【作者】 张丽红；

【导师】 王占友；

【作者基本信息】 中国医科大学 ， 人体解剖与组织胚胎学， 2008， 博士

【摘要】 前言阿尔茨海默病(Alzheimer’s disease,AD)是主要发生在老年人的一种以进行性痴呆为临床特征的神经退行性疾病。随着人口的逐渐老龄化,AD的发病率呈逐年上升趋势,给社会和家庭带来了沉重的负担,由于缺乏有效的治疗手段,AD已成为危害人类健康的主要致死性疾病之一。AD的典型病理特征包括:β-淀粉样蛋白(β-amyloid,Aβ)在脑内病理性沉积形成老年斑、神经原纤维缠结(neurofibrillary tangles,NFT)和大脑淀粉样血管病(cerebral amyloid angiopath,CAA)。Aβ是AD发生和发展的中心环节,体内的Aβ由β-淀粉样前体蛋白(amyloidprecursor protein,APP)经β-和γ-分泌酶水解而成。到目前为止,AD的具体发病机制还不是十分清楚,但越来越多的证据表明,锌离子在AD的发病和病理过程中起着关键性的作用。锌离子能够通过链接相邻Aβ分子的第13位上的组氨酸而导致Aβ聚集;与Aβ生成密切相关的γ-分泌酶为锌结合蛋白,细胞内的锌离子具有抑制γ-分泌酶裂解APP向细胞外分泌Aβ的作用;口服金属螯合剂可以明显抑制APP转基因小鼠脑内老年斑的形成。因此,调节细胞内外金属离子稳态,维持Aβ生成与降解的动态平衡,已经成为目前AD治疗学的一个重要策略。锌离子不能自由通过细胞膜,特定的转运体和膜通道参与锌的转运和代谢。锌转运体(zinc transporter,ZnT)是参与脑锌代谢的重要蛋白家族之一,目前至少拥有7个成员(ZnT1-7),除ZnT2外均在脑内表达,其主要功能是将锌离子转运出细胞或聚集在细胞器内:ZnT1定位在细胞膜上,负责将细胞内的锌离子向细胞外转运;ZnT3定位在突触小泡膜上,其功能是将锌离子转运并聚集在突触小泡内;ZnT4定位在胞质小泡膜上,可将细胞质内的锌离子转运到胞质小泡内;ZnT5-7定位在高尔基复合体,参与高尔基复合体内外的锌离子转运过程。最新的研究表明,ZnT1,4,6在AD病人大脑内的表达明显增强,而敲除ZnT3基因的APP转基因惺?其脑内Aβ老年斑数量、血管淀粉样改变均明显减少,提示ZnT与锌离子共同参与了AD的发病和病理过程。本研究对锌离子和ZnT在AD和APP/PS1转基因小鼠脑内的表达变化、定位分布及与Aβ的相关性进行系统分析,应用RNA干扰技术(RNA interference,RNAi)技术探讨ZnT1基因沉默阻抑APP转染细胞Aβ分泌的效果,对深入探讨脑锌代谢紊乱与AD病理生理机制具有重要意义。实验方法采用AD病人尸检脑组织、APP/PS1转基因小鼠及稳定转染APPsw、APP基因的SH-SY5Y细胞(APPsw细胞、APP细胞)为研究对象,应用浸入式金属自显影技术(autometallography,AMG)检测AD病人和APP/PS1转基因小鼠脑内锌含量的变化,应用免疫荧光双标和激光共聚焦扫描显微技术检测ZnTs在AD病人和APP/PS1转基因小鼠脑内的定位分布及其与Aβ在老年斑内的位置关系,应用Western Blot技术检测APP/PS1转基因小鼠大脑皮层及海马内ZnTs的表达变化,应用免疫共沉淀技术(co-immunoprecipitation,co-IP)检测APP/PS1转基因小鼠脑内ZnTs和Aβ的蛋白相关性,应用RNAi技术阻抑ZnT1基因在稳定转染APPsw、APP基因的SH-SY5Y细胞的表达,并应用RT-PCR和Western Blot技术检测RNAi对ZnT1的基因沉默效果,应用Zinquin荧光技术、Western Blot技术、免疫荧光双标技术、ELISA技术、MTT技术检测ZnT1-RNAi对细胞锌离子转运、APP表达、Aβ分泌和细胞活力的影响。实验结果1、锌离子在AD病人及APP/PS1转基因小鼠脑内的分布AMG结果显示,AMG阳性的老年斑广泛分布于AD病人和APP/PS1转基因小鼠大脑皮质和海马等脑区,在血管壁及其周围也可见到明显的呈棕黑色的AMG阳性反应产物。AMG阳性的老年斑呈圆形或不规则形,大小不等,在AD病人大脑中多具有明显的致密核心,而在APP/PS1转基因小鼠大脑内多呈中空状。2、ZnTs在AD病人及APP/PS1转基因小鼠脑内的分布免疫组织化学结果显示,ZnTs免疫阳性反应产物呈棕黄色,在大脑皮层及海马内均可见ZnTs阳性的老年斑分布,斑块大小不等,形状为圆形或不规则形状,边界较清晰。高倍镜下可见ZnT1和ZnT4广泛分布到整个老年斑内,ZnT3、ZnT5及ZnT6主要分布在斑块周围变性的神经元突起内,而ZnT7则主要表达于老年斑的中心部位。此外,ZnTs还广泛表达于淀粉样变性的血管壁及其周围,其中以ZnT3染色最深。免疫荧光双标的共聚焦激光扫描结果显示,在AD病人和APP/PS1转基因小鼠脑内,Aβ免疫阳性的老年斑广泛分布于大脑皮层及海马的齿状回、CA1-CA3区域,几乎所有Aβ阳性的老年斑均有不同程度的ZnTs表达,即ZnTs和Aβ共存于老年斑内。高倍镜观察可见在APP/PS1转基因小鼠脑内,Aβ主要分布于老年斑的核心,而不同的ZnTs在老年斑内的位置分布存在一定差异(同免疫组织化学染色结果),ZnT3免疫荧光除了分布在大脑皮层和海马的Aβ老年斑中,还可见于海马苔藓纤维和淀粉样变性的血管壁及其周围。3、ZnTs在APP/PS1转基因小鼠大脑皮层及海马内的表达变化Western Blot结果显示,ZnTs在APP/PS1转基因小鼠大脑皮层和海马内的表达均明显高于野生型对照小鼠。其中ZnT1,3,4,5,6,7在大脑皮层的表达量分别是野生型小鼠的196.6%,201.9%,150.3%,134.2%,162.0%,122.9%;在海马的表达量分别是野生型小鼠的280.1%,398.6%,277.1%,168.2%,142.2%,282.3%。4、ZnTs与Aβ蛋白相关性分析Co-IP结果显示,应用Aβ抗体对APP/PS1转基因小鼠大脑皮层蛋白进行免疫共沉淀,经过琼脂糖凝胶电泳,未见ZnTs条带。5、RNAi对ZnT1基因的沉默效果RT-PCR结果显示,RNAi可在mRNA水平明显抑制APPsw细胞的ZnT1表达,干扰后24h即可出现ZnT1的表达下降,干扰后48h为抑制效果最佳时间点,ZnT1表达量仅为对照组的22%。Western Blot结果显示,RNAi可明显抑制APPsw细胞的ZnT1蛋白表达,干扰后48h的抑制率达80%。6、ZnT1-RNAi对锌离子转运的影响Zinquin荧光染色结果显示,在APPsw细胞,ZnT1-RNAi后48h,可见锌离子在细胞内的聚集明显高于未干扰的对照组细胞。7、ZnT1-RNAi对APP表达的影响Western Blot结果显示,RNAi可明显抑制APPsw、APP细胞的APP蛋白表达,干扰后48h,APPsw、APP细胞的APP蛋白表达量分别为对照组的58%和57%。免疫荧光双标和荧光显微镜观察结果显示,RNAi可明显抑制APPsw细胞的ZnT1和APP表达,与对照组相比,二者的免疫荧光均明显减弱。8、ZnT1-RNAi对Aβ分泌量的影响Elisa结果显示,RNAi可明显抑制APPsw、APP细胞的Aβ分泌,干扰48h后,APPsw和APP细胞培养液内的Aβ含量分别降低49%和37%。9、锌离子对细胞活力的影响MTT结果显示,正常培养的APPsw、APP和空质粒转染的SH-SY5Y细胞(NEO细胞)活力几乎没有差别;10μM氯化锌处理可分别降低APPsw、APP细胞活力19.7%和13.9%,而对NEO细胞活力没有影响;50μM氯化锌处理可分别降低APPsw、APP、NEO细胞活力48.5%、42.7%、30%;100μM氯化锌处理可分别降低APPsw、APP、NEO细胞活力66.6%、56.5%、46.1%。10、ZnT1-RNAi对细胞活力的影响MTT结果显示,正常培养条件下,ZnT1-RNAi对细胞活力几乎没有影响,在50μM氯化锌处理的情况下,ZnT1-RNAi可分别提高APPsw、APP细胞活力14.4%、10.5%。结论1、AD病人和APP/PS1转基因小鼠脑内的老年斑和淀粉样变性的血管壁及其周围组织富含锌离子。AMG技术对检测游离锌离子具有高度特异性和敏感性,是研究锌离子在AD病人和APP/PS1转基因小鼠脑内分布的有利手段。2、ZnTs与Aβ共表达于老年斑和淀粉样变性的血管壁及其周围。不同ZnTs在老年斑内的定位分布具有差异性。ZnTs在APP/PS1转基因小鼠大脑皮层和海马的表达均高于野生型小鼠。3、ZnT1-RNAi可明显降低稳定转染APPsw,APP基因的SH-SY5Y细胞的APP表达和Aβ分泌。4、ZnT1-RNAi可提高稳定转染APPsw,APP基因的SH-SY5Y细胞在高锌环境下的细胞活力。更多还原

【Abstract】 PrefaceAlzheimer’s disease（AD）is a disease cinically characterized by progressive intellectual deterioration.With the gradual ageing of the population,the morbidity of AD increased year by year.It had brought heavy burden to the society and family and become one of the fatal disease that hazard to human health.AD is pathologically characterized by senile plaques（SP）formed by pathological deposition ofβ-amyloid（Aβ）,neurofibrillary tangles（NFT）and cerebral amyloid angiopathy（CAA）.Aβis the key factor in the pathologic process of AD and generated from the amyloid precursor protein（APP）by a proteolytic activity ofβ-andγ-secretase. By now,the pathogenes of AD is not very clear,but more and more evidences suggested that zinc played a key role in the pathogenes and pathologic process of AD. Zinc ions can trigger a deposition of Aβby connecting the 13^thamino acides between the adjecent Aβmolecules.γ-secretase is a zinc binding protein,and cytoplasmic zinc has the function to decrease the extracellular secretion of insoluble Aβby inhibitting theγ-secretase cracking APP.Metal chelating agents have been shown to inhibit the formation of amyloid plaques in APP transgenic mouse brains.Therefore,the regulation of metal ion homeostasis and maintain of the formation and degradation of Aβhave become an important therapeutic strategy of AD.Zinc cannot travel across biological membranes by passive diffusion.Specific membrane transporters and channels involved in its transfer and metabolism.Zinc transporter（ZnT）is one of the important protein family involved in zinc metabolism of brain.Until now,seven members of the ZnT family（ZnT1-7）have been characterized and except for ZnT2,all the ZnTs are expressed in the brain.ZnT family members are responsible for the extrusion of zinc outside the cytoplasm to the extracellular space or intracellular organelles.ZnT1,an ubiquitous zinc transporter localized on the plasma membrane, serves an essential function of zinc efflux from the cell.ZnT3 is mainly localized in the membranes of zinc-rich synaptic vesicles and involved in the release of zinc ions in the synaptic vesicles.ZnT4 is localized on the intracellular vesicular membrane and functions to increase vesicular zinc concentration.ZnT5,ZnT6 and ZnT7 are localized on the Golgi apparatus and believed to facilitate the translocation of the cytoplasmic zinc into the Golgi apparatus.Recently,it has been reported that there were significant alterations in the expression of ZnT1,ZnT4 and ZnT6 in AD patient brains,and genetic ablation of ZnT3 in the Tg2576 Alzheimer mouse model inhibited the formation of amyloid plaques and CAA,indicating the roles of ZnT and zinc ions in the pathogenes and pathologic process of AD.In summary,it has scientific significance to analyse the expression and distribution of zinc ions and ZnT and correlation between ZnT and Aβin the AD brain, as well as the further study about zinc metabolism and its correlation to the pathophysiological mechanism of AD.MethodsAD patient brains,APP/PS1 transgenic mice and SH-SY5Y cells stable transfected APPsw or APP gene were used for the present study.The levels of free zinc ions in AD patient brains and APP/PS1 transgenic mice brains were evaluated by immersion autometallography（AMG）.The distribution patterns of ZnTs in these brains and the positional relation between ZnTs and Aβwere detected by double immunofluorescence and confocal laser scanning microscopy.The changes of ZnTs expression levels in the APP/PS1 transgenic mouse cerebral cortex and hippocampus were studied by western blot analyses.Co-immunoprecipitation（co-IP）was used to detect the molecular correlation between Aβand ZnTs in the APP/PS1 transgenic mice brains.RNA interference（RNAi）technology was used to inhibite the expression of ZnT1 gene in SH-SY5Y cells stably transfected APPsw or APP gene.The gene silencing effect of ZnT1-RNAi was detected by RT-PCR and Western Blot.Zinquin fluorescence technology,Western Blot,double immunofluorescence techniques, ELISA technology and MTT were used to detect the effects of ZnT1 RNAi on zinc ions transport,APP expression,Aβsecretion and activity of cells. Results1.Distribution of zinc ions in the human AD and APP/PS1 transgenic mice brains.AMG results showed that AMG-positive plaques were widely distributed in the cerebral cortex and hippocampus of APP/PS1 transgenic mouse.Brown black AMG positive reaction products could be seen clearly in the vascular wall and its surrounding. The AMG-positive plaques were round or irregular in shape and different in size and morphology.In the AD patients,a majority of the AMG-stained plaques had a dence core,while in the APP/PS1 transgenic mice,most plaques were rosette-shaped with a non-zinc stained interior.2.Abundant expression of ZnTs in SP and amyloid angiopathic vesselsImmunohistochemimistry results revealed that ZnTs immunopositive reaction products were brown.ZnTs-positive plaques were round or irregular and distributed throughout the cortex and hippocampus with vary size and clear boundary.At higher magnification,ZnT1 and ZnT4 were extensively expressed in all parts of the plaques. ZnT3,ZnT5 and ZnT6 were expressed prominently in the degenerating neurites in the peripheral part of the plaques,while ZnT7 was present in the core of the plaques.Our data also showed an abundant expression of ZnTs in the amyloid angiopathic vessels. ZnT3 immunoreactivity was the most intense.Double-immunofluorescence staining for Aβand one of the ZnTs showed that the Aβ-positive plaques were widely distributed in the APP/PS1 transgenic mouse cerebral cortex and the DG and CA1-CA3 region of the hippocampus,ZnTs were expressed in most of the Aβcontaining plaques,i.e.Aβand ZnTs protein were co-expressed in the senile plaques.At higher magnification,the Aβwas located in the core of plaques,and ZnTs,however,exhibited different staining patterns（similar to immunohistochemical results）.Apart from senile plaques,an intense ZnT3 fluorescence was also seen in the hippocampal mossy fibers and the wall of amyloid angiopathic vessels.3.Altered expression of ZnTs in the APP/PS1 transgenic mouse cerebral cortex and hippocampusWestern blot results showed that the expressions of ZnTs were increased in the hippocampus and cortex of APPswe/PS1dE9 transgenic mice.The expressions of ZnT1, 3,4,5,6 and 7 were 196.6%,201.9%,150.3%,134.2%,162.0%,122.9%of wild-type control mice in the cerebral cortex;280.1%,398.6%,277.1%,168.2%,142.2%, 282.3%in the hippocampus.4.Molecular Correlation Analysis of ZnTs and AβCo-IP results showed that using Aβantibodies to perform the co-immunoprecipitation of ZnTs and Aβin the APP/PS1 transgenic mouse cerebral cortex,after agarose gel electrophoresis,no ZnTs band could be seen.5.The effect of RNAi to silence genes ZnT1RT-PCR results show that RNAi significantly inhibited the expression of ZnT1 at mRNA level in SH-SY5Y cells stable transfected APPsw gene.The inhibitory effect beginning to show 24 h after RNAi and 48 h after RNAi was the most effective time, the expression of ZnT1 account for only 22%of control group.Western Blot results showed that RNAi could significantly inhibit ZnT1 expression in APPsw-cells,and the inhibiting rate can reach 80%48 h after RNAi.6.The effect of ZnT1-RNAi on zinc ion transportZinquin staining results showed that the aggregation of zinc ions in the SH-SY5Y cells stable transfected APPsw gene（APPsw cells）was significantly higher than that of the control group 48 h after RNAi.7.The effect of ZnT1-RNAi on the expression of APPWestern Blot results showed that RNAi could significantly inhibit the APP expression in SH-SY5Y cells stable transfected APPsw or APP gene,the expression of APP account for only 58%or 57%of control group in SH-SY5Y cells stable transfected APPsw or APP gene 48 h after RNAi.Double immunofluorescence and fluorescence microscopy showed that RNAi could significantly inhibit the APP expression in SH-SY5Y cells stable transfected APPsw gene,the immunofluorescence of them was significantly weakened compared with control group.8.The effect of ZnT1-RNAi on AβsecretionElisa results showed that RNAi significantly inhibitted Aβsecretion,the amount of Aβin the culture medium was decreased by 49%and 37%respectively in SH-SY5Y cells stable transfected APPsw and APP gene 48 h after RNAi.9.Effect of zinc ions on the activity of cellsMTT results show that there are almost no activity difference among normal cultured SH-SY5Y cells stable transfected APPsw gene,APP gene or empty vector （NEO）.The cell activity were reduced by 19.7%or 13.9%in SH-SY5Y cells stable transfected APPsw or APP gene after 10μM zinc chloride treatment.The cell activity can be reduced by 48.5%,42.7%,30%in APPsw,APP or NEO cells after 50μM zinc chloride treatment.The cell activity can be reduced by 66.6%,56.5%,46.1%in APPsw, APP or NEO cells after 100μM zinc chloride treatment.10.Effect of ZnT1-RNAi on the activity of cellsMTT results show that ZnT1-RNAi did not affect cell viability at normal cultured state,and can improve cell viability of APPsw,APP cell cultured with 50μM zinc chloride by 14.4%and 10.5%respectively.Conclusion1.Zinc ion is enriched in the senile plaques and the wall and the vicinity of CAA changed vessels of AD patients and APP/PS1 transgenic mice brain.AMG,a high specificity and sensitivity technology to detect free zinc ions,is a favorable means to study the distribution of zinc ions in the AD patients and APP/PS1 transgenic mice brain.2.ZnTs and Aβare co-expressed in the senile plaques and the wall and the vicinity of CAA changed vessels.Different ZnTs has different localization in the senile plaques. The expression of ZnTs in the cerebral cortex and hippocampus of APP/PS1 transgenic mice is higher than that in wild-type mice.3.ZnT1-RNAi could significantly reduce the APP expression and Aβsecretion in SH-SY5Y cells stable transfected APPsw or APP gene.4.ZnT1-RNAi can improve the viability of SH-SY5Y cells stable transfected APPsw or APP gene in the environment of high zinc level.更多还原