【作者】 李洪玉；

【导师】 邹原；

【作者基本信息】 大连医科大学 ， 生理学， 2008， 硕士

【摘要】 目的:阿尔茨海默病(Alzheimer’s disease, AD)目前是老年人群痴呆的主要原因,随着人口老龄化的加剧,全球发病人数日益增多。由于AD严重影响患者的生活质量并给社会和家庭带来沉重的负担,因此对于该病的防治显得尤为重要。AD的临床表现主要为进行性智能减退,病理特征为老年斑(SP)、神经元纤维缠结(NFTs)、海马锥体细胞颗粒空泡变性和神经元的缺失。SP的形成与β-样淀粉蛋白(Aβ)过量产生和沉积有关。到目前为止,它们与神经细胞退行性变性之间的关系尚不十分清楚。NFT的主要成分是过度磷酸化的Tau蛋白,它聚积在退行性变性神经元的胞体并与AD患者临床痴呆程度正相关[1],且Tau蛋白的病理改变出现在痴呆症状之前并独立于Aβ异常[2]。Tau蛋白是一种微管相关蛋白,主要存在于神经元,具有促进微管组装和维持微管稳定的功能。在AD患者脑中Tau蛋白丧失了正常生理功能,从而缠结形成NFTs。已知有多种蛋白磷酸酯酶,如PP-2A,PP-l参与Tau蛋白的AD样异常磷酸化。茶多酚(tea polyphenol, TP)是从绿茶中提取出来的一种多酚类化合物,其在B-环和C-环上的酸性羟基有很强的供氢能力,是天然的抗氧化剂。其抗氧化能力明显优于强氧化剂维生素C、维生素E[3]。大量研究已证实,TP在抑菌、抗病毒、抑制肿瘤、防治心血管疾病等方面具有良好功效,同时还具有益肝保肾作用。且有文献证实,TP在体外培养的海马神经元中能够减轻Aβ诱导的神经毒性[4],而长期给小鼠EGCG(表没食子儿茶素没食子酸酯,茶多酚的主要成分)可以诱导海马APP水平的下降[5],此外TP还能够抑制Aβ原纤维的形成、延长和脱稳定性[6]。但是TP是否对于AD中Tau蛋白的过度磷酸化具有保护作用,目前尚无文献报道。本实验通过冈田酸(okadaic acid, OA)诱导大鼠海马神经元Tau蛋白过度磷酸化实验模型,研究茶多酚对OA致海马神经元Tau蛋白过度磷酸化的作用及其大鼠学习记忆的影响。方法:选用冈田酸(okadaid acid, OA)来诱导Tau蛋白过度磷酸化。健康雄性SD大鼠共60只,体重250-300g。将大鼠随机分为6组,每组10只。包括正常对照组(normal),溶媒对照组(vehicle-control),OA损伤组(OA model)和100 mg/kg、250 mg/kg和625mg/kg不同浓度茶多酚预处理组(TP)。除正常对照组大鼠外,其它各组大鼠分别灌胃给予等量生理盐水或不同浓度TP。灌胃至第21天时对各组大鼠进行空间学习与记忆能力训练和行为学测试。至第27天时,向右海马背侧(前囟后3.8mm,中线旁开2.5mm,深度3mm)定向注射OA 1.5μL(0.473μg,10% DMSO),于10min内缓慢注射,留针5min。溶媒对照组施以等体积10%DMSO。OA注射后第二天即第28天时,进行水迷宫考试,考试后处死大鼠,收集标本。制作大鼠右侧脑组织石蜡切片,免疫组织化学染色观察脑组织中海马处磷酸化的Tau蛋白表达分布情况。Western blot方法检测脑海马内磷酸化的Tau蛋白的表达并进行定量分析。结果:1.TP降低OA致海马神经元过度磷酸化Tau蛋白的表达免疫组化结果显示, OA损伤组大鼠海马神经元胞体和突起的Tau-pSer396表达较正常对照组和溶媒对照组大鼠海马区显著增多,呈现棕黄色染色,TP预处理组的Tau-pSer396阳性表达较OA损伤组减少,其中以250mg/kg和625mg/kg剂量组的作用较明显。Western blot实验进一步证实免疫组化的结果,定量分析表明,OA损伤组Tau-pSer396的表达量比正常组和溶媒对照组增加,而TP预处理各组则比OA损伤组降低,且250 mg/kg和625 mg/kg剂量组降低明显,具有统计学意义(p<0.01)。2.TP改善OA致大鼠空间记忆障碍随着训练次数的增加,各组大鼠逃避潜伏期逐渐缩短,在给予OA注射前TP预处理的三个组大鼠与OA损伤组大鼠相比其逃避潜伏期差异有显著性(p<0.01)。TP预处理的三个组和OA损伤组大鼠在给予OA前后穿越平台的次数经Kruskal-Wallis H检验有显著性差异(p<0.05)。在总时间一定的情况下,OA损伤组大鼠给予OA后,在目的象限停留时间及在目的象限游泳距离占总游泳距离的百分比显著降低(p<0.01),而TP预处理的三个组大鼠在OA注射后在目的象限停留时间及在目的象限游泳距离占总游泳距离的百分比无显著变化。此外,OA注射后,TP预处理的三个组大鼠较OA损伤组大鼠,在目的象限停留时间及在目的象限游泳距离占总游泳距离的百分比显著增加(p<0.01)。上述结果表明,冈田酸(OA)使得大鼠空间记忆出现障碍,表现为穿越平台次数减少,目的象限停留时间缩短,在目的象限游泳距离占总游泳距离的百分比减少;而茶多酚(TP)预处理能够改善大鼠记忆障碍,增强学习能力,即缩短逃避潜伏期,增加大鼠穿越平台的次数及在目的象限停留的时间,增加在目的象限游泳距离占总游泳距离的百分比并表现出一定的搜索策略。结论:冈田酸能够诱导大鼠海马神经元Tau蛋白过度磷酸化并引起大鼠空间记忆损伤,茶多酚能够减轻Tau蛋白的过度磷酸化,改善大鼠记忆损伤状况。更多还原

【Abstract】 Objectives:Alzheimer’s disease (AD) is the most common cause of dementia in the elderly population. With the increasing of the population aging and incidence of AD, heavy burden was brought to society and family due to poor life quality in AD patients. The clinical manifestations of AD is a smart diminish and AD is characterized by the presence of two histo- pathological hallmark brain lesions, extracellular deposits ofβ-amyloid in neuritic plaques and intracellular neurofibrillary tangles (NFTs). The latter is composed of bundles of paired helical filaments (PHF), the major protein subunit of which is the microtubule-associated protein Tau. Tau in PHF is different from that in normal neurons. It is abnormally hyperphosphorylated, aggregated into filaments, and does not bind to microtubules or stimulate microtubule assembly. Evidence from several studies has indicated that the hyperphosphorylation of Tau is responsible for its loss of biological activity and its resistance to proteolytic degradation, and probably plays a key role in neurofibrillary degeneration in AD. Many studies have suggested that the number of NFTs is correlated with the degree of dementia in AD.Green tea is a drink made from the steamed and dried leaves of the Camellia sinesis plant, a natural oxidation inhibitor, a shrub native to Asia. It is a beverage that is widely consumed in China, Japan, and other Asian nations and that is becoming more popular in Western countries. Recently, green tea has attracted attention for its healthy benefits, particularly with respect to its potential for preventing and treating cancer, cardiovascular diseases, inflammatory diseases, and neurodegenerative diseases in humans. Some references suggest that TP can inhibition of amyloid fibril formation in vitro and protect neurons againstβ-amyloid-induced toxicity in vitro. However, it is still unclear whether the TP has a protective effect in the hyperphosphorylation of Tau protein of AD. The aim of this study is to explore the function of tea polyphenols (TP) on the Tau protein hyperphosphorylation in hippocampal neurons in rats, which is induced by okadaic acid (OA).Methods: We chose OA as the inducer of Tau protein hyper- phosphorylation. Healthy male SD rats were divided randomly into six groups (n=10): normal group, vehicle-control group, model group treated with OA, and three TP groups, pretreated with TP in 100 mg/kg、250 mg/kg and 625mg/kg. At the 21st day of the rats of TP-pretreated groups intragastric administration, we operated ethology (water maze) test. At the 27th day, the rats were anesthetized with 2% of sodium pentobarbital (40 mg / kg, ip), according to rat brain stereotactic map, restrained in a stereotaxis apparatus and microinjected OA (soluble in 10% DMSO) 1.5μL (0.473μg) into the right dorsal hippocampus (A: -3.8 mm, L: 2.5 mm from bregma, and V: 3mm) within 10min, staying for 5min. The vehicle-control group was microinjected 10%DMSO 1.5μL in the same position. At the 28th day, we made the exzamination of water maze, then sacrificed the rats and collected the samples. Paraffin slides were used for immunohistochemistry analysis of hyperphosphorylation of Tau protein. Hippocamp were prepared for western blot analysis of hyperphosphorylation of Tau protein. Quantified analysis of Western blot was performed later.Result:1. TP attenuates the expression of hyperphosphorylation of Tau induced by OA.Immunohistochemistry staining indicated that there is strong positive hyperphosphorylation of Tau granules in the OA group and abundant brown positive hyperphosphorylation of Tau granules were detected in the hipocamp of TP pretreated-groups, but weaker than OA group, especially in 250 and 625mg/kg TP groups.Furthermore, Western blot analysis showed that the average relative photodensity was obviously increased in the OA group compared with the normal group. With TP pretreated, hyperphosphorylation of Tau expression was decreased, and a significant difference between TP and OA group still could be seen (P < 0.01). 2. TP improves OA-induced spatial memory impairment in ratsWith the increasing of training number of time, the escape latency of each group shortened gradually. And there was a significant difference between the TP pre-treared groups and OA model group before the microinjection (P<0.01). There was a significant difference on the number of crossing platform among each group before and after OA microinjection. The total time was the same and there was a significant difference about the time that the rats stayed in the target quadrant of OA model group between before and after OA microinjection (P<0.01). After OA microinjection, there was a significant difference between the TP pre-treared groups and OA model group about the time that the rats stayed in the target quadrant (P<0.01). After OA microinjection, the percentage of swimming distance in target quadrant in total time of each group was smaller than that before OA microinjection, and there was a significant decreasingof OA model group (P<0.01). After OA microinjection, there was a significant difference between the TP pre-treared groups and OA model group about the percentage of swimming distance in target quadrant (P<0.01). These results suggested that OA can make rats space and memory impairment, in the manifestation of the reduction in the number of cross platform, shorten the time and swimming distance of staying in the target quadrant. But pretreated with TP can improve this impairment, showing that shortening the escape latency, increasing the number of rats across the platform and the time staying in the target quadrant and the swimming distance. The rats reasched the plat, according to some strategy.Conclusions: Our data suggested that OA can make Tau hyperphosphorylation and memory impairment, and TP can decrease the hyperphosphorylation and improve the impairment.更多还原