【作者】 吴秀奎；

【导师】 邹原；

【作者基本信息】 大连医科大学 ， 生理学， 2009， 硕士

【摘要】 目的:阿尔茨海默病(Alzheimer’s disease, AD)是一种老年人常见的神经系统退行性疾病,是痴呆最常见的病因。目前AD的病因学和发病机制尚不清楚,也没有特异的早期诊断AD的生物学标志及有效的治疗措施,因此,AD的防治仍为世界性的医学难题。大量研究证实Aβ纤维在大脑皮质的堆积形成老年斑是AD病理发生早期的关键性事件。此外,临床病理研究发现AD患者的痴呆症状严重程度与脑组织中神经原纤维缠结(NFTs)的数量呈正相关,NTFs的主要成分为过度磷酸化的tau蛋白,提示tau蛋白异常过度磷酸化与AD的发病机制直接相关。Tau蛋白是神经细胞主要的微管相关蛋白,对促进微管形成、稳定微管系统起着重要作用。在AD患者脑中,聚集成PHF的过度磷酸化的tau蛋白(PHF-tau)明显增加。冈田酸(okdaic acid, OA)是蛋白磷酸酯酶PP-2A与PP-1的抑制剂,在离体培养的海马神经元中能够诱导tau蛋白过度磷酸化;同时,OA在体内能促进Aβ沉积,造成神经元退化,突触缺失及记忆障碍,产生类AD样病理特征。绿茶多酚(green tea polyphenols, GTPs)是从绿茶中提取出来的一种多酚类化合物,研究表明GTPs对癌症、心血管疾病、炎性疾病及神经变性疾病等均具有防治作用。但GTPs对AD中的tau蛋白的过度磷酸化是否具有抑制作用尚未有文献报道。本实验室前期研究证实GTPs能够减轻OA诱导的大鼠海马神经元tau蛋白的过度磷酸化,改善大鼠记忆损伤状况。因此,我们在前期实验的基础上,通过OA损伤大鼠原代海马神经元,在离体细胞水平上进一步观察并探讨GTPs的神经保护作用。方法:选用出生24小时内的SD大鼠,无菌分离并培养海马神经元,倒置显微镜进行形态学观察,培养至第7天,经冷丙酮固定后,尼氏染色鉴定神经元。将实验组分为6组,分别为正常对照组(normal),DMSO对照组(vehicle-control),OA损伤组(OA model)和GTPs保护组(OA+GTPs,GTPs浓度分别为5μg/ml,15μg/ml,25μg/ml)。将海马神经元培养7天后,分别将等体积的培养液、含DMSO(终浓度<0.01%)的培养液,终浓度为10 nmol/L的OA以及OA (10 nmol/L)和终浓度分别为5μg/ml,15μg/ml,25μg/ml的绿茶多酚加入培养海马神经元中,37℃、5% CO2孵育12小时。倒置显微镜下观察OA及OA+GTPs作用于海马神经元后细胞的形态学变化;MTT微量比色法检测OA及OA+GTPs对海马神经元的存活率影响;分光光度法检测细胞上清液中乳酸脱氢酶(LDH)含量的变化。免疫细胞化学法观察Ser396位点的磷酸化tau蛋白的定位与表达分布情况,Western blot方法检测海马神经元内磷酸化的tau蛋白的表达并进行定量分析。结果:1.海马神经元鉴定:原代培养大鼠海马神经元,倒置显微镜进行形态学观察,尼氏染色鉴定神经元,细胞胞浆内蓝紫色颗粒及明显的空泡状核,为神经元所特有,占细胞总数90%以上。细胞的数量与状态可用于本研究。2.细胞形态学变化:正常海马神经元胞体呈椭圆,轮廓清晰,轴突交错成网,而OA模型组(10 nmol/L)的海马神经元明显减少,胞体呈不规则圆形,轴突明显退化、缩短、断裂;GTPs处理组中,低剂量保护组(OA 10 nmol/L+GTPs 5μg/ml)与OA没有明显差别,中、高剂量保护组(OA 10 nmol/L+GTPs 15μg/ml、OA 10 nmol/L+GTPs 25μg/ml)较OA组细胞数增多,可见轴突缩短、断裂现象明显减少,神经元胞体之间的轴突仍可见连接成网状结构。3.细胞存活率及LDH:OA 10nmol/L损伤及不同浓度GTPs与OA共同孵育作用于海马神经元后,MTT检测细胞存活率,上清检测LDH含量,可见OA模型组比正常对照组及DMSO组细胞存活率明显降低(P<0.05 ),LDH含量明显增多(P<0.01);GTPs保护组较OA模型组存活率明显提高(P<0.05 ),LDH漏出明显减少(P<0.01),以GTPs 15μg/ml及25μg/ml剂量组明显。4.海马神经元过度磷酸化tau蛋白的表达:免疫细胞化学结果显示,OA模型组海马神经元胞体和突起的pTau-Ser396表达较正常对照组和DMSO对照组显著增多,呈棕黄色染色,GTPs保护组的pTau-Ser396阳性表达较OA模型组明显减少。Western blot实验进一步证实免疫细胞化学的结果,定量分析表明,OA模型组pTau-Ser396的表达量比正常组和DMSO对照组增加,而GTPs保护组表达量则比OA模型组降低,尤其以GTPs 15μg/ml及25μg/ml剂量组明显,具有统计学意义(p<0.01)。结论:冈田酸能够诱导大鼠原代海马神经元tau蛋白过度磷酸化并引起神经元死亡,轴突断裂、损伤,降低细胞存活率,绿茶多酚能够减轻冈田酸的毒性损伤,提高细胞存活率,降低tau蛋白过度磷酸化程度。更多还原

【Abstract】 Objectives:Alzheimer’s disease (AD) is a progressive neurodegenera- tive disease and the most common form of dementia. The neuropathological hallmarks of AD include extensive neuronal loss, the presence of numerous neurofibrillary tangles (NFTs), and senile plaques (SP) in the brain. The major constituent of a senile plaque is Aβand the NFTs are composed of hyperphosphorylated tau protein. So far, the etiology and mechanisms of AD still have been unknown clearly, and no objective and specific biomarkers can be used for early diagnosis and efficient cure. Accordingly, strategies that oppose proteolytic processing of APP into Aβpeptides and ensuing cerebral amyloidosis have remained a primary focus of AD research. Clinical researches found that the formation of NFTs is closely correlated with the temporal and spatial progression of AD pathology and other tauopathies. In the case of AD and various other tauopathies, tau has been found to be abnormally phosphorylated or dephosphorylated at specific residues by several possible neuronal kinases/phosphotases both in vitro and in vivo, which is hypothesized to ultimately lead to conformational abnormality, dysfunction, and aggregation. Okadaic acid (OA) an inhibitor of protein phosphotase 2A (PP-2A) and PP-1, has been a useful tool to study AD. Treatment neurons with OA in vitro or in vivo induced aberrant hyperphosphorylation of tau or neuronal death or both. Previous studies showed that apoptosis was involved in neuronal death induced by OA or other inhibitors of PP in vitro.Tea polyphenols are natural plant flavonoids present in the leaves and stem of tea plant. The green tea polyphenols comprise (-)-epigallocatechin- 3-gallate (EGCG), (-)-epigallocatechin, (-)-epicatechin, (-)-gallocatechin, and catechin. Many biological functions of tea polyphenols have been reported, including anti-inflammatory, anticarcinogenic, and antioxidant effects. However, whether green tea polyphenol can reduce the OA induced toxicity and inhibit the tau hyperphosphorylation has been unknown. Our previous studies (unpublished) indicated that OA can induce tau protein hyperphosphorylation and cause memory impairment, while GTPs can reduce the hyperphosphorylation and attenuate the impairment. Therefore, according to our previous data and references, the aim of present study is to explore the effects of green tea polyphenols (GTPs) on Okadaic acid induced toxicity and tau phosphorylation in cultured primary hippocampal neurons.Methods: Hippocampal cell cultures were prepared from newly born(less than 24 hours) Sprague-Dawley rats and neurons were cultured in CO2 incubator with 37℃and 5% CO2 .The experiments were performed using 7-day-old cultures, at which time the hippocampal neurons are fully different- tiated. On the 7th day, hippocampal neurons were fixed in cold acetone and identified by thionine staining. For experiments, neurons were divided into 6 groups which respectively added normal culture medium, culture medium contained DMSO(<0.01%), 10 nmol/L OA, and OA co-treatment with three different concentration GTPs(5μg/ml, 15μg/ml, 25μg/ml). Neurons were visualized and photographed by using phase-contrast microscopy. Cell viability was evaluated 12 h later using the MTT colorimetric assay, whereas the extent of neuronal injury was measured by using lactate dehydrogenase (LDH) release as a marker for membrane leakage and cell death. Immuno- cytochemistry was used to detect the distribution of hyperphosphorylated tau protein, while Western blot were used to analyze the expression of hyperpho- sphorylated tau protein.Results:1. Hippocampal neurons were approximately 90% of the total cells in the cultured system.2. Normal healthy neurons were defined as having a cell body that was phase bright, with a smooth contour, round to oval shape, and multiple neuritic processes. After exposure for 12 h to OA(10 nmol/L) treatment, degenerating neurons were characterized by marked fragmentation and degeneration of neurites and a round irregular cell body, whereas OA co- treatment with GTPs exhibited less impairment, especially the OA+GTPs 15μg/ml and OA+GTPs 25μg/ml groups.3. The MTT colorimetric assay and LDH release revealed that a 12 h exposure to OA(10 nmol/L) is neurotoxic to hippocampal cells(about 30% decrease in MTT values vs. control and approximately 50% increase in LDH release vs. control).Both assays revealed that the toxic effect of OA was reduced, in a dose-dependent manner, by a co-treatment with GTPs. These effects are significant at 15μg/ml and 25μg/ml.4. Immunocytochemistry staining showed that tau protein hyperpho- sphorylation at Ser 396 (PHF-13)-positive neuritic processes appeared very clearly and PHF-13 immunoreactivity was marked increased in cell bodies after exposure to OA(10 nmol/L) for 12 h. Whereas, the groups co-treated with GTPs was obviously reduced the PHF-13 immunoreactivity, especially at the dose of 15μg/ml and 25μg/ml. Moreover, examination of the tau in a total extract of cells by using Western blot suggested that average relative density was obviously increased in the OA group, while the groups of OA co-treated with GTPs decreased obviously when compared with OA group, especially at 15μg/ml and 25μg/ml (P<0.01).Conclusions:Our results demonstrate that treatment of cultured primary hippocampal neurons with OA (10 nmol/L) induced cell neurotoxicity and hyperpho- sphorylation of tau, while GTPs can attenuate these neurotoxic effects.更多还原