【作者】 秦秀军；

【导师】 牛侨；

【作者基本信息】 山西医科大学 ， 劳动卫生与环境卫生学， 2020， 博士

【摘要】 目的:通过转人ApoE4基因和铝暴露联合干预,模拟遗传危险因素和环境危险因素联合作用,研究二者对小鼠学习记忆能力的影响,以及神经元形态和突触结构可塑性的影响,分析二者间是否存在交互作用;同时,通过检测Aβ含量,APP、ApoER2、LRP1、VLDLRs基因和蛋白的表达情况,tau磷酸化水平,探讨二者联合作用对认知损伤的可能机制。方法:本实验采用3种雄性小鼠,ApoE基因敲除小鼠（KO）、野生型小鼠（WT）和ApoE4人源化小鼠（KI）,分别设对照组和染铝组。染铝组通过腹腔注射给予麦芽酚铝（Al,40μmol/kg,每染毒5天,间歇2天,共60天）,对照组给予同体积的生理盐水（NS）。染毒结束后,采用连接酶检测反应检测ApoE4人源化小鼠是否稳定遗传了ApoE4基因;电感耦合等离子体质谱法测定脑铝含量;Morris水迷宫和新物体识别实验检测动物学习和记忆能力的变化。通过HE染色、Golgi染色以及电镜实验,观察动物海马组织病理学的结构变化、神经元形态结构的变化、树突棘密度的变化、突触结构的变化。通过Elisa法检测Aβ₄₀和Aβ₄₂的含量,通过RT-PCR和Western-blot法检测APP、ApoER2、LRP1、VLDLRs等基因和蛋白的表达情况。同时,用Western-blot法检测tau-5、pThr181、pThr231、pSer262和pSer396蛋白含量。结果:1.实验中所使用ApoE4人源化小鼠在rs429358、rs7412对应位点的碱基均为CC/CC,证明小鼠均稳定遗传了人ApoE4基因。脑铝含量检测结果显示,染铝组动物脑铝含量（KO+Al:35.34±5.4μg/g、WT+Al:33.62±4.00μg/g、KI+Al:35.56±8.72μg/g）均明显高于对照组（KO+NS:6.52±2.34μg/g、WT+NS:5.75±1.20μg/g、KI+NS:5.80±1.83μg/g）,差异有统计学意义（P<0.01）。Morris水迷宫实验结果显示,动物类型和干预措施（是否染铝）对动物逃避潜伏期无交互作用（P>0.05）,而各自主效应有统计学意义（P<0.05）,LSD法进行两两比较显示ApoE4、铝均可延长逃避潜伏期;穿越平台位置次数结果（KO+NS:4.63±0.92次、WT+NS:2.75±1.28次、KI+NS:2.38±0.92次;KO+Al:1.75±0.71次、WT+Al:1.50±0.53次、KI+Al:1.25±0.89次）显示,动物类型和干预措施有交互作用（P<0.05）,各自的单独效应也有统计学意义（P<0.05）,两两比较结果表明ApoE4、铝可减少动物穿越平台位置次数,并存在交互作用。新物体识别实验结果,T2测试期KI+Al组小鼠探索新物体和已熟悉物体时间相近,无统计学意义（P>0.05）,其余各组均有统计学意义（P<0.05）;辨别指数结果（KO+NS:55.1±10.7、WT+NS:46.7±8.7、KI+NS:40.7±5.9;KO+Al:41.9±9.8、WT+Al:26.8±9.4、KI+Al:0.8±16）分析表明,动物类型和干预措施有交互作用（P<0.01）,各自的单独效应也有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可降低动物辨别指数,并存在交互作用。2.HE染色结果显示,各组动物海马神经元细胞大小、形态均正常,未观察到萎缩、凋亡等改变;Golgi染色,KI+Al组动物可观察到有神经元断裂。树突交叉点个数（距胞体20μm处,KO+NS:7.20±0.64、WT+NS:5.44±0.33、KI+NS:5.35±0.31;KO+Al:5.29±0.75、WT+Al:4.60±0.2、KI+Al:4.15±0.28）和树突棘密度（KO+NS:0.72±0.07个/μm、WT+NS:0.57±0.06个/μm、KI+NS:0.39±0.05个/μm;KO+Al:0.53±0.06个/μm、WT+Al:0.34±0.05个/μm、KI+Al:0.26±0.04个/μm）结果分析显示,动物类型和干预措施有交互作用（P<0.05）,各自的单独效应也有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可减少树突交叉点个数和树突棘密度,并存在交互作用。电镜实验,突触后致密区（PSD）厚度（KO+NS:41.02±2.31 nm、WT+NS:35.17±2.75 nm、KI+NS:33.19±3.49 nm;KO+Al:31.80±2.01 nm、WT+Al:26.07±3.98 nm、KI+Al:19.67±2.06 nm）和突触界面曲率（KO+NS:26.70±7.95、WT+NS:17.34±1.96、KI+NS:6.40±1.16;KO+Al:8.96±1.54、WT+Al:4.54±1.05、KI+Al:2.57±0.89）结果分析显示,动物类型和干预措施有交互作用（P<0.05）,各自的单独效应也有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可降低PSD厚度和突触界面曲率,并存在交互作用。3.Elisa法检测Aβ含量,动物类型和干预措施对Aβ₄₀含量无交互作用（P>0.05）,各自主效应也无统计学意义（P>0.05）,而Aβ₄₂测定结果（KO+NS:7.01±3.75 pg/ml、WT+NS:11.06±4.23 pg/ml、KI+NS:13.13±6.92 pg/ml;KO+Al:14.10±7.69 pg/ml、WT+Al:16.29±6.50 pg/ml、KI+Al:29.10±15.06 pg/ml）显示,动物类型和干预措施有交互作用（P<0.01）,各自的单独效应也有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可增加脑内Aβ₄₂含量,并存在交互作用。基因和蛋白测定结果,动物类型和干预措施对APP基因和蛋白表达无交互作用（P>0.05）,但各自主效应有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可增加APP基因和蛋白的表达;ApoER2基因,动物类型和干预措施无交互作用（P>0.05）,但各自主效应有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可降低ApoER2基因表达,ApoER2蛋白（KO+NS:1.000±0.000、WT+NS:0.764±0.091、KI+NS:0.516±0.082;KO+Al:0.788±0.074、WT+Al:0.660±0.084、KI+Al:0.284±0.053）,动物类型和干预措施有交互作用（P<0.05）,各自的单独效应也有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可降低ApoER2蛋白表达,并存在交互作用;LRP1基因和蛋白,动物类型和干预措施无交互作用（P>0.05）,但各自主效应有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可降低LRP1基因和蛋白表达;VLDLRs基因和蛋白,动物类型和干预措施无交互作用（P>0.05）,但各自主效应有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可降低VLDLRs基因和蛋白表达。4.tau蛋白检测结果:动物类型和干预措施对tau-5蛋白表达（KO+NS:1.000±0.000、WT+NS:1.327±0.098、KI+NS:1.538±0.210;KO+Al:1.405±0.150、WT+Al:1.747±0.305、KI+Al:2.339±0.460）有交互作用（P<0.05）,各自单独效应也有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可增加tau-5蛋白的表达,并存在交互作用;pThr181蛋白（KO+NS:1.000±0.000、WT+NS:1.434±0.350、KI+NS:1.905±0.418;KO+Al:1.638±0.292、WT+Al:2.22±0.500、KI+Al:3.438±0.719）,动物类型和干预措施有交互作用（P<0.01）,各自单独效应也有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可增加pThr181蛋白的表达,并存在交互作用;pThr231蛋白（KO+NS:1.000±0.000、WT+NS:1.543±0.409、KI+NS:2.215±0.470;KO+Al:1.722±0.338、WT+Al:2.549±0.571、KI+Al:4.176±0.577）,动物类型和干预措施有交互作用（P<0.01）,各自单独效应也有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可增加pThr231蛋白的表达,并存在交互作用;pSer262蛋白,动物类型和干预措施无交互作用（P>0.05）,而动物类型主效应有统计学意义（P<0.01）,两两比较结果表明ApoE4可增加pSer262蛋白表达;pSer396蛋白（KO+NS:1.000±0.000、WT+NS:1.305±0.107、KI+NS:1.564±0.254;KO+Al:1.325±0.192、WT+Al:1.687±0.404、KI+Al:2.423±0.490）,动物类型和干预措施有交互作用（P<0.05）,各自单独效应也有统计学意义（P<0.01）,两两比较结果表明ApoE4、铝可增加pSer396蛋白的表达,并存在交互作用。结论:1.ApoE4单独作用,可引起小鼠脑内Aβ₄₂含量升高,APP基因和蛋白表达升高,ApoER2、LRP1、VLDLRs基因和蛋白表达下降,脑内t-tau和p-tau水平升高,神经元复杂程度和突触结构可塑性降低,最终影响小鼠的学习和记忆能力,本实验进一步证实了ApoE4是AD（Alzheimer’s disease）的危险遗传因素。2.亚慢性铝暴露,可引起小鼠脑内铝含量升高,Aβ₄₂含量升高,APP基因和蛋白表达升高,ApoER2、LRP1、VLDLRs基因和蛋白表达下降,脑内t-tau和p-tau水平升高,神经元复杂程度和突触结构可塑性降低,小鼠学习和记忆能力下降,也进一步证实了铝是AD的危险环境因素。3.通过析因分析,ApoE4和铝联合作用对AD动物的影响主要表现为,升高脑内Aβ₄₂含量,影响神经元的形态结构、复杂程度以及突触的结构可塑性,降低ApoER2蛋白含量,升高t-tau和p-tau水平,增加tau231,tau396位点的磷酸化,降低小鼠的认知功能。4.本实验首次从动物实验角度,证实了遗传因素（ApoE4）和环境因素（铝）可促进AD的进程发展,其机制可能为,二者联合作用阻断了ApoER2的受体循环,使转录后ApoER2的表达降低,影响Aβ的清除,升高t-tau和p-tau水平,从而损伤神经元的形态结构和突触的结构可塑性,进而影响动物的学习和记忆能力,最终促进AD进程的发展。更多还原

【Abstract】 Objective:In order to investigate combined effect of genetic risk factor ApoE4 and environmental risk factor aluminium on study and memory,and plasticity of neuronal phenotype and synapse,combined effect was stimulated by intervention of transgenic ApoE4 gene and aluminium exposure in mice,and interaction between two factors was analyzed.Moreover,to discuss the potential mechanism of cognitive damages by the combined effect of ApoE4 gene and aluminium through determination of the contents of Aβ,the gene and protein expression of APP,ApoER2,LRP1,and VLDLRs,and the level of tau phosphorylation.Methods:Three kinds of male mice were used in this experiment:ApoE knock-out mice（KO）,wild type mice（WT）,and transgenic knock-in mice that express the human ApoE4（KI）.The control group and the aluminium-exposed group has been set.The aluminium-exposed group was given aluminium-maltolate（40μmol/kg）by intraperitoneal injection consecutively for 5 days,and pausing for 2 days,and the exposure duration lasted for 60days.The control group was injected with same volume of saline solution.After the end of the experiment,a ligase test was used to evidence whether ApoE4 gene was stably inherited in the transgenic ApoE4 mice.The aluminium content in brain was determined by Inductively Coupled Plasma Mass Spectrometry.Morris water maze and new object recognition tests were used to detect changes in learning and memory ability of animals.Through HE staining,Golgi staining,and electron microscope experiments,the changes of the hippocampal histopathological structure,the neuronal morphological structure,the dendritic spine density,and the synaptic structure were observed.The content of Aβ₄₀0 and Aβ₄₂2 was detected by Elisa method,and the expression of APP,ApoER2,LRP1,VLDLRs and other genes and proteins were detected by RT-PCR and western-blot.At the same time,the protein contents of tau-5,pThr181,pThr231,pSer262 and pSer396were detected by western-blot.Results:1.The bases at corresponding site of rs429358,rs7412 in the transgenetic ApoE4mice that express the human ApoE4 used in the experiment were all CC/CC.This has proved that all mice have stably inherited in the ApoE4 gene of human.The contents of aluminium in the hippocampus in the aluminium-exposed mice were 35.34±5.40μg/g（KO）,33.62±4.00μg/g（WT）,35.56±8.72μg/g（KI）,respectively,and higher than the control group,6.52±2.34μg/g（KO）,5.75±1.20μg/g（WT）,5.80±1.83μg/g（KI）.The differences were statistically significant（P<0.01）.Morris water maze experiment results showed that ApoE4 gene and aluminium exposure have no interaction on the escape latency of the animals（P>0.05）,and their main effects were statistically significant（P<0.05）.The pairwise comparison by using LSD method showed that both ApoE4 and aluminium could extend the escape latency.The number of crossing the platform in the aluminium-exposed mice were 1.75±0.71（KO）,1.50±0.53（WT）,1.25±0.89（KI）,respectively,and less than the control group,4.63±0.92（KO）,2.75±1.28（WT）,2.38±0.92（KI）.Statistical analysis showed that there was an interaction between the animal type and the intervention（P<0.05）,and the individual effects were also statistically significant（P<0.05）.The results from pairwise comparison showed that ApoE4 and aluminium could reduce the number of times that animals cross the platform,and the interaction was existed.Results of the new object recognition test showed that the time used in the KI+Al group of mice in the T2 test period were similar for exploring new objects and familiar objects,and there was no statistical significance（P>0.05）,and other remaining groups all had statistical significance（P<0.05）.The discrimination index in the aluminium-exposed mice were 41.9±9.8（KO）,26.8±9.4（WT）,0.8±16（KI）,respectively,and lower than the control group,55.1±10.7（KO）,46.7±8.7（WT）,40.7±5.9（KI）.Statistical analysis showed that there was an interaction between animal types and interventions（P<0.01）,and their individual effects were also statistically significant（P<0.01）.The pairwise comparison results showed that ApoE4 and aluminium could reduce the animal discrimination index and the interaction was existed.2.The results of HE staining showed that the size and morphology of hippocampal neurons in each group were normal,and no changes such as atrophy and apoptosis were observed.Golgi staining showed that neurons were fractured in the KI+Al group.The number of dendritic intersections at a distance of 20μm from the cell body in the aluminium-exposed mice were 5.29±0.75（KO）,4.60±0.20（WT）,4.15±0.28（KI）,respectively,less than the control group,7.20±0.64（KO）,5.44±0.33（WT）,5.35±0.31（KI）,and the dendritic spine density in the aluminium-exposed mice were 0.53±0.06（KO）,0.34±0.05（WT）,0.26±0.04（KI）,respectively,lower than the control group,0.72±0.07（KO）,0.57±0.06（WT）,0.39±0.05（KI）.Statistical analysis showed that there was an interaction between animal types and interventions（P<0.05）,and their individual effects were also statistically significant（P<0.01）.The pairwise comparison results showed that ApoE4 and aluminium could reduce the number of dendritic intersections and dendritic spine density with an interaction.The electron microscope experiments,the thickness of the postsynaptic density（PSD）in the aluminium-exposed mice were31.80±2.01 nm（KO）,26.07±3.98 nm（WT）,19.67±2.06 nm（KI）,respectively,less than the control group,41.02±2.31 nm（KO）,35.17±2.75 nm（WT）,33.19±3.49 nm（KI）,and the curvature of the synaptic interface in the aluminium-exposed mice were 8.96±1.54（KO）,4.54±1.05（WT）,2.57±0.89（KI）,respectively,less than the control group,26.70±7.95（KO）,17.34±1.96（WT）,6.40±1.16（KI）.Statistical analysis showed that the animal type and the intervention all had an interaction effect（P<0.05）,and the individual effects were also statistically significant（P<0.01）.The comparison of the results in pairs showed that ApoE4 and aluminium could reduce PSD thickness and curvature of synaptic interface with interaction.3.Elisa method detected Aβcontent that animal types and interventions had no interaction on Aβ₄₀0 content（P>0.05）,and their main effects were not statistically significant（P>0.05）.The content of Aβ₄₂2 in the aluminium-exposed mice were14.10±7.69 pg/ml（KO）,16.29±6.50 pg/ml（WT）,29.10±15.06 pg/ml（KI）,respectively,and higher than the control group,7.01±3.75 pg/ml（KO）,11.06±4.23 pg/ml（WT）,13.13±6.92 pg/ml（KI）.Statistical analysis showed that there was an interaction in animal types and interventions（P<0.01）,and their individual effects were also statistically significant（P<0.01）.The results of pairwise comparison showed that ApoE4 and aluminium could increase Aβ₄₂2 content in the brain.The results of gene and protein determination showed that there was no interaction on animal types and interventions with APP gene and protein expression（P>0.05）,but their main effects were statistically significant（P<0.01）.The pairwise comparison results showed that ApoE4 and aluminium could increase the expression of APP genes and proteins.There was no interaction in animal types and interventions with ApoER2 gene（P>0.05）,but their main effects were statistically significant（P<0.01）.The pairwise comparison results showed that ApoE4and aluminium could reduce ApoER2 gene expression.The expression of ApoER2protein in the aluminium-exposed mice were 0.788±0.074（KO）,0.660±0.084（WT）,0.284±0.053（KI）,respectively,and lower than the control group,1.000±0.000（KO）,0.764±0.091（WT）,0.516±0.082（KI）.Statistical analysis showed that there was an interaction in animal types and interventions（P<0.05）,and their individual effects were also statistically significant（P<0.01）.The pairwise comparison results showed that ApoE4 and aluminium could reduce ApoER2 protein expression with interaction effect.For LRP1 gene and protein,there was no interaction between animal types and intervention（P>0.05）,but their main effects were statistically significant（P<0.01）.The pairwise comparison results showed that ApoE4 and aluminium could reduce LRP1 gene and protein expression.For VLDLRs genes and proteins,there was no interaction between animal types and interventions（P>0.05）,but their main effects were statistically significant（P<0.01）.The pairwise comparison showed that both ApoE4 and the aluminium could reduce VLDLRs gene and protein expression.4.The expression of tau-5 protein in the aluminium-exposed mice were 1.405±0.150（KO）,1.747±0.305（WT）,2.339±0.460（KI）,respectively,and higher than the control group,1.000±0.000（KO）,1.327±0.098（WT）,1.538±0.210（KI）.Statistical analysis showed that there was an interaction in animal types and interventions（P<0.05）,and their individual effects were also statistically significant（P<0.01）.The results of pairwise comparison showed that ApoE4 and aluminium could increase tau-5 protein expression with interactions.The expression of pThr181 protein in the aluminium-exposed mice were 1.638±0.292（KO）,2.22±0.500（WT）,3.438±0.719（KI）,respectively,and higher than the control group,1.000±0.000（KO）,1.434±0.350（WT）,1.905±0.418（KI）.Statistical analysis showed that there was an interaction in animal types and interventions（P<0.01）,and their individual effects were also statistically significant（P<0.01）.The pairwise comparison showed that ApoE4 and aluminium could increase the expression of pThr181 protein with an interaction.The expression of pThr231 protein in the aluminium-exposed mice were 1.722±0.338（KO）,2.549±0.571（WT）,4.176±0.577（KI）,respectively,and higher than the control group,1.000±0.000（KO）,1.543±0.409（WT）,2.215±0.470（KI）.Statistical analysis showed that there was an interaction in animal types and interventions（P<0.01）,and their individual effects were also statistically significant（P<0.01）.The pairwise comparison results showed that ApoE4 and aluminium could increase the expression of pThr231 protein with an interaction.There is no interaction among pSer262 protein,animal types and interventions（P>0.05）,and the main effect of animal types were statistically significant（P<0.01）.The pairwise comparison results showed that ApoE4 could increase the expression of pSer262 protein.The expression of pSer396 protein in the aluminium-exposed mice were 1.325±0.192（KO）,1.687±0.404（WT）,2.423±0.490（KI）,respectively,and higher than the control group,1.000±0.000（KO）,1.305±0.107（WT）,1.564±0.254（KI）.Statistical analysis showed that there was an interaction in animal types and interventions（P<0.05）,and their individual effects were also statistically significant（P<0.01）.The pairwise comparison results showed that ApoE4 and aluminium could increase the expression of pSer396protein with an interaction.Conclusion:1.The single action of ApoE4 could cause rise in Aβ₄₂2 content in mice brain,rise in APP gene and protein expression,decline in ApoER2,LRP1,VLDLRs gene and protein expression,rise in t-tau and p-tau levels in the brain,and decline in neuronal complexity and synaptic structural plasticity.Finally it would affect the learning and memory ability of mice.This experiment further confirmed that ApoE4 is a risk genetic factor for AD.2.Subchronic aluminium exposure could cause rise in aluminium content in the brain,rise in Aβ₄₂2 content,rise in APP gene and protein expression,decline in ApoER2,LRP1,VLDLRs gene and protein expression,and rise in levels of brain t-tau and p-tau,decline in neuronal complexity and plasticity of synaptic structures,and decline in learning and memory ability in mice.Also it has further confirmed that aluminium is a dangerous environmental factor for AD.3.Through factorial analysis,the effects of the combined effect of ApoE4 and aluminium on AD animals were mainly as follows:rise the content of Aβ₄₂2 in the brain,affect the morphology,complexity and structural plasticity of synapses,reduce the content of ApoER2 protein and increase in t-tau and p-tau levels,increase the phosphorylation of tau231 and tau396 sites,and reduce cognitive function in mice.4.From the perspective of animal experiments at the first time,this experiment confirmed that genetic factors（ApoE4）and environmental factors（aluminium）could enhance the development of AD.The mechanism may be the combination of the two factors that block the ApoER2 receptor cycle and make the expression of post-transcriptional of ApoER2 decline to affect the clearance of Aβ,rise the levels of t-tau and p-tau,thereby damages the morphology and synaptic plasticity of neurons,which in turn affects the learning and memory abilities of animals,and finally enhance the development of AD process.更多还原