【作者】 陈志则；

【导师】 张传汉；

【作者基本信息】 华中科技大学 ， 麻醉学， 2010， 博士

【摘要】 研究背景和目的近年来,伴随着临床急救水平的进一步提高,很多心肺复苏、心肌梗塞、窒息、休克等危重病人抢救成功率逐渐提高,而这些疾病所造成的全脑缺血损伤以及缺血再灌注后损伤已成为研究热点。其中,脑缺血及再灌注后如何减少神经元死亡一直是临床研究的难点。大量学者经过一系列脑缺血动物模型研究,分别从形态学、生化等多角度证实凋亡存在于脑缺血再灌注损伤中。因此,研究如何有效抑制凋亡是减轻脑缺血再灌注损伤的一个重要环节。研究认为,Cdh1为细胞周期末期促进复合物(anaphase promoting complex, APC)的调节亚基之一,发现在增殖细胞中,其起到调控细胞周期的作用,是通过泛素化降解特定的底物蛋白来实现的。随着最近一些年来的研究发现,Cdh1在成熟神经元中有大量表达,并且具有调节突触传递、神经元存活以及神经元轴突生长等的作用。然而,目前尚不清楚关于APC-Cdh1在神经损伤等中枢神经系统其他方面的作用。Almeida等研究发现APC-Cdhl具有抑制神经元凋亡的作用。推测APC-Cdhl的活性降低与Cyclin B的异常积聚可能是阿尔茨海默病及其他神经退行性疾病中神经元凋亡的机制之一。当前,在我国国内关于细胞周期末期促进复合物(anaphase promoting complex, APC)之调节亚基Cdhl在脑缺血再灌注损伤中的表达变化,以及对APC-Cdhl的表达进行干预在脑缺血再灌注损伤中的作用以及机制的研究还很少。本研究将首先通过建立大鼠全脑缺血再灌注损伤模型,探讨脑缺血再灌注损伤后APC-Cdh1的表达变化与神经元凋亡及神经功能改变的关系。然后通过下调APC-Cdh1活性,实现对脑缺血后神经元凋亡的影响,从在体实验的角度探讨APC-Cdhl在缺血性脑损伤中的作用及机制。继而为选择APC-Cdhl作为脑保护新策略提供实验依据。研究方法与结果1.大鼠全脑缺血再灌注损伤后神经元凋亡及认知功能的研究方法：取健康成年雄性SD大鼠80只,体重260-300 g,随机分成假手术组(SH组)、缺血再灌注组(R组),每组各40只。根据再灌注的不同时间点分为1d、3d、7d三个亚组。假手术组只电凝椎动脉而不结扎两侧颈总动脉,缺血再灌注组采用改良4-VO法建立大鼠全脑缺血再灌注损伤模型,缺血时间为7min。根据分组于不同时间点取大鼠脑部制作冰冻切片,尼氏染色观察海马神经元的形态和海马神经元的存活情况,TUNEL法检测海马CA1区凋亡细胞,分别计算神经元密度和凋亡指数。两组大鼠建立模型后第七天后开始,进行Morris水迷宫测试大鼠的学习记忆功能。结果：尼氏染色显示,假手术组海马神经元蓝染,细胞排列整齐,海马形态完整,CA1区神经细胞4-5层,排列整齐、细胞核大而圆,核仁清楚,亚甲胺蓝染色显示尼氏体丰富。缺血再灌注组第1d、3d、7d亚组海马CA1区可见神经细胞脱失明显,排列紊乱,胞体形状不规则。尼氏体减少甚至消失、核变小、深染固缩。与假手术组比较,缺血再灌注组海马CA1区神经元密度显著下降(p<0.05); TUNEL法检测显示,假手术组海马区偶见凋亡神经元。缺血再灌注组第1d、3d、7d亚组海马CA1区神经元大量凋亡。与假手术组相比,缺血再灌注组第1,3,7天亚组凋亡指数明显增加,差异有显著统计学意义(P<0.01)。Morris水迷宫检测：Morris水迷宫检测发现,与假手术组相比,缺血再灌注组大鼠训练阶段第2—4天的寻台时间明显延长,差异有统计学意义(P<0.05),而第五天的记忆测试寻台潜伏期也明显长于假手术组,差异有显著统计学意义(P<0.01)。2.大鼠全脑缺血再灌注损伤后APC-Cdhl的表达变化方法：健康雄性SD大鼠60只,体重260～300 g,随机分成假手术组(SH组)、模型组(R组)。根据再灌注的不同时间点分为1d、3d、7d三个亚组。模型组采用改良4-VO法建立SD大鼠全脑缺血再灌注损伤模型,假手术组只电凝椎动脉而不结扎两侧颈总动脉,缺血时间为7min。根据分组在损伤后不同时间点,取大鼠脑部制作冰冻切片,免疫组化染色检测Cdh1的表达部位并且采用实时荧光定量PCR检测大鼠海马组织APC-Cdh1mRNA的表达以及采用Western Blot检测大鼠海马组织APC-Cdhl蛋白的表达。结果：免疫组化检测显示APC-Cdhl在海马区及皮层区中均有大量表达。荧光定量PCR显示,对照组中,术后不同时点Cdhl mRNA表达比较,差异无统计学意义(P>0.05)；与对照组比较,模型组第1d亚组Cdh1 mRNA表达减少,差异有统计学意义(P<0.05),第3d亚组及第7d亚组Cdhl mRNA表达升高,差异有统计学意义(P<0.05)；模型组中,与第1d亚组比较,第3d亚组Cdh1 mRNA表达升高,差异有统计学意义(P<0.05),与第3d亚组比较,第7d亚组Cdh1 mRNA表达降低,差异有统计学意义(P<0.05)。Western Blot显示,与对照组相比,缺血再灌注组术后第1d组APC-Cdhl蛋白表达减少,术后第3d显著升高,术后7d又降低,差异有统计学意义(p<0.05)。3.慢病毒介导的Cdhl-siRNA在大鼠脑缺血再灌注损伤后的表达及功能方法：150只雄性SD大鼠,体重260～300 g,随机分成生理盐水组(A组,n=50)、空慢病毒组(B组,n=50)和重组慢病毒(C组,n=50),三组分别注射生理盐水、空慢病毒和重组慢病毒。药物注射3天后,各组大鼠均采用改良4-VO法建立SD大鼠全脑缺血模型,缺血时间为7min。建立模型七天后取大鼠脑部制作冰冻切片,以及取大鼠海马组织,分别用荧光定量PCR检测海马组织Cdh1 mRNA表达和Western Blot检测Cyclin B的变化。TUNEL法检测海马CA1区凋亡细胞,计算各组大鼠的凋亡指数。建立模型七天后,三组大鼠进行Morris水迷宫测试大鼠的学习记忆功能的变化。结果：荧光定量PCR显示,与A组和B组比较,C组的Cdh1 mRNA表达明降低,差异有统计学意义(P<0.05)。Western Blot显示,与A组和B组比较,C组CyclinB的表达升高,差异有统计学意义(P<0.05)。TUNEL法检测显示：与A组和B组比较,C组凋亡指数显著增加(P<0.05)。Morris水迷宫检测显示,与A组和B组比较,C组大鼠学习阶段即第一天至第四天大鼠寻台时间明显延长,而其记忆测试潜伏期也明显长于A组或B组,差异有统计学意义(P<0.05)。研究结论1.成功地构建了全脑缺血再灌注损伤模型。采用改良4-VO法建立SD大鼠全脑缺血再灌注损伤模型后海马神经元凋亡显著增加,认知功能明显下降。2. APC-Cdhl在大鼠正常脑组织以及全脑缺血再灌注损伤后有大量表达。而且,大鼠全脑缺血再灌注损伤后,在各不同的时点,大鼠脑组织中APC-Cdhl的表达明显增高。推测APC-Cdhl可能与中枢神经系统的损伤有着密切的关系,参与全脑缺血再灌注损伤的病理生理过程。3.大鼠海马组织注射慢病毒介导的Cdhl RNA干扰后,下调了Cdhl的表达,大鼠全脑缺血再灌注损伤后大鼠海马组织Cyclin B的表达明显增高。APC-Cdhl可能是通过Cyclin B堆积介导缺血性神经元凋亡的。研究总结本课题首先通过建立改良4-VO全脑缺血再灌注损伤模型,揭示了全脑缺血再灌注后对大鼠海马神经元造成损伤,海马神经元出现迟发性死亡,大鼠的学习记忆能力下降,认知功能明显降低。然后在建立大鼠全脑缺血再灌注后,发现的大鼠海马区APC-Cdh1的表达升高。提示APC-Cdh1可能参与全脑缺血再灌注损伤等病理生理过程。然后通过脑立体定向注射Cdhl RNA干扰慢病毒载体至大鼠的海马后,在相应的时间点建立改良4-VO全脑缺血再灌注损伤模型。观察慢病毒介导的Cdh1 RNA干扰对全脑缺血再灌注损伤的影响,发现Cdhl的表达下调,Cyclin B表达升高,大鼠的学习记忆能力下降,认知功能明显降低。本实验初步研究了APC-Cdh1在全脑缺血再灌注后在脑组织中的表达特点,以及全脑缺血再灌注后的作用。进一步探讨利用对APC-Cdh1的干预,为脑缺血再灌注损伤治疗及脑保护提供新的选择和实验依据。更多还原

【Abstract】 Background and objectiveRecent years, accompanied with the improvement of the clinical emergency ability, the successful rate of rescuing patients undergoing cardiopulmonary resuscitation, myocardial infarction, asphyxia, shock and other critical conditions is increased gradually. The cerebral ischemia and ischemia-reperfusion injury caused by these diseases has become a hotspot. Among them, the main difficulty in clinical research lies in how to reduce neuronal death after cerebral ischemia and reperfusion. Through a series of researches on cerebral ischemia animal model, a large number of researchers confirmed that the apoptosis exist in cerebral ischemia-reperfusion injury in a morphology or a biochemistry way. How to inhibit apoptosis will be the key to reduce cerebral ischemia-reperfusion injury.The major ubiquitin ligase required for mitosis is the anaphase-promoting complex/cyclosome (APC/C). This unusually complex E3 ubiquitin ligase targets cell-cycle-related proteins such as cyclins and securin for degradation by the proteasome in mitosis and meiosis.The APC/C is regulated by phosphorylation, as well as by various activators and inhibitors that alter its substrate specificity at different phases of the cell cycle. Such tight control ensures appropriately timed degradation of key cell-cycle regulators.Cdhl-APC plays important roles in function and neuronal survival.,patterning synaptic development and the regulation of axonal growth.However there was few report about the feature of APC-Cdhl expression in central nervous system after cerebral ischemia reperfusion injury and the role of APC-Cdhl intervention in isc hemic cerebral injury.In our study, we first established a model of global cerebral ischemia and reperfusion injury, then and we investigated the relationship between the expression of APC-Cdhl, the neuronal apoptosis and the neurological functions changes. Later we used lentivius mediated RNAi vector to reduce the expression of APC-Cdhl and to investigate the effect of APC-Cdhl in ischemic brain injury and the possible mechanism. This study provided an experimental evidence for selecting the APC-Cdhl as a new strategy for brain protection.Methods and Results1. The changes of cognitive function and neural cell apoptosis in rats with cerebral ischemia reperfusion injuryMethods 80 male Sprague-Dawley rats were randomly divided into Sham-operated group (SH) and ischemia reperfusion group (IR). The rats of ischemia reperfusion group were induced by modified four-vessel occlusion (4-VO). In sham group, the vertebral arteries of the rats were permanently electrocauterized and the bilateral common carotid arteries were not electrocauterized. Both common arteries were occluded for seven minutes in IR group. According to the time points of ischemia reperfusion, the two groups were divided into 1d,3d,7d three subgroups respectively. At different time points, fresh frozen sections were prepared from brain tissue of the two group rats. Nissl dyeing was used to observe surviving hippocampus neurons. And neuronal apoptosis was detected by TUNEL staining. Neurons density and apoptotic index were calculated.Water maze was employed and analyzed by computer image analysis system. Seven days after the operation, learning and memory of the rats in two groups were tested by Morris water maze. Results 1). Nissl dyeing:In SH group, the hippocampal neurons were stained blue.The cell arranges were in neat rows. The forms were a complete hippocampus. CA1 area of nerve cells arranged in 4 to 5 layers and large round nucleus, clear nucleolus.Abundant Nissl bodies were showed by azomethine blue staining. In ischemia reperfusion group, the hippocampal neurons were apparent disorganized and irregular. The number of Nissl bodies were reduced or even disappeared. Nuclears became smaller and showed stained chromatin condensation.Compared with Sham group, the neuron density in ischemia reperfusion group was significantly lower (p<0.05).2). The results of TUNEL method showed that in Sham group, apoptosis of hippocampus neurons were occasionally in the CA1 area. But in ischemia reperfusion group, there were a large number of apoptosis hippocampus neurons in the CA1 area. Compared with Sham group, the apoptotic index of hippocampal neurons in ischemia reperfusion group was significantly higher (p<0.05).3). In Morris water maze tests, the incubation period of seeking the platform and the learning and memorizing ability in ischemia reperfusion group were significantly longer than sham-operated group(p<0.05).2. The feature of APC-Cdhl expression in central nervous system after cerebral ischemia reperfusion injury.Methods 60 male Sprague-Dawley rats were randomly divided into Sham-operated group (SH) and ischemia reperfusion group (IR). The rats in ischemia reperfusion group were induced by modified four-vessel occlusion (4VO). The methods were same to the first study. At different time points, fresh frozen sections were prepared from brain tissue of the two group rats.The expression of Cdhl was examined in the hippocampus by immunohistochemistry.At different times after the operations, the expression of APC-Cdhl of hippocampus was observed by quantitive real time PCR and Western Blot.Results The immuno-staining showed that Cdhl was highly expressed in cerebral cortex and hippocampus.Compared with SH group, the expression of Cdhl mRNA was significantly decreased at 1 day after injury and increased obviously at 3 days, but decreased again at 7 days. Compared with control group, the expression of Cdhl protein was significantly decreased at 1 day after injury and increased obviously at 3 days, but decreased again at 7 days(p<0.05).3. Expression and function of lentivirus-mediated Cdhl-SiRNA in cerebral ischemia-reperfusion injury in ratsMethods 150 male Sprague-Dawlcy rats were divided into group A(n=50), group B(n=50)and group C(n=50). The rats of 3 groups received injection with normal saline, lentivirus vector and recombinant lentivirus respectively. At 3 days after injection, all rats were implemented with global brain ischemia model by modified four-vessel occlusion (4-VO). The methods were same to the first study.Seven days after the surgery,the expression of Cdhl and Cyclin B were examined by quantitive real time PCR and Western Blot respectively. Apoptosis was examined using TUNEL staining method. The behavior was evaluated by Morris water maze.Results The expression of Cdhl mRNA in group C was significantly lower than those in group A and B (p<0.05), but the expression of Cyclin B in group C was higher (p<0.05). In addition,the results of TUNEL method showed that compared with group A and B, the apoptotic index of hippocampal neurons in group C was significantly increased(p<0.05). In Morris water maze tests, compared with group A and B the incubation period of seeking the platform and the learning and memorizing ability in group C was significantly longer(p<0.05).Conclusions1. The number of apoptotic neurons in hippocampus was increased and the cognitive ability was decreased after global cerebral ischemia reperfusion injury in rats 2. This result indicated that APC-Cdhl may participate in the pathophysiology of global cerebral ischemia-reperfusion injury and play an important role in the injury of central nervous system.3. APC-Cdhl may be mediated by Cyclin B accumulation in ischemic neuronal apoptosis.SummaryThis study shows that global cerebral ischemia-reperfusion injury model was successfully established by modified 4-VO. The hippocampus in rats suffered from delayed neuronal death and the apoptotic cells were increased. The learning and memory of rats were declined. This model could cause the hippocampus injury.Then we found the expression of APC-Cdhl in the rat hippocampus was increased after global cerebral ischemia-reperfusion injury. This observation suggests that APC-Cdhl may be involved in pathophysiology change of cerebral ischemia-reperfusion injury.Last Cdhl RNA interference lentiviral vector was injected to the rat hippocampus by Stereotactic injection.After three days,modified 4-VO cerebral ischemia-reperfusion injury model was established. Seven days later,we observed that the expression of Cdhl decreased and the expression of Cyclin B increased.The learning and memory of rats decreased.The results suggested that the cerebral ischemia-reperfusion injury could be aggratated by lentivirus-mediated Cdhl RNA interference.Our study exhibited the differential expression of Cdhl in global cerebral ischemia-reperfusion injury model and furtherly explored the effect of the APC-Cdhl intervention for the treatment of cerebral ischemia-reperfusion injury.lt could provided a new option and an experimental evidence for cerebral protection.更多还原