CN105494263B - 一种产生ho-1/app/psen1三转基因阿尔茨海默病小鼠模型的方法 - Google Patents
一种产生ho-1/app/psen1三转基因阿尔茨海默病小鼠模型的方法 Download PDFInfo
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Abstract
本发明公开了一种产生HO‑1/APP/PSEN1三转基因阿尔茨海默病小鼠模型的方法。本发明利用转基因技术用HO‑1基因代替APP/PSEN1/TAU三转基因模型中的tauP301L突变基因,建立了HO‑1/APP/PSEN1三转基因AD小鼠模型,该转基因小鼠符合阿尔茨海默病的病理状态。经行为学、形态学、分子生物学检测,HO‑1/APP/PSEN1转基因小鼠在认知功能、脑组织病理及分子标志方面均符合阿尔茨海默病特征性表型,可作为阿尔茨海默病动物模型。并且,HO‑1/APP/PSEN1三转基因小鼠是一种既能涵盖阿尔茨海默病表型又符合阿尔茨海默病病理发生机制的动物模型。并且,HO‑1/APP/PSEN1三转基因小鼠较APP/PSEN1双转基因小鼠的阿尔茨海默病表型更为显著。本发明可用于阿尔茨海默病发病机制探讨、药物新靶点发现及临床前药效评价等。
Description
技术领域
本发明涉及一种生物技术,具体涉及建立一种生物模型的方法,也是建立一种疾病的生物模型的方法。
背景技术
阿尔茨海默病(AD)模型是研究AD发病机制、治疗策略必不可少的工具,目前建立AD模型的最佳方法是转基因技术。
现有阿尔茨海默病转基因模型主要转入的是家族性AD(FAD)中的突变基因,最常用的是:β-淀粉样前体蛋白(APP)和早老素(PSEN)的突变基因。
由于鼠类Aβ多肽即使是在过表达的状态下也很难在体内高水平聚集形成明显的斑块(Jankowsky,J.L.,Younkin,L.H.,Gonzales,V.,Fadale,D.J.,Slunt,H.H.,Lester,H.A.,Younkin,S.G.,and Borchelt,D.R.(2007)Rodent Ab modulates the solubilityand distribution of amyloid deposits in transgenic mice.J.Biol.Chem.282,22707–22720.),因此为了在小鼠转基因模型中获得老年斑(senile plaque,SP)病理,通常会将人源APP的突变基因转入小鼠体内。常用的APP突变型有:瑞典双突变型(K670N以及M671L),伦敦突变型(V717I),印第安纳突变型(V717F),荷兰突变型(E693Q)等,这些位点突变使APP易于被分泌酶切割生成Aβ。
早老素(PSEN1/2)参与构成γ-分泌酶复合体,其基因突变出现在FAD中,可促进γ-分泌酶裂解APP生成Aβ(Newman,M.,Musgrave,I.F.,and Lardelli,M.(2007)Alzheimerdisease:amyloidogenesis,the presenilins and animalmodels.Biochim.Biophys.Acta 1772,285–297.)。阿尔茨海默病转基因模型中常联合应用PSEN和APP,以增强和提前Aβ斑块的形成。
尽管APP/PSEN转基因鼠具备了一些重要的AD表现型,但是却缺少神经纤维缠结(neurofibrillary tangles,NFPs)这个重要的病理标志。因此为了获得神经纤维缠结人们又向模型中转入了tau蛋白突变基因,从而使APP/PSEN/TAU三转基因小鼠获得了AD所有主要的表现型:包括老年斑;神经纤维缠结;神经元丢失;认知功能障碍。
但是事实上阿尔茨海默病与tau基因突变并无联系,FAD中也并不存在tau基因突变(Ballatore,C.,Lee,V.M.,and Trojanowski,J.Q.(2007)Tau-mediatedneurodegeneration in Alzheimer’s disease and relateddisorders.Nat.Rev.Neurosci.8,663–672.Giannakopoulos,P.,Gold,G.,Kovari,E.,vonGunten,A.,Imhof,A.,Bouras,C.,and Hof,P.R.(2007)Assessing the cognitive impactof Alzheimer disease pathology and vascular burden in the aging brain:TheGeneva experience.Acta Neuropathol.113,1–12.)。目前AD转基因模型中广泛应用的tauP301L突变型来源于前颞颥痴呆帕金森症(frontal temporal dementia withParkinsonism,FTDP-17),该突变导致tau蛋白结合微管的性能降低,促使FTDP-17脑中形成明显的神经纤维缠结(Lee,V.M.,Goedert,M.,and Trojanowski,J.Q.(2001)Neurodegenerative tauopathies.Annu.Rev.Neurosci.24,1121–1159.Roberson,E.D.(2006)Frontotemporal dementia.Curr.Neurol.Neurosci.Rep.6,481–489.)。
也就是说,AD中的神经纤维缠结并不是由tau基因突变引起的,而是存在其他的诱导因素。虽然APP/PSEN/TAU三转基因动物是目前最佳的AD模型,具有很多应用价值,但是它并不符合AD真实的病理发生机制。如果用这样的模型研究AD发病机制可能会得出与实际不符的结论,用其开发治疗靶点或作临床前药效评价,可能收不到预期的疗效。
因此,开发既能够涵盖AD表现型,又能够真实模拟阿尔茨海默病的模型,对于阿尔茨海默病的研究十分必要。
发明内容
本发明的目的在于通过转基因技术建立HO-1/APP/PSEN1三转基因AD小鼠模型,以获得一种可稳定遗传、既能涵盖阿尔茨海默病表型,又符合阿尔茨海默病真实病理状态的动物模型。该模型将可用于基因在体功能分析、疾病发病机制探讨、药物新靶点发现及临床前药效评价等研究,具有十分重要的科学意义和临床价值。
本发明的目的是通过以下技术手段实现的:
一种产生HO-1/APP/PSEN1三转基因阿尔茨海默病小鼠模型的方法,其是将mHO-1转基因小鼠与APP/PSEN1双转基因小鼠杂交,对获得的子代小鼠进行鉴定,获得阳性HO-1/APP/PSEN1三转基因阿尔茨海默病小鼠模型。
在本发明中,所述的对获得的子代小鼠进行鉴定,优选包括按照阿尔茨海默病的分子、病理、行为学特征对HO-1/APP/PSEN1转基因小鼠进行鉴定。
在本发明中,APP/PSEN1双转基因小鼠可购买自美国国立Jackson动物中心(BarHarbor,ME,USA)。
在本发明中,优选的,所述的mHO-1转基因小鼠的受精卵通过以下方法制备得到:
(1)将小鼠血红素加氧酶1(heme oxygenase 1)编码基因Hmox1的cDNA插入pCAG载体EcoRⅠ酶切位点,获得的含有Hmox1的cDNA的pCAG重组载体,命名为pCAG-mHO-1;
(2)将包括CMV增强子,chickenβ-actin启动子,mHO-1cDNA编码序列,终止信号rabbitβ-globin poly(A)在内的一段序列从载体pCAG-mHO-1中切下,纯化线性DNA片段;
(3)制备显微注射用的DNA溶液;
(4)应用显微注射的方法将构建的基因序列注入到小鼠的成活的受精卵中,即得。
在本发明中,优选的,步骤(2)是通过SalI以及DraI酶切将包括CMV增强子,chickenβ-actin启动子,mHO-1cDNA编码序列,终止信号rabbitβ-globin poly(A)在内的一段序列从载体pCAG-mHO-1中切下,纯化线性DNA片段。
目前APP/PSEN/TAU三转基因模型是能够复制出所有阿尔茨海默病病理特征的最佳模型,它虽然通过引入突变的tau基因获得了神经纤维缠结(NFTs)病理,但事实上阿尔茨海默病中的NFTs并不是由tau基因突变引起的,而是存在其他诱导因素。本发明利用转基因技术用HO-1基因代替APP/PSEN/TAU三转基因模型中的tauP301L突变基因,建立了HO-1/APP/PSEN1三转基因AD小鼠模型,该转基因小鼠是符合阿尔茨海默病的病理状态的。经行为学、形态学、分子生物学检测,HO-1/APP/PSEN1转基因小鼠在认知功能、脑组织病理及分子标志方面均符合阿尔茨海默病特征性表型,可作为阿尔茨海默病动物模型。并且,HO-1/APP/PSEN三转基因小鼠是一种既能涵盖阿尔茨海默病表型又符合阿尔茨海默病病理发生机制的动物模型。该模型可用于基因在体功能分析、疾病发病机制探讨、药物新靶点发现及临床前药效评价等研究。
附图说明
图1为构建Tg(mHO-1)小鼠转基因片段结构示意图;
图2为外源mHO-1转基因片段的mRNA表达水平;
其中,L:低表达系;H1:高表达系1;H2:高表达系2;
图3为Tg(mHO-1)小鼠总HO-1的mRNA表达水平;
图4为Tg(mHO-1)小鼠总HO-1的蛋白表达水平;
图2,图3,图4通过检测外源mHO-1转基因片段的表达水平,以RT-PCR和westernblot方法检测Tg(mHO-1)转基因小鼠总HO-1基因RNA和蛋白表达水平,对Tg(mHO-1)转基因小鼠进行鉴定。
图5为杂交获得Tg(HO-1/APP/PSEN1)转基因小鼠方法示意图;
将Tg(mHO-1)转基因小鼠与Tg(APP/PSEN1)小鼠(2TG)杂交,经筛选获得双阳性子代,即Tg(HO-1/APP/PSEN1)转基因小鼠(3TG)。
图6为水迷宫实验训练头5天中逃逸潜伏期的检测结果;
图7为水迷宫实验中各组小鼠游泳路线示踪;
图8为水迷宫实验第6天各组小鼠的目标象限路程百分比;
图9为水迷宫实验第6天各组小鼠的目标象限时间;
图6,图7,图8,图9用Morris水迷宫实验检测评价Tg(HO-1/APP/PSEN1)转基因小鼠学习记忆和空间认知能力。经验证,Tg(HO-1/APP/PSEN1)三转基因小鼠的学习和空间记忆能力与野生型小鼠相比显著降低,并且较Tg(APP,PSEN1)小鼠下降得更为明显。
图10为Aβ42免疫组化法检测小鼠脑组织中的老年斑;
图11为比较Aβ42免疫组化法检测小鼠脑组织中老年斑个数;
图10,图11通过免疫组织化学检测Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中的老年斑病理表现。结果表明,Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中出现大量老年斑(Aβ斑块),并且老年斑个数多于Tg(APP/PSEN1)小鼠。
图12为总tau蛋白免疫组化法检测小鼠脑组织中的神经缠结病理;
图13为电镜下观察Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中神经元微管解聚现象和神经纤维缠结现象;
图12,图13通过免疫组织化学和电镜方法检测Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中的神经纤维缠结病理表现。免疫组化结果可见Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中出现大量神经纤维缠结病理现象。电镜下可见神经元微管解聚,胞浆内可见异常聚集的神经纤维。
图14为免疫组化法检测小鼠脑组织中的Aβ寡聚体病理;
图15为免疫组化法检测小鼠脑组织中的tau寡聚体病理;
图14,图15通过免疫组织化学检测Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中的Aβ寡聚体和tau寡聚体病理表现;免疫组化结果可见Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中Aβ寡聚体和tau寡聚体大量表达,明显高于野生型和Tg(APP/PSEN1)小鼠,并且,Aβ寡聚体和tau寡聚体高表达的神经元发生核固缩、细胞变性病理现象。
图16为western blot法检测小鼠脑海马组织中的总tau蛋白含量;
图17为western blot法检测小鼠脑海马组织中过度磷酸化异常聚集的tau蛋白含量;
图18为western blot法检测小鼠脑海马组织中可溶性的tau蛋白寡聚体含量;
图16,图17,图18通过western blot方法检测不同形式的tau蛋白:总tau蛋白、tau-PHF以及tau寡聚体,在Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中的表达水平。结果可见,Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中3种形式的tau蛋白的表达明显高于野生型小鼠。而Tg(APP/PSEN1)小鼠较野生型小鼠只有总tau蛋白表达升高,而tau蛋白的病理形式(tau-PHF和tau寡聚体)的表达水平并未升高。说明Tg(HO-1/APP/PSEN1)转基因小鼠具备了阿尔茨海默病的tau病理特征,并且在tau病理的表现上优于Tg(APP/PSEN1)小鼠。
图19为western blot法检测小鼠脑海马组织中的Aβ42蛋白含量
图20为western blot法检测小鼠脑海马组织中可溶性的Aβ寡聚体含量
图19,图20通过western blot方法检测Aβ42蛋白和Aβ寡聚体,在Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中的表达水平。结果可见,Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中Aβ42蛋白和Aβ寡聚体的表达明显高于野生型小鼠和Tg(APP/PSEN1)小鼠。说明Tg(HO-1/APP/PSEN1)转基因小鼠具备了阿尔茨海默病的Aβ病理特征,并且在Aβ病理的表现上优于Tg(APP/PSEN1)小鼠。
具体实施方式
下面结合具体实施例和附图来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
实施例1:Tg(mHO-1)转基因小鼠模型的构建
1、获得目的基因:
设计含有EcoRI限制性内切酶位点的引物,在小鼠基因组中扩增血红素加氧酶1(heme oxygenase 1(mHO-1),Gene ID:15368)基因的cDNA序列,采用EcoRI限制性内切酶酶切扩增得到的产物,将酶切后的产物插入pCAGG载体的EcoRI位点,获得的含有Hmox1的cDNA的pCAG重组载体,命名为pCAG-mHO-1。mHO-1cDNA基因由chickenβ-actin启动子和CMV增强子启动,mHO-1连接rabbitβ-globin poly(A)终止信号。pCAG-mHO-1质粒经SalI以及DraI酶切,获得线性转基因序列,构建的Tg(mHO-1)小鼠转基因片段结构示意图如图1所示,纯化回收用于显微注射。
2、产生Tg(mHO-1)转基因小鼠的受精卵,进而建立Tg(mHO-1)转基因小鼠模型
将线性化的目的基因片段注射入小鼠受精卵的原核中,使其与小鼠基因组整合,随着细胞不断分裂,每个细胞将携带该片段。将经显微注射后成活的受精卵植入假孕鼠的输卵管的壶腹部,产生F0代转基因小鼠,该小鼠基因组中整合有外源性mHO-1表达序列。仔鼠出生3周后,剪去小鼠尾尖,以PCR的方法检测外源mHO-1转基因片段的mRNA水平进行转基因小鼠的鉴定,得到Tg(mHO-1)转基因小鼠。以RT-PCR和western blot方法检测Tg(mHO-1)转基因小鼠总HO-1基因RNA和蛋白表达水平。
结果:
外源mHO-1转基因片段的mRNA表达水平如图2所示,Tg(mHO-1)小鼠总HO-1的mRNA表达水平如图3所示,Tg(mHO-1)小鼠总HO-1的蛋白表达水平如图4所示。以上结果表明,Tg(mHO-1)转基因小鼠中外源mHO-1、总HO-1基因RNA和蛋白表达水平相较于野生型小鼠均有所提高,说明Tg(mHO-1)转基因小鼠模型构建成功。
实施例2:产生Tg(HO-1/APP/PSEN1)转基因小鼠
杂交获得Tg(HO-1/APP/PSEN1)转基因小鼠方法示意图如图5所示:将实施例1构建的Tg(mHO-1)转基因小鼠与APP/PSEN1双转基因AD小鼠模型(Tg(APP/PSEN1),2TG)杂交,获得HO-1/APP/PSEN1三转基因阳性子代,即Tg(HO-1/APP/PSEN1)转基因小鼠(3TG)。Tg(APP/PSEN1)小鼠(stock number:004462)来源于美国国立Jackson动物中心(Bar Harbor,ME,USA),由南京大学—南京生物医药研究院引种。
杂合后的子代提取鼠尾DNA经PCR筛选三转基因的杂合子,APP/PSEN1引物(美国国立Jackson动物中心提供),HO-1引物如下表1所示:
表1
实施例3:Tg(HO-1/APP/PSEN1)转基因小鼠认知功能特征
Morris水迷宫(Morris R.Developments of a water-maze procedure forstudying spatial learning in the rat.J Neurosci Methods.1984May;11(1):47-60.)是检测阿尔茨海默病海马认知功能的经典实验,通过水迷宫行为学实验评价Tg(HO-1/APP/PSEN1)转基因小鼠学习记忆和空间认知能力。经验证,Tg(HO-1/APP/PSEN1)三转基因小鼠的学习和空间记忆能力与野生型小鼠(WT)相比显著降低,并且较Tg(APP/PSEN1)小鼠(2TG)下降得更为明显。
结果:
水迷宫实验训练头5天中逃逸潜伏期的检测结果如图6所示,水迷宫实验中各组小鼠游泳路线示踪如图7所示,水迷宫实验第6天各组小鼠的目标象限路程百分比如图8所示,水迷宫实验第6天各组小鼠的目标象限时间如图9所示。
从上述结果可以看出:Tg(HO-1/APP/PSEN1)三转基因小鼠的学习和空间记忆能力与野生型小鼠相比显著降低,并且较Tg(APP/PSEN1)小鼠下降得更为明显。
实施例4:Tg(HO-1/APP/PSEN1)转基因小鼠脑组织形态学特征
斑块病理(Aβ累积,老年斑)和缠结病理(tau蛋白累积,神经纤维缠结)是经典的阿尔茨海默病病理评价指标。通过免疫组织化学和电镜方法检测以上指标在Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中的病理表现。
结果:
1、Aβ42免疫组化法检测小鼠脑组织中的老年斑的情况如图10所示,比较Aβ42免疫组化法检测小鼠脑组织中老年斑个数情况如图11所示。
从以上结果可以看出:Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中出现大量老年斑(Aβ斑块),并且老年斑个数多于Tg(APP/PSEN1)小鼠(2TG)。
2、由于tau蛋白过度磷酸化将从微管上解离下来,丧失结合微管,保持微管结构的功能。并且过度磷酸化的tau蛋白会发生聚集,在细胞内形成神经纤维缠结的病理现象。总tau蛋白免疫组化法检测小鼠脑组织中的神经缠结病理结果如图12所示。电镜下观察Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中神经元微管解聚现象和神经纤维缠结现象如图13所示。
免疫组化结果可见Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中出现大量神经纤维缠结病理现象。电镜下可见神经元微管解聚,胞浆内可见异常聚集的神经纤维。
3、目前的研究显示,细胞溶质中的可溶性的Aβ寡聚体和tau寡聚体是Aβ和tau蛋白的主要毒性形式(Alonso AD,Li B,Grundke-Iqbal I,Iqbal K.Polymerization ofhyperphosphorylated tau into filaments eliminates its inhibitoryactivity.Proc Natl Acad Sci USA.2006;23:8864–8869.Wittmann CW,Wszolek MF,Shulman JM,Salvaterra PM,Lewis J,Hutton M,et al.Tauopathy in Drosophila:neurodegeneration without neurofibrillary tangles.Science.2001;293:711–714.Oddo S,Vasilevko V,Caccamo A,Kitazawa M,Cribbs DH,LaFerla FM.Reduction ofsoluble Abeta and tau,but not soluble Abeta alone,ameliorates cognitivedecline in transgenic mice with plaques and tangles.J Biol Chem.2006;281:39413–39423.),因此检测Aβ寡聚体和tau寡聚体,对于评价阿尔茨海默病模型具有重要价值。
免疫组化法检测小鼠脑组织中的Aβ寡聚体病理结果如图14所示,免疫组化法检测小鼠脑组织中的tau寡聚体病理结果如图15所示。
免疫组化结果可见Tg(HO-1/APP/PSEN1)转基因小鼠(3TG)脑组织中Aβ寡聚体和tau寡聚体大量表达,明显高于野生型和Tg(APP/PSEN1)小鼠(2TG)。并且,Aβ寡聚体和tau寡聚体高表达的神经元发生核固缩、细胞变性病理现象。
实施例5:Tg(HO-1/APP/PSEN1)转基因小鼠脑组织分子标志表达特征
Aβ蛋白和tau蛋白,包括Aβ42、Aβ寡聚体、总tau蛋白、tau-PHF以及tau寡聚体,是经典的阿尔茨海默病脑组织分子评价指标。这里通过western blot方法检测以上指标在Tg(HO-1/APP/PSEN1)转基因小鼠脑组织中的表达水平。
western blot法检测小鼠脑海马组织中的总tau蛋白含量结果如图16所示,western blot法检测小鼠脑海马组织中过度磷酸化异常聚集的tau蛋白含量结果如图17所示,western blot法检测小鼠脑海马组织中可溶性的tau蛋白寡聚体含量结果如图18所示,western blot法检测小鼠脑海马组织中的Aβ42蛋白含量结果如图19所示,western blot法检测小鼠脑海马组织中可溶性的Aβ寡聚体含量结果如图20所示。
以上结果表明,Tg(HO-1/APP/PSEN1)转基因小鼠(3TG)脑组织中的AD分子标志表达明显高于野生型和Tg(APP/PSEN1)小鼠(2TG)。
Claims (3)
1.一种产生HO-1/APP/PSEN1三转基因阿尔茨海默病小鼠模型的方法,其特征在于是将mHO-1转基因小鼠与APP/PSEN1双转基因小鼠杂交,对获得的子代小鼠进行鉴定,获得阳性HO-1/APP/PSEN1三转基因阿尔茨海默病小鼠模型;
所述的mHO-1转基因小鼠的受精卵通过以下方法制备得到:
(1)将小鼠血红素加氧酶1(heme oxygenase 1)编码基因Hmox1的cDNA插入pCAG载体EcoRⅠ酶切位点,获得的含有Hmox1的cDNA的pCAG重组载体,命名为pCAG-mHO-1;
(2)将包括CMV增强子,chickenβ-actin启动子,mHO-1cDNA编码序列,终止信号rabbitβ-globin poly(A)在内的一段序列从载体pCAG-mHO-1中切下,纯化线性DNA片段;
(3)制备显微注射用的DNA溶液;
(4)应用显微注射的方法将构建的基因序列注入到小鼠的成活的受精卵中,即得。
2.如权利要求1所述的方法,其特征在于所述的对获得的子代小鼠进行鉴定包括按照阿尔茨海默病的分子、病理、行为学特征对HO-1/APP/PSEN1转基因小鼠进行鉴定。
3.如权利要求1所述的方法,其特征在于步骤(2)是通过SalI以及DraI酶切将包括CMV增强子,chickenβ-actin启动子,mHO-1cDNA编码序列,终止信号rabbit β-globin poly(A)在内的一段序列从载体pCAG-mHO-1中切下,纯化线性DNA片段。
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