【作者】 胡薇；

【导师】 张国华；

【作者基本信息】 中国医科大学 ， 法医学， 2009， 硕士

【摘要】 前言我国是一个人口大国,而且是一个老龄化进程最快、老龄人口众多的国家。我国又是一个具有悠久历史、多种多样酒文化并存的国家。由于特殊的社会和历史原因,酒精中毒的情况经常发生。慢性酒精中毒(chronic alcoholism)是以对乙醇的成瘾为特征,由于经常反复饮酒对乙醇产生耐受,当其戒酒时出现脱瘾(withdrawal)的神经症候。慢性酒精中毒引起神经系统及其他器官的严重损害,其中神经系统的损害最为严重。在法医学实践中,有时会遇到酒精中毒的案例,常与交通事故、重要器官组织功能障碍等有关。阿尔茨海默病(Alzheimer’s disease,AD)是一种渐进性的神经变性性疾病,表现为全面的认知功能障碍。主要病理学改变是由来源于淀粉样前体蛋白(amyloid precursor protein,APP)的β-淀粉样肽(beta-amyloid,Aβ)构成的淀粉样斑块和过度或异常磷酸化的Tau蛋白构成的神经纤维缠结。阿尔茨海默病的发病受很多因素影响,包括环境因素、不良饮食习惯、药物种类、中毒等。慢性酒精中毒是否会导致脑内APP和Aβ蛋白的增多从而引发阿尔茨海默病的发生,目前尚缺乏系统的理论和实验研究。神经细胞特异性烯醇化酶(Neuron-specific enolase,NSE)是神经细胞的标志性蛋白,特异性的存在于神经元和神经内分泌细胞中,占脑内全部可溶性蛋白的1.5%,神经元损伤或坏死后NSE进入血液。本研究在建立小鼠慢性酒精中毒的动物模型基础上,应用组织病理学和免疫组织化学等技术,研究小鼠慢性酒精中毒后,脑组织神经细胞中APP和Aβ的表达,并应用SPSS统计软件对观察到的实验结果进行统计学分析,探讨慢性酒精中毒脑神经细胞中APP和Aβ的含量变化以及与阿尔茨海默病发病之间的因果关系。材料和方法一、动物模型的建立及分组雄性健康9周龄昆明小鼠42只,随机分为30天组,60天组,每组又分10%酒精组、20%酒精组和对照组,其中对照组各5只,10%酒精组各8只,20%酒精组各8只。实验组将无水乙醇(色谱纯)用饮用矿泉水配成浓度为10%、20%酒精溶液作为实验小鼠饮用水,对照组采用饮用矿泉水代替。实验结束后,直接断头处死动物,迅速提取小鼠脑组织,4%多聚甲醛/PBS pH7.4固定液固定,石蜡包埋、制片。二、实验方法(1)应用HE染色法观察小鼠慢性酒精中毒后脑神经细胞的病理组织学形态变化;(2)应用免疫组织化学染色法观察小鼠慢性酒精中毒后脑神经细胞内神经元特异性烯醇化酶表达的变化;(3)应用免疫组织化学染色法观察小鼠慢性酒精中毒后脑神经细胞内APP和Aβ表达情况;(4)采用Motic Images Advanced 3.2图像分析系统计算免疫组织化学染色阳性细胞数和细胞内阳性反应产物的光密度;(5)应用SPSS统计学软件对实验数据进行统计分析。实验结果(1)各实验组小鼠出现明显的慢性中毒表现,且体重明显下降;(2) HE染色见慢性酒精中毒的小鼠脑组织水肿,神经细胞内尼氏体消失或边聚,神经细胞周围间隙增宽;(3)神经元特异性烯醇化酶免疫组织化学染色见该酶蛋白含量增加;(4)对照组可见少量APP免疫组织化学染色阳性的神经细胞,染色产物分布于细胞胞浆内,呈棕黄色。实验组APP染色阳性细胞明显增多,表达增强,且不同浓度不同时间段阳性表达强度不同。实验组脑神经细胞胞浆内出现明显的Aβ阳性染色产物,且高浓度长时间给药组阳性细胞数明显增多,表达增强。使用Motic Image Advanced 3.2图像分析系统计算各组平均积分光密度,并采用SPSS软件进行统计分析,发现实验组和对照组间有明显差异。结论1.慢性酒精中毒可引起实验小鼠脑神经细胞呈退行性改变;2.慢性酒精中毒可引起实验小鼠脑神经细胞神经元特异性烯醇化酶含量增多和分布异常;3.慢性酒精中毒可引起实验小鼠脑神经细胞胞浆内产生Aβ蛋白,且APP蛋白的表达明显增强。4.慢性酒精中毒可能是诱发阿尔茨海默病发病的有关因素之一。更多还原

【Abstract】 IntroductionChina is a country with a large population and the aging process is very fast. China is a long history,and diverse cultures co-exist wine in the country.Because of the particular social and historical reasons,alcoholism frequently is the case.Chronic alcoholism is characterized by addiction,as is often repeated in drinking on the ethanol tolerance,when it occurs when alcohol withdrawal(withdrawal) symptoms of the nerve.Nervous system caused by chronic alcoholism and serious damage to other organs,including nervous system damage to the most serious.Practice in forensic science,and sometimes encounter cases of alcoholism are often associated with traffic accidents,major organ dysfunction and other related organizations.Alzheimer’s disease(AD) is a progressive neurodegenerative disease,manifested as a comprehensive cognitive impairment.The main histopathological changes is derived from amyloid precursor protein(APP) ofβ-amyloid peptide(Aβ) consisting of amyloid plaque and excessive or abnormal phosphorylation of Tau protein composition neurofibrillary tangles.The incidence of Alzheimer’s disease are subject to many factors,including environmental factors,poor dietary habits,drug types,such as poisoning.Chronic alcoholism lead to brain APP and Aβprotein in order to trigger an increase in the incidence of Alzheimer’s disease,is still a lack of systematic theoretical and experimental studies.Neuron-specific enolase(NSE) is a sign of nerve cell protein,the existence of specificity in neurons and neuroendocrine cells in the brain of all soluble protein accounted for 1.5%of neuronal damage or NSE into the blood after necrosis.In this study,mice in the establishment of animal models of chronic alcoholism, based on the application of histopathological and immunohistochemical techniques, research in mice after chronic alcoholism and brain nerve cells in the expression of APP and Aβ,and the application of SPSS statistical software of the observed experimental results were statistically analyzed to explore neurons in chronic alcoholism APP and Aβ,as well as the content changes in the incidence of Alzheimer’s disease and the causal link between them.Materials and Methods1.Establishment of animal model and packet42 9-week-old Kunming mice were randomly divided into 30-day group,the 60-day groups was divided into 10%alcohol group,20%alcohol group and the control group,of which five of the control group,10%ethanol group 8,and 20%alcohol group 8.The experimental group will be anhydrous ethanol(HPLC pure) in drinking water to match the concentration of 10%,20%alcohol solution as the drinking water of mice in the control group used in place of drinking water.After the end of the experiment, animals were killed directly decapitation,rapid extraction of mouse brain tissue,4% paraformaldehyde/PBS pH7.4 fixed,paraffin-embedded,producer.2.Methods(1) HE staining to observe the application of the morphological characteristics of organizations.(2) the use of Nissl staining of nerve cells circumstances Nissl bodies.(3) Application of neuron-specific enolase staining patterns characteristic of neurons.(4) immunohistochemical staining APP and Aβin the brain tissues.(5) the use of Motic Images Advanced 3.2 image analysis system to calculate immunohistochemical staining positive cells and expression of the area.(6) the application of SPSS statistical software for statistical analysis of experimental dataResult(1) The experimental group of mice significantly the performance of chronic poisoning,and decreased body weight. (2) HE staining of chronic alcoholism see the mice brain edema,nerve cells in the disappearance of Nissl bodies or margination,nerve cells around the gap widened.(3) neuron-specific enolase immunohistochemistry see increase in enzyme protein content.(4) the control group shows that a small number of APP positive immunohistochemical staining of nerve cells,a product of the distribution of staining in the cell cytoplasm,showing brown yellow.Experimental group,APP staining positive cells markedly increased expression of different concentrations and different time periods of different intensity of positive expression.Experimental group,nerve cells showed a visible product of Aβ-positive staining,and high concentrations of prolonged treatment group the number of positive cells markedly increased expression.Using Motic Image Advanced 3.2 image analysis system to calculate the average integrated optical density,and the use of SPSS software for statistical analysis and found that the experimental group and the control group there was significant difference.Conclusion(1) Chronic alcoholism can cause brain cells of mice degeneration.(2) Chronic alcoholism can cause brain cells of mice neuron-specific enolase increase in content and distribution of abnormalities.(3) Chronic alcoholism can cause brain cells of mice produce Aβprotein,and APP protein expression was significantly enhanced.(4) Chronic alcoholism may be induced by Alzheimer’s disease,one of the relevant factors.更多还原