CN114561428B - 一种阿尔茨海默病动物模型、构建方法及应用 - Google Patents
一种阿尔茨海默病动物模型、构建方法及应用 Download PDFInfo
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Abstract
本发明属于动物模型构建技术领域,公开了一种阿尔茨海默病动物模型、构建方法及应用,通过利用嵌合小鼠/人淀粉样蛋白前体蛋白以及突变的人PSEN1基因,两者均在小鼠Prion启动子的控制下针对CNS神经元,构建成APP/PS1双转基因AD模型小鼠;运用CRISPR‑Cas9技术将Hepcidin基因全身性敲除,构建APPswe/PS1dE9+HAMP‑/‑(+)三转基因动物模型。本发明构建的模拟人AD疾病的小鼠模型表现出明显的脑内老年斑沉积,铁离子聚集等多种人AD患者临床表现,稳定性强,遗传稳定,与人类AD疾病表现类似,可为研究AD发病机制以及基因治疗提供经济、简单、可靠的动物模型。
Description
技术领域
本发明属于动物模型构建技术领域,尤其涉及一种阿尔茨海默病动物模型、构建方法及应用。
背景技术
目前,阿尔茨海默病(Alzheimer’s disease,AD)是一种以进行性认知功能障碍和行为损害为特征的由多种病因引起的中枢神经系统退行性病变,然而,到目前为止,仍不能在早期阶段确诊AD,且该病的病因和发病机制仍未完全研究清楚。此外,它的发病与年龄密切相关,如今老龄化速度在加快,AD患病率也在逐渐增加,已成为危害人们健康的重大疾病。因此,深入揭示AD的发病机制,寻找有效而特异的治疗药物迫在眉睫。
动物模型,尤其是小鼠动物模型,是研究人类发病机制、筛选药物治疗靶点和开发治疗手段的主要工具。应用于构建AD动物模型的方法较为多样,且各具特点,其中转基因动物模型是目前研究AD较为理想且应用广泛的模型,能表征多个病理特点或较早出现的病理改变,表现与AD患者类似的临床症状,因此转基因动物模型具有非常广阔的研究及应用前景。如今有单转基因与多基因突变转基因动物模型:包括APP/PS1双转基因小鼠、APP/Tau双转基因小鼠、TAPP小鼠和APP/PS1/Tau三转基因小鼠。目前研究铁调素在AD中的作用均是通过体内或体外注射的方法,缺乏相应的动物模型。所以本技术领域亟需解决如何获得一种更符合疾病病理特征,可为研究AD的发病机制和筛选药物提供疾病动物模型的技术问题。
通过上述分析,现有技术存在的问题及缺陷为:目前研究铁调素在AD中的作用均是通过体内或体外注射的方法,缺乏相应的动物模型。
发明内容
针对现有技术存在的问题,本发明提供了一种阿尔茨海默病动物模型、构建方法及应用,尤其涉及一种Hepciden基因敲除的APP/PS1三转基因动物模型的构建方法及应用。
本发明是这样实现的,一种阿尔茨海默病动物模型的构建方法,所述阿尔茨海默病动物模型的构建方法包括以下步骤:
通过利用嵌合小鼠/人淀粉样蛋白前体蛋白以及突变的人PSEN1基因,两者均在小鼠Prion启动子的控制下针对CNS神经元,构建成APP/PS1双转基因AD模型小鼠;运用CRISPR-Cas9技术将Hepcidin基因全身性敲除,从而构建APPswe/PS1dE9+HAMP-/-(+)三转基因动物模型。
进一步,所述阿尔茨海默病动物模型的构建方法还包括基于CRISPR-Cas9系统设计靶向小鼠Hepciden基因的sgRNA。
进一步,所述小鼠/人淀粉样蛋白前体蛋白为Mo/HuAPP695swe。
进一步,所述人PSEN1基因为PS1-dE9。
进一步,用于PCR鉴定的特异性引物包括:Hepciden-F和Hepciden-R,APP/PS1-P1和APP/PS1-P2,以及WT-F1和WT-R。
进一步,所述引物Hepciden-F的核苷酸序列为SEQ ID NO:1,所述APP/PS1的核苷酸序列为SEQ ID NO:2,所述WT的核苷酸序列为SEQ ID NO:3。
本发明的另一目的在于提供一种实施所述的阿尔茨海默病动物模型的构建方法构建得到的阿尔茨海默病动物模型。
本发明的另一目的在于提供一种所述的阿尔茨海默病动物模型在分析阿尔茨海默病的发病机制中的应用。
本发明的另一目的在于提供一种所述的阿尔茨海默病动物模型在筛选治疗阿尔茨海默病的药物中的应用。
本发明的另一目的在于提供一种所述的阿尔茨海默病动物模型在非诊断方法和非治疗方法中的应用。
结合上述的所有技术方案,本发明所具备的优点及积极效果为:本发明提供的阿尔茨海默病动物模型的构建方法,通过在正常饮食喂养的成年APP/PS1小鼠,利用CRISPR-Cas9技术将铁调素Hepcidin基因全身性敲除。本发明的构建提供了一种阿尔茨海默病动物模型,该模型稳定性强且稳定遗传,以及该动物模型在研究阿尔茨海默病的发病机制和在筛选治疗药物中的应用。本发明提供了一种Hepciden基因敲除的APP/PS1三转基因小鼠模型,可为研究AD的发病机制和筛选药物提供疾病动物模型的技术问题。
本发明提供了一种通过Hepciden基因敲除的APP/PS1三转基因小鼠模型模拟人AD疾病的方法,利用CRISPR-Cas9基因敲除技术,敲除Hepciden基因;Hepciden基因敲除后的APP/PS1+Hepciden-/-三转基因小鼠均表现出明显的脑内老年斑沉积,铁离子聚集等多种人AD患者临床表现。本发明构建的模拟人AD疾病的小鼠模型,该模型稳定性强稳定遗传,与人类AD疾病表现类似,可为进一步研究AD发病机制以及基因治疗提供经济、简单、可靠的动物模型。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对本发明实施例中所需要使用的附图做简单的介绍,显而易见地,下面所描述的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下还可以根据这些附图获得其他的附图。
图1是本发明实施例提供的阿尔茨海默病动物模型的构建方法流程图。
图2是本发明实施例提供的构建的Hepciden基因敲除的APP/PS1三转基因小鼠基因检测鉴定电泳图;其中,1、3、5、6为杂合APP/PS1+Hepciden-/+三转基因小鼠,51、54、62、64为纯合APP/PS1+Hepciden-/-三转基因小鼠。
图3是本发明实施例提供的水迷宫实验结果示意图。
图4是本发明实施例提供的旷场实验结果示意图。
图5是本发明实施例提供的石墨炉原子吸收光谱技术检测小鼠脑铁含量的结果示意图。
图6是本发明实施例提供的免疫组化检测小鼠海马组织Aβ的表达示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
针对现有技术存在的问题,本发明提供了一种阿尔茨海默病动物模型、构建方法及应用,下面结合附图对本发明作详细的描述。
如图1所示,本发明实施例提供的阿尔茨海默病动物模型的构建方法包括以下步骤:
S101,基于CRISPR-Cas9系统设计靶向小鼠Hepciden基因的sgRNA;
S102,利用嵌合小鼠/人淀粉样蛋白前体蛋白突变的人PSEN1基因,在小鼠Prion启动子的控制下针对CNS神经元构建成APP/PS1双转基因AD模型小鼠;
S103,运用CRISPR-Cas9技术将Hepcidin基因全身性敲除,从而构建APPswe/PS1dE9+HAMP-/-(+)三转基因动物模型。
下面结合具体实施例对本发明的技术方案作进一步描述。
实施例:基因型检测
APP/PS1+Hepciden-/-(+)纯合或杂合三转基因模型小鼠配繁获得的F1代基因型鉴定PCR结果对应的扩增产物大小为野生型455bp,突变型759bp与142bp,455bp为野生型等位基因条带,759bp为Hepciden突变等位基因条带,142bp为APP/PS1突变等位基因条带,三条带同时出现表明小鼠同时携带野生型与突变型基因,即小鼠为杂合子APP/PS1+Hepciden-/+三转基因小鼠,若只出现759bp与142bp两条带表明小鼠只携带突变型基因,即小鼠为纯合子APP/PS1+Hepciden-/-三转基因小鼠。
用于PCR鉴定特异性引物如下:
SEQ ID NO:1:
Hepciden-F:5’-CTGCTCATACCAAGGGGTCCAGG-3’
Hepciden-R:5’-AAAGGATCCGGGCGTGGTAGTAC-3’
SEQ ID NO:2:
APP/PS1-P1:5’-ATGGTAGAGTAAGCGAGAACACG-3’
APP/PS1-P2:5’-GGATCTCTGAGGGGTCCAGT-3’
SEQ ID NO:3:
WT-F1:5’-GATCTGGAGTTTATGTAGCAGCCTGG-3’
WT-R:5’-AAAGGATCCGGGCGTGGTAGTAC-3’
图2本发明实施例构建的Hepciden基因敲除的APP/PS1三转基因小鼠基因检测鉴定电泳图,1、3、5、6为杂合APP/PS1+Hepciden-/+三转基因小鼠,51、54、62、64为纯合APP/PS1+Hepciden-/-三转基因小鼠。
本发明提供了通过Hepciden基因敲除的APP/PS1三转基因小鼠模型模拟人AD疾病的方法。利用CRISPR-Cas9基因敲除技术,敲除Hepciden基因。Hepciden基因敲除后的APP/PS1+Hepciden-/-三转基因小鼠均表现出明显的脑内老年斑沉积,铁离子聚集等多种人AD患者临床表现。本发明构建的模拟人AD疾病的小鼠模型,该模型稳定性强稳定遗传,与人类AD疾病表现类似,可为进一步研究AD发病机制以及基因治疗提供经济、简单、可靠的动物模型。
下面结合试验对本发明的技术效果作详细的描述。
如图3所示,水迷宫:实验分为3组,6月龄C57小鼠,APP/PS1小鼠和APP/PS1/Hepcidin各10只。
实验方法:采用Morris水迷宫对各组小鼠进行行为认知能力测试。Morris水迷宫主要由圆形水池和自动录像及分析系统组成。圆形水池直径120cm、高50cm,平台直径为10cm,在整个实验中保持操作者位置及周围环境的相对稳定。
定位航行试验:历时5d,每日分上、下午两个时段,固定时间为每日上午9:00-11:00,下午14:00-16:00进行。每个时段分别从4个不同的入水点入水,将小鼠面向池壁放人水中,记录2min内寻找到平台所用的时间,即逃避潜伏期。若小鼠入水后2min内未能找到平台,则将其置于平台上并停留10s,记录逃避潜伏期为120s。每次训练时间间隔为60s。
空间探索实验:用于测量动物对平台空间位置准确记忆能力,即记忆保持能力。第6天撤除平台,任选一个入水点将动物放入水中,动物在水中游泳120s,测量120s内小鼠跨过原平台的次数,即为平台区域穿越次数,并记录小鼠在平台取悦停留时间。
如图4所示,旷场实验:实验分为3组,6月龄C57小鼠,APP/PS1小鼠和APP/PS1/Hepcidin各10只。
实验方法:使用一敞箱,规格为83cm(长)×83cm(宽)×40cm(高),敞箱内壁及底部均为白色。利用隔板将敞箱分为四个等大方形空间,每个方形空间内分为16个等大小方格。实验开始时,将4只小鼠同时放入每个方形空间中央,以SMART3.0小动物视频追踪系统记录分析小鼠5min内水平运动总距离、中央区进入总次数等指标。每只小鼠结束测试后,清除旷场底部粪便与尿液,并用75%酒精擦拭整个旷场及隔板,避免气味干扰。待75%酒精全部挥发后进行下一组小鼠实验。所有小鼠完成旷场实验后要经过1周的实验遗忘期。
如图5所示,实验分为3组,6月龄C57小鼠,APP/PS1小鼠和APP/PS1/Hepcidin各10只。石墨炉原子吸收光谱技术检测小鼠脑铁含量:准确称取待消解样品,精确至0.0001g(0.1mg),置于100mL高脚烧杯中,加入硝酸:高氯酸(4:1)20mL,瓶口盖一凹皿,避光消化10小时,微波消解,在通风橱中置于电热板上中温加热,沸腾后保持微沸,消解至无色透明,高温加热挥完余酸,冷却后加入去离子水10mL,加热至近干,用2%HNO3溶解残渣,移入比色管,定容至10mL(10mL容量瓶),平行作空白。Fe标准溶液(国家标准物质研究中心,1000μg/mL)2%HN03,配制至所需浓度。Fe标准溶液浓度系列如下(μg/L):10、20、30、40。采用石墨炉原子吸收分光光谱法测定样品中铁含量。首先上机测定各浓度Fe标准溶液,绘制出标准曲线。然后依次测定各样品管,得出各样品管的铁含量。
如图6所示,实验分为3组,6月龄C57小鼠,APP/PS1小鼠和APP/PS1/Hepcidin各10只。免疫组化检测小鼠海马组织Aβ的表达:各组动物端头取脑组织,4%多聚甲醛体外固定24h,放入30%蔗糖溶液,沉淀组织在冷冻切片机-25℃下,20μm连续切片,-20℃冰箱保存。各组选取10张小鼠脑组织冰冻切片做免疫组化,一抗为兔抗小鼠Aβ,二抗为抗兔生物素化,操作严格按照说明书进行。显微成像系统拍照后,每张切片选取3个视野用Image-Pro Plus软件测得平均光密度值。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,都应涵盖在本发明的保护范围之内。
序列表
<110> 董贤慧,高维娟,贺小平,张天赐,刘珊,孙宁宁,刘晓晓,李佳明,王雅欣,林一璨,张博,郭一诺,张鑫萌,张红军,李佳琦
<120> 一种阿尔茨海默病动物模型、构建方法及应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ctgctcatac caaggggtcc aggaaaggat ccgggcgtgg tagtac 46
<210> 2
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggtagagt aagcgagaac acgggatctc tgaggggtcc agt 43
<210> 3
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gatctggagt ttatgtagca gcctggaaag gatccgggcg tggtagtac 49
Claims (7)
1.一种阿尔茨海默病动物模型的构建方法,其特征在于,所述阿尔茨海默病动物模型的构建方法通过利用嵌合小鼠/人淀粉样蛋白前体蛋白以及突变的人PSEN1基因,两者均在小鼠Prion启动子的控制下针对CNS神经元,构建成APP/PS1双转基因AD模型小鼠;运用CRISPR-Cas9技术将Hepcidin基因全身性敲除,杂交繁殖,从而构建APPswe/PS1dE9+HAMP-/-(+)三转基因动物模型;
所述阿尔茨海默病动物模型的构建方法还包括基于CRISPR-Cas9系统设计靶向小鼠Hepciden基因的sgRNA。
2.如权利要求1所述的阿尔茨海默病动物模型的构建方法,其特征在于,所述小鼠/人淀粉样蛋白前体蛋白为Mo/HuAPP695swe。
3.如权利要求1所述的阿尔茨海默病动物模型的构建方法,其特征在于,所述人PSEN1基因为PS1-dE9。
4.如权利要求1所述的阿尔茨海默病动物模型的构建方法,其特征在于,用于PCR鉴定的特异性引物包括:Hepciden-F和Hepciden-R,APP/PS1-P1和APP/PS1-P2,以及WT-F1和WT-R。
5.如权利要求4所述的阿尔茨海默病动物模型的构建方法,其特征在于,所述引物Hepciden-F的核苷酸序列为SEQ ID NO:1,所述APP/PS1的核苷酸序列为SEQ ID NO:2,所述WT的核苷酸序列为SEQ ID NO:3。
6.一种如权利要求1~5任意一项所述的阿尔茨海默病动物模型在分析阿尔茨海默病的发病机制中的应用。
7.一种如权利要求1~5任意一项所述的阿尔茨海默病动物模型在筛选治疗阿尔茨海默病的药物中的应用。
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Citations (3)
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---|---|---|---|---|
CN105274144A (zh) * | 2015-09-14 | 2016-01-27 | 徐又佳 | 通过CRISPR/Cas9技术得到敲除铁调素基因斑马鱼的制备方法 |
CN105494263A (zh) * | 2015-12-25 | 2016-04-20 | 哈尔滨医科大学 | 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 |
CN112899280A (zh) * | 2021-04-09 | 2021-06-04 | 中国药科大学 | 基于CRISPR/Cas9基因编辑技术建立的AD细胞模型及其构建方法和应用 |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105274144A (zh) * | 2015-09-14 | 2016-01-27 | 徐又佳 | 通过CRISPR/Cas9技术得到敲除铁调素基因斑马鱼的制备方法 |
CN105494263A (zh) * | 2015-12-25 | 2016-04-20 | 哈尔滨医科大学 | 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 |
CN112899280A (zh) * | 2021-04-09 | 2021-06-04 | 中国药科大学 | 基于CRISPR/Cas9基因编辑技术建立的AD细胞模型及其构建方法和应用 |
Non-Patent Citations (4)
Title |
---|
astrocyte hepcidin amelioraters neuronal loss through attenuating brain iron deposition and oxidative stress in APP/PS1 mice;Yong Xu等;《Free Raical Biology and Medicine》;第158卷;84-95 * |
CRISPR/Cas9基因编辑技术在阿尔兹海默病研究中的应用;孙谕莹等;《生命科学》;第33卷(第7期);912-920 * |
淫羊藿、黄芪、葛根有效组分复方对APPswe/PS1dE9双转基因AD模型小鼠海马CA3区hepcidin表达的影响;马冬雪等;中国药理学通报;20191028;第35卷(第11期);1523-1527 * |
阿尔茨海默病转基因动物模型:如何更接近病理特征?;董贤慧;柴锡庆;;中国组织工程研究(46);8075-8082 * |
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