠局部脑缺血再灌注后缺血侧皮质巢蛋白(nestin)的表达及空间学习记忆能力障碍的作用。
方法：线栓法制作大鼠大脑中动脉阻塞(MCA O)再灌注模型,HUK治疗组于术后2周内腹腔注射HUK,第15天开始各组大鼠采用Morris水迷宫装置评价大鼠的空间学习记忆能力,尼氏染色观察缺血侧皮质神经元数量的变化,免疫组化染色检测缺血侧皮质nestin的表达。
结果：脑缺血组大鼠在定向航行试验和空间探索试验中均表现出明显的空间认知功能障碍。在定向航行试验中,HUK中、高剂量治疗组大鼠平均逃避潜伏期与缺血组比较明显缩短(P<0.05)。在空间探索试验中,HUK中、高剂量治疗组大鼠原平台象限停留时间百分比以及穿过原平台位置次数均大于脑缺血组(P<0.05)。缺血侧皮质尼氏染色结果显示,HUK中、高剂量治疗组大鼠缺血侧皮质存活神经元数量明显高于脑缺血组(P<0.05)。缺血侧皮质nestin免疫组化染色结果显示,HUK中、高剂量治疗组大鼠缺血侧皮质nestin的光密度值明显高于脑缺血组(P<0.05)。
结论：HUK可以明显改善大鼠局部脑缺血再灌注后的空间学习记忆能力,其机制可能与HUK增加缺血侧皮质nestin的表达,促进神经再生有关。
Part I Effect of human urinary kallidinogenase on neuronal apopto-sis at focal cerebra injured region after ischemia-reperfusion in rats
Objective:To investigate the effect of human urinary kallidinogenase (HUK) on the expression of caspase-3, apoptosis-inducing factor (AIF), Survivin and neuronal apoptosis in perifocal areas of cerebra after ischemia-reperfusion (I/R) in rats.
Methods:Wistar rats were randomly divided into sham group,model group and HUK group.The middle cerebral artery occlusion/reperfusion (MCAO/R) model was established by thread ligation. After operation in sham group,2 h ischemia and 6h,24h, 48h,72h reperfusion in model and HUK group,the expression of caspase-3,AIF, Survivin positive cells and neuronal apoptosis in the ischemic cortex were detected by immunohistochemistry and in situ apoptotic detection (TUNEL method).
Results:The caspase-3,AIF,Survivin and Tunel positive neurons expresses in perifocal areas at different time points after I/R. The trends of expressed caspase-3, AIF,Survivin and Tunel positive neurons in HUK group and model group were basi-coincidence. The peak of expressed caspase-3,AIF and Tunel positive neurons were at 24h in perifocal areas after I/R.The peak of expressed Survivin was at 48h in perifocal areas. The optical density value of Caspase-3,AIF and Tunel positive neurons in HUK group at each time were significantly decreased compared with model groups,and the optical density value of Survivin was increased compared with model groups (P<0.05).
Conclusions:HUK play protective function for the cerebral tissues after I/R by increasing the expression of Survivin, reducing the expression of caspase-3,AIF and tardive neuronal apoptosis in posse.
Part II Effect of human urinary kallidinogenase on function of spatial learning and memory in rats and nestin expression in peri-infarction cortex after focal cerebral ischemia-reperfusion
Objective:To investigate the effects of human urinary kallidinogenase (HUK) on the ability of spatial learning and memory in rats and the expression of nestin in peri-infarction cortex after focal cerebral ischemia-reperfusion.
Methods:The focal cerebral Ischemia-reperfusion model was established by introducing an intraluminal filament into the right middle cerebral artery of the rats, then HUK was administered introperitoneally after the operation and afterward once a day for 2 weeks. The spatial learning and memory function was studied by morris water maze test,Then Nissl staining technique was conducted for observing the morphology of neurons in peri-infarction cortex,the nestin expression in the peri-infarction cortex was measured by immunohistochemistry.
Results:The ischemia-control group exhibited seriously spatial learning and memory deficits in both place navigation trail and spatial probe trial.In place navigation trial, the mean values of escape latency of middle-dose and higher-dose HUK groups were shorter than those of ischemia-control group (P<0.05).In spatial probe trial, there were significant difference in the time percentage of spending in the former platform quadrant and frequency of crossing the former platform site between middle and higher-dose HUK groups and ischemia-control group(P<0.05). Nissl staining analysis showed that the number of neurons in the peri-infarction cortex of middle and higher-dose HUK groups significantly increased as compared with that of ischemia-control group (P<0.05).Immunohistochemical analysis showed that nestin expression in the peri-infarction cortex of middle and higher-dose HUK groups significantly increased as compared with that of ischemia-control group (P<0.05).
Conclusion:HUK improves the rats spatial learning and memory ability after focal cerebral ischemia-reperfusion, this may be the results of the increasing expression of nestin and the proliferation of neural stem cells after focal cerebral ischemia.
目的：观察人尿激肽原酶(HUK)对大鼠脑缺血再灌注(I/R)后大脑缺血侧皮质缺血灶周围区Caspase-3.凋亡诱导因子(AIF)和Survivin的时相表达及其对神经细胞凋亡的影响。
方法：将Wistar大鼠随机分为假手术组、模型组及HUK干预组,应用线栓法建立大鼠大脑中动脉阻塞(MCAO)再灌注模型。假手术组于术后,模型组、HUK干预组于缺血2h再灌注6h、24h、48h、72h,应用免疫组化技术和原位末端标记法(TUNEL)检测不同时相各组缺血灶周围脑区Caspase-3. AIF. Survivin和TUNEL阳性细胞的表达。
结果：大鼠I/R后在缺血灶周围区各个时间点均可以见到Caspase-3.AIF.Survivin和TUNEL阳性细胞的表达,HUK组与模型组相比较,两组Caspase-3.AIF.Survivin和TUNEL阳性细胞的表达趋势基本一致,Caspase-3.AIF和TUNEL的表达高峰在I/R后24h,Survivin的表达高峰在I/R后48h。HUK组各时间点Caspase-3、AIF和TUNEL阳性细胞的光密度值较模型组明显降低,Survivin的光密度值较模型组明显升高(均为P<0.05).
结论：HUK对大鼠脑缺血再灌注损伤有保护作用,其作用机制可能与HUK抑制Caspase-3.AIF的表达,促进Survivin的表达,减轻迟发性神经元的凋亡有关。关键词：人尿激肽原酶；细胞凋亡；Caspase-3；AIF；Survivin；脑缺血再灌注
第二部分人尿激肽原酶对大鼠脑缺血再灌注后空间学习记忆功能及皮质巢蛋白表达的影响
目的：探讨人尿激肽原酶(HUK)对大鼠局部脑缺血再灌注后缺血侧皮质巢蛋白(nestin)的表达及空间学习记忆能力障碍的作用。
方法：线栓法制作大鼠大脑中动脉阻塞(MCA O)再灌注模型,HUK治疗组于术后2周内腹腔注射HUK,第15天开始各组大鼠采用Morris水迷宫装置评价大鼠的空间学习记忆能力,尼氏染色观察缺血侧皮质神经元数量的变化,免疫组化染色检测缺血侧皮质nestin的表达。
结果：脑缺血组大鼠在定向航行试验和空间探索试验中均表现出明显的空间认知功能障碍。在定向航行试验中,HUK中、高剂量治疗组大鼠平均逃避潜伏期与缺血组比较明显缩短(P<0.05)。在空间探索试验中,HUK中、高剂量治疗组大鼠原平台象限停留时间百分比以及穿过原平台位置次数均大于脑缺血组(P<0.05)。缺血侧皮质尼氏染色结果显示,HUK中、高剂量治疗组大鼠缺血侧皮质存活神经元数量明显高于脑缺血组(P<0.05)。缺血侧皮质nestin免疫组化染色结果显示,HUK中、高剂量治疗组大鼠缺血侧皮质nestin的光密度值明显高于脑缺血组(P<0.05)。
结论：HUK可以明显改善大鼠局部脑缺血再灌注后的空间学习记忆能力,其机制可能与HUK增加缺血侧皮质nestin的表达,促进神经再生有关。
Part I Effect of human urinary kallidinogenase on neuronal apopto-sis at focal cerebra injured region after ischemia-reperfusion in rats
Objective:To investigate the effect of human urinary kallidinogenase (HUK) on the expression of caspase-3, apoptosis-inducing factor (AIF), Survivin and neuronal apoptosis in perifocal areas of cerebra after ischemia-reperfusion (I/R) in rats.
Methods:Wistar rats were randomly divided into sham group,model group and HUK group.The middle cerebral artery occlusion/reperfusion (MCAO/R) model was established by thread ligation. After operation in sham group,2 h ischemia and 6h,24h, 48h,72h reperfusion in model and HUK group,the expression of caspase-3,AIF, Survivin positive cells and neuronal apoptosis in the ischemic cortex were detected by immunohistochemistry and in situ apoptotic detection (TUNEL method).
Results:The caspase-3,AIF,Survivin and Tunel positive neurons expresses in perifocal areas at different time points after I/R. The trends of expressed caspase-3, AIF,Survivin and Tunel positive neurons in HUK group and model group were basi-coincidence. The peak of expressed caspase-3,AIF and Tunel positive neurons were at 24h in perifocal areas after I/R.The peak of expressed Survivin was at 48h in perifocal areas. The optical density value of Caspase-3,AIF and Tunel positive neurons in HUK group at each time were significantly decreased compared with model groups,and the optical density value of Survivin was increased compared with model groups (P<0.05).
Conclusions:HUK play protective function for the cerebral tissues after I/R by increasing the expression of Survivin, reducing the expression of caspase-3,AIF and tardive neuronal apoptosis in posse.
Part II Effect of human urinary kallidinogenase on function of spatial learning and memory in rats and nestin expression in peri-infarction cortex after focal cerebral ischemia-reperfusion
Objective:To investigate the effects of human urinary kallidinogenase (HUK) on the ability of spatial learning and memory in rats and the expression of nestin in peri-infarction cortex after focal cerebral ischemia-reperfusion.
Methods:The focal cerebral Ischemia-reperfusion model was established by introducing an intraluminal filament into the right middle cerebral artery of the rats, then HUK was administered introperitoneally after the operation and afterward once a day for 2 weeks. The spatial learning and memory function was studied by morris water maze test,Then Nissl staining technique was conducted for observing the morphology of neurons in peri-infarction cortex,the nestin expression in the peri-infarction cortex was measured by immunohistochemistry.
Results:The ischemia-control group exhibited seriously spatial learning and memory deficits in both place navigation trail and spatial probe trial.In place navigation trial, the mean values of escape latency of middle-dose and higher-dose HUK groups were shorter than those of ischemia-control group (P<0.05).In spatial probe trial, there were significant difference in the time percentage of spending in the former platform quadrant and frequency of crossing the former platform site between middle and higher-dose HUK groups and ischemia-control group(P<0.05). Nissl staining analysis showed that the number of neurons in the peri-infarction cortex of middle and higher-dose HUK groups significantly increased as compared with that of ischemia-control group (P<0.05).Immunohistochemical analysis showed that nestin expression in the peri-infarction cortex of middle and higher-dose HUK groups significantly increased as compared with that of ischemia-control group (P<0.05).
Conclusion:HUK improves the rats spatial learning and memory ability after focal cerebral ischemia-reperfusion, this may be the results of the increasing expression of nestin and the proliferation of neural stem cells after focal cerebral ischemia.
引文
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