【作者】 李岳美；

【导师】 鄢文海；

【作者基本信息】 郑州大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 目的阿尔茨海默病(Alzheimer’s disease,AD)是一种渐进性神经系统退行性疾病,严重危害老年人的生活质量甚至生命,其发病率随着世界人口的老龄化进程呈迅速上升趋势。AD主要病理特征表现为在神经细胞之间出现大量的淀粉样斑块(senile plaques,SPs)沉积和神经细胞内出现神经元纤维缠(neural fibrillary tangles,NFTs)。NFTs是神经元异常聚集形成的不溶性复合物,主要由过度磷酸化的神经细胞骨架蛋白Tau蛋白组成。AD的主要病因之一是由于体内APP蛋白异常代谢产物β-淀粉样蛋白(β-amyloid peptide,Aβ)沉积于脑内并对神经细胞产生毒性作用,引起神经元主要是胆碱能神经元变性和凋亡、神经元突触和皮层神经元胆碱能受体丧失、脑皮层神经元减少以及皮层小动脉血管淀粉样变性。近年来对AD的发病机制尤其是Aβ毒性机制的相关研究一直是热点,但各种治疗方法都不甚理想。4.1蛋白是细胞膜骨架的一个基本成分,其家族包括4.1R(主要在红细胞中表达)、4.1N(神经元特异表达)、4.1G(广泛表达)和4.1B(广泛表达,以脑表达为主),它们都具有一些相同的结构域:SABD结构域、FERM结构域或称为膜结合结构域和CTD结构域。研究发现4.1N广泛表达于中枢神经系统和外周神经元中,并在胚胎早期神经元的有丝分裂过成中发现。4.1蛋白家族成员不仅具有维持细胞膜及各种细胞器膜结构、调节多种跨膜转运蛋白的功能和膜结合酶类的活性,并在蛋白质分选、调控细胞有丝分裂和介导细胞信号转导过程中起关键作用。Aβ的神经毒性作用被证明在AD发生发展过程中起着关键作用。而APP基因突变导致APP蛋白酶切位点的改变或APP基因过表达产生过多APP蛋白能够生成过量聚集态具有神经毒性的Aβ。有研究表明AD患者脑内4.1蛋白与NFTs共定位,并可与APP的羧基端序列(Y687 KFFEQMQN)相结合,这一区域非常接近β-分泌酶的水解位(Met671-Asp672)及APP锚定的必需片段(Tyr653-664),而β-分泌酶被认为是Aβ产生的限速酶,因此,4.1蛋白很可能影响APP的代谢及Aβ的生成,AD患者脑内有可能存在4.1家族基因突变或蛋白表达异常。本实验研究APP转基因小鼠脑组织中4.1家族成员在细胞定位、蛋白及mRNA水平的表达特性,探索APP蛋白及其代谢物Aβ与4.1蛋白家族之间的关系,进一步探讨AD的发病机制,为寻找更有效防治AD和其他Aβ毒性相关疾病的方法奠定理论依据。方法采用PCR技术对提取的新生APP转基因(人APP695/770)小鼠鼠尾DNA进行APP基因的检测,根据APP转基因鼠是否携带APP基因,将其分为APP(+)组和APP(-)组。取12月龄APP转基因小鼠脑组织,采用免疫组织化学染色和刚果红染色分别对APP蛋白、Aβ多肽及SPs检测。采用免疫组织化学、Western-blot及RT-PCR等技术,分别从细胞定位、蛋白及mRNA水平检测APP(+)组和APP(-)组小鼠的海马、皮层及小脑神经细胞中4.1蛋白家族成员的表达,采用独立样本t检验统计学方法分析各脑区两组之间的差异,P＜0.05表示有统计学意义。结果1.对新生APP转基因小鼠的APP DNA进行PCR检测,将APP DNA高表达组称为APP阳性鼠,APP DNA无表达组称为APP阴性鼠。2.免疫组织化学染色结果显示,12月龄APP转基因阳性小鼠海马和皮层神经细胞胞浆高表达APP蛋白,神经细胞胞浆及细胞间质高表达Aβ,而阴性小鼠无表达;刚果红染色结果显示APP转基因阳性小鼠皮层间质及海马齿状回内可见大小不一的淡红色着色团块状同质性无定形物质,而阴性小鼠未见。3.免疫组织化学法对APP(+)组和APP(-)组小鼠脑切片染色,DAB染色结果:显示两组中均有4.1家族蛋白的表达。APP(+)组:蛋白4.1N主要在大脑皮层神经细胞和海马神经细胞胞膜、轴突部位分布,两脑区细胞间质内亦可阳性着色斑块;蛋白4.1B主要在皮层神经细胞胞膜分部,并在海马齿状回内强表达;蛋白4.1G主要在皮层和海马神经细胞膜周表达,海马齿状回强表达;蛋白4.1R主要在皮层和海马神经细胞膜表达,在海马齿状回强表达。APP(-)组中:蛋白4.1N未出现着色斑块;4.1B、4.1R和4.1G在海马齿状回内无明显表达(P＜0.05,n=6)。4.Western-blot检测脑组织中4.1蛋白家族各成员的表达,4.1N、4.1G、4.1B和4.1R在APP(+)与APP(-)组小脑、海马和皮层组织中均有表达,前者较后者表达量明显增高,差异有统计学意义(P＜0.05,n=6)。5.RT-PCR检测4.1N、4.1G、4.1B和4.1R的mRNA在APP(+)与APP(-)组小脑、海马及皮层组织中均有表达,但组间表达无明显差异(P＞0.05,n=6)。结论APP转基因阳性鼠与阴性鼠脑组织中4.1蛋白家族在mRNA表达水平无明显差异,蛋白表达水平在阳性鼠明显高于阴性鼠,提示APP转基因鼠脑内4.1蛋白翻译或代谢过程异常,这种异常可能是APP高表达引起,同时4.1蛋白的代谢异常可能也参与APP蛋白的异常代谢以及Aβ生成。更多还原

【Abstract】 PURPOSEAlzheimer’s disease(AD) is a progressivly degenerative disease of the neural system,seriously affact- ting the quality of life of older people and even their lives,with the aging of the world’s population,the incidence of the process was rising.The main pathological features of AD is a large number of amyloid senile plaques(SPs) deposite between the neurons and neural fibrillary tangles(NFTs) in neurons of the brain.NFTs are insoluble compounds of the abnormal accumulation of neurons,mainly formainted by the hyperphosphorylation of protein Tau,which is a neuron cell skeleton protein.Studies showed that one major reason of AD was the abnormal metabolites of the protein APP,Which is namedβ-amyloid peptide(Aβ) depositting in the brain and neuron cells.Because Aβcunld produce toxic which effects on neurons and affact theirs degenerat-ion,or cause them to wither and death,especially the cortical neurons and neuron synapses of cholinergic receptor loss,reduction in cerebral cortical neurons,and caused cortical arterioles of the vascular amyloidosis.In recent years,the pathogenesis of the toxic mechanism of Aβ-related research on AD has been a hot spot and made great achievements,but has not yet been clarified,the clinical treatment is not ideal.Protein 4.1 is an essential element of membrane skeleton proteins,its family including protein 4.1R(red cell expression),4.1N(neuron-specific expression),4.1G (wide expression)and 4.1B(wide expression,the expression of brain-based),they have some of the same domain:SABD domain,FERM domain or the membrane binding domain and the CTD domain.4.1N was found expressed widely in the central nervous system and peripheral neurons,and were found in the mitosition of early embryonic neurons.Family protein 4.1 members not only have the functions of maintainning the cell membrane structure,regulating avariety of transmembrane protein function and activity of membrane-bound enzymes,but also play a key role in the procession of protein sorting,cell mitosion regulation and cell signal transduction.It was proved the neurotoxicity of Aβplays a critical role in the process of the occurrence and development of AD lesions.The over-expression of APP can produce excessive aggregation of neurotoxic and the APP gene mutations results in the changing in restriction sites,leading to the overexpression of Aβ.Research shows that brain protein 4.1 with neurofibrillary tangles have a total of positioning in AD patients,the former have a combination sequence(Y687 KFFEQMQN) with the protein APP in carboxy-terminal,which is close to the bit(Met671-Asp672) ofβ-secretase hydrolysis and the necessary anchoring fragment(Tyr653-664) of protein APP,andβ-secretase is considered to be the rate limiting enzyme of Aβproduce. Therefore,the 4.1 protein may affect the metabolism of APP and the generation of Aβ, There may be mutation or abnormal protein expression of 4.1 family gene existence in AD patients brain.In this study,we explore the relationship between protein APP and 4.1 protein family,also the relationship between the metabolites Aβof APP and 4.1 protein family by study the cell position,the level of protein and gene expression characteristics of 4.1 family members in APP transgenic mouse brain tissue,in order to explore the pathogenesis better prevention and treatment of of AD and other Aβ-related disease.METHODSWe extracted DNA of new born APP transgenic(human APP695/770) mice tail,using the PCR technology to detect the APP gene,according to whether APP transgenic mice carrying APP gene,the APP transgenic mice are divided into two groups:APP(+) group and APP(-) group.From 12-month-old APP transgenic mouse brain tissue to detect the expression of APP and Aβby immunohistochemical staining and Congored staining.We study in order to detecte the expression in protein and mRNA level of 4.1 protein family members in the hippocampus,cortex,and cytoplasm neural cells of APP(+) group and APP(-) mice by immunohistochemistry,Western-blot and RT-PCR technology.Statistic analysis:all the experimental results were analyzed by spss 10.0 software with independent samples t statistical methods test,the differences between the two groups in every brain regions to P＜0.05 indicated statistical significance.RESULTS1.The identification results of DNA by PCR technology showed that the high expression of APP DNA in APP transgenic mice known as APP positive mice group the,but no APP DNA expression in APP transgenic mice known as the APP negative mice.2.Immunohistochemistry results showed that there are high expression of protein APP in cytoplasmic of noural cell,and Aβhigh expressed in cytoplasmic and stromal of hippocampus and cortex cells of 12-month-old APP transgenic positive mice,but no expression in APP transgenic negative mice;the results of Congo red staining showed that pink colored homogeneous mass of amorphous material can be seen in the positive APP transgenic mice cortex and hippocampus interstitial,but no expression in negative APP transgenic mice.3.The expression of the family proteins 4.1 by DAB immunohistochemical staining of APP(+) group and APP(-) group mice brain tissue showed that the two groups both have the expression of protein 4.1 family members.APP(+) group:the protein 4.1N express in the cerebral cortex and hippocampus neurol cell membrane, connecting parts of axons and cell distribution of interstitial plaque can also be positive staining in the two brain regions.Protein 4.1B was filamentous distribution mainly in cerebral cortex and hippocampus neuron cells membrane,and strong expression in dentate gyru;Protein 4.1G mainly express in the cerebral cortex and hippocampal neural cells membrane,and express strongly in hippocampal dentate gyrus.Protein 4.1R mainly express in the cortex and hippocampal neurons memb- rane,strong expression in hippocampus.APP(-) group:every protein 4.1 family member shows a weak positive staining(P＜0.05 n=6).4.The results of Western-blot detection showed that 4.1 protein family member 4.1N,4.1G,4.1B and 4.1R express in the cerebellum,hippocampus and cortex tissues in mice brain tissue of the APP(+) and APP(-) groups,the former express higher than the latter,the difference had statistically significant(P＜0.05,n=6).5.The results of RT-PCR detection showed that mRNA of 4.1 protein family member 4.1N,4.1G,4.1B and 4.1R express in the cerebellum,hippocampus and cortex tissue in mice brain tissue of the APP(+) and APP(-) groups,but the expression of the two groups has no difference(P＞0.05,n=6).CONCLUSIONThere are no significant differences in the level of gene expression of protein 4.1 family members between the negative and positive APP transgenic mice brain tissue.But in the level of protein expression,the 4.1 family members express significantly higher in positive APP transgenic mice than in the negative APP transgenic mice,these suggesting that there may be abnormalities in 4.1 protein translation or metabolic process in APP transgenic mice brain,such abnormalities may be caused by protein APP high expression,while 4.1 protein abnormalities metabolic may also be involved in abnormal metabolism of protein APP and Aβgeneration.更多还原