CN110679549B - 一种阿尔茨海默病小鼠模型的构建方法 - Google Patents
一种阿尔茨海默病小鼠模型的构建方法 Download PDFInfo
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Abstract
本发明公开了一种阿尔茨海默病小鼠模型的构建方法，采用基因型FVB‑Fgf14Tg(tetO‑MAPT*P301L)4510Kha/JlwsJ雌性小鼠，于小鼠4月龄时脑注射截短tau蛋白或者表达截短tau蛋白的载体后饲养2‑4个月，所述截短tau蛋白氨基酸序列如SEQ ID NO:1所示。本发明成功构建了阿尔茨海默病小鼠模型，构建的小鼠模型只有海马区出现tau病理，其他脑区未见tau病理，本发明将构建周期缩短至6个月，节省了50％以上的建模时间。
Description
技术领域
本发明属于生物医药领域，具体涉及一种阿尔茨海默病小鼠模型的构建方法
背景技术
阿尔茨海默病(Alzheimer’s Disease，简称AD)，俗称老年痴呆，是老年人中常见的一种神经退行性疾病，表现为学习记忆障碍，失语、失用、失认，人格行为改变等全面性痴呆。AD有两大神经病理学特征：细胞外的由β-淀粉样蛋白(β-amyloid,Aβ)聚积成的淀粉样斑块(amyloid plaques)和细胞内的主要由异常过度磷酸化tau蛋白形成的神经纤维缠结(neurofibrillary tangle,NFT)。迄今为止，针对Aβ的所有临床试验并无明显效果(Iqbal,K.,Liu,F.,Gong,C.X.,2018.Recent developments with tau-based drugdiscovery.Expert Opin Drug Discov 13,399-410.)。越来越多的研究提示，人脑中tau病理传播是导致AD病程进展的关键原因之一，抑制tau病理可能对延缓或阻止AD病理传播起到积极作用(Iqbal et al.,2018)。现有技术从AD患者脑中提取的异常过度磷酸化tau或体外表达重组tau蛋白经肝素诱导形成的聚集物，注射到小鼠脑海马区，诱发小鼠tau蛋白发生类AD样tau病理,并向相关脑区传播，可作为AD的疾病模型小鼠，用于AD相关致病机制研究，药物开发和筛选等(Clavaguera et al.,2009Clavaguera,F.,Bolmont,T.,Crowther,R.A.,Abramowski,D.,Frank,S.,Probst,A.,Fraser,G.,Stalder,A.K.,Beibel,M.,Staufenbiel,M.,Jucker,M.,Goedert,M.,Tolnay,M.,2009.Transmission and spreadingof tauopathy in tra nsgenic mouse brain.Nat Cell Biol 11,909-913.Hu,W.,Zhang,X.,Tung,Y.C.,Xie,S.,Liu,F.,Iqbal,K.,2016.Hyperphosphorylation determines boththe spread and the morphology of tau pathology.Alzheimers Dement 12,1066-1077.)。
现有技术制作AD的tau病理小鼠模型主要有三种方式。第一种方式选用野生型小鼠，如C57/B6J，FVB等品系小鼠。在小鼠4月龄时，注射AD病人脑中提取的病理性tau或体外表达重组的tau聚集物后，需要9-11个月可在小鼠脑中检测到诱发性tau病理。模型制作周期相对较长，共需13至15个月(Hu,W.,Zhang,X.,Tung,Y.C.,Xie,S.,Liu,F.,Iqbal,K.,2016.Hyperphosphorylation determines both the spread and the morphology oftau pathology.Alzheimers Dement 12,1066-1077.)。
第二种方式选择转基因小鼠，如3XTg(美国Jackson实验室小鼠编号034830，基因型B6；129-Psen1tm1Mpm Tg(APPSwe,tauP301L)1Lfa/Mmjax)，12个月大时注射AD病人脑中提取的病理性tau后6周，可以检测到诱导性tau病理；其缺点在于模型制作周期仍然过长，超过一年，显然不适合大规模药物筛选(Dai,C.L.,Hu,W.,Tung,Y.C.,Liu,F.,Gong,C.X.,Iqbal,K.,2018.Tau passive immunization blocks seeding and spread of Alzheimerhyperphosphorylated Tau-induced pathology in 3x Tg-AD mice.Alzheimers ResTher 10,13.)。
第三种方式是在鼠脑中表达人的tau蛋白突变体，制作转基因小鼠，如P301L(Taconic公司小鼠编号2508，基因型Tg(Prnp-MAPT*P301L)JNPL3Hlmc)和P301S(美国Jackson实验室小鼠编号008169，基因型B6；C3-Tg(Prnp-MAPT*P301S)PS19Vle/J)等。这些小鼠分别在4.5月龄和6月龄时就产生tau病理，但因为转基因在全脑的神经元均有表达，后期无法观察海马神经元中tau病理向其他神经元的扩散传播。(Terwel,D.,Lasrado,R.,Snauwaert,J.,Vandeweert,E.,Van Haesendonck,C.,Borghgraef,P.,Van Leuven,F.,2005.Changed conformation of mutant Tau-P301L underlies the moribundtauopathy,absent in progressive,nonlethal axonopathy of Tau-4R/2N transgenicmice.J Biol Chem 280,3963-3973.)
因此建立一个造模时间短，且仅在海马神经元中产生tau病理的小鼠模型是现有科研工作者们仍然需要攻克的难题。
发明内容
本发明针对现有技术不足，通过选择特定的tau蛋白以及小鼠，获得的模型鼠海马区出现tau病理，其他脑区未见tau病理，且成功缩短了造模周期。
本发明具体技术方案如下：
一种阿尔茨海默病小鼠模型的构建方法，采用基因型FVB-Fgf14Tg(tetO-MAPT*P301L)4510Kha/JlwsJ雌性小鼠，于小鼠4月龄时脑注射截短tau蛋白或者表达截短tau蛋白的载体后饲养2-4个月(优选饲养2个月)，所述截短tau蛋白氨基酸序列如SEQ ID NO:1所示。
上述基因型FVB-Fgf14Tg(tetO-MAPT*P301L)4510Kha/JlwsJ小鼠为美国Jac kson实验室小鼠，编号为15815(基因型FVB-Fgf14Tg(tetO-MAPT*P301L)4510Kha/JlwsJ)。该转基因小鼠含有tet反应元件和小鼠prion蛋白启动子序列，指导人tau蛋白突变体tau P301L表达。在没有tet诱导时，该小鼠的prion蛋白启动子使大脑中tau P301L中度表达，但不会导致tau病理。
本发明所述氨基酸序列如SEQ ID NO:1所示的人截短tau蛋白为全长tau蛋白氨基酸序列的第151-391位的蛋白片段。
上述方法，小鼠4月龄时脑注射截短tau蛋白或者表达截短tau蛋白的载体后采用常规的饲养方式自由摄取水和食物。室温20度，12小时光照/黑暗循环环境。
上述方法，优选表达人截短tau蛋白的载体为腺病毒，进一步优选为腺病毒AAV9载体。
本发明另一目的在于提供氨基酸序列如SEQ ID NO:1所示的截短tau蛋白在快速构建阿尔茨海默病小鼠模型中的应用，采用基因型FVB-Fgf14Tg(tetO-MAPT*P301L)4510Kha/JlwsJ雌性小鼠造模。
阿尔茨海默病(Alzheimer s disease AD)是一种神经退行性疾病其主要病理的特征有神经细胞内的神经元纤维缠结NFTs、神经细胞外的淀粉样斑块SP和大量神经细胞的凋亡丧失等。NFTs的主要组成成份即是Tau蛋白。目前已知AD病人脑中tau蛋白K44-E45，R230-T231(Park SY,Ferreira A.The generation of a 17kDa neurotoxic fragment:analternative mechanism by which tau mediates beta-amyloid-inducedneurodegeneration.J Neurosci.2005；25:5365–5375),A152-T153,E391-I392,D421-S422(B.Kovacech1 and M.Novak1,Tau Truncation is a Productive PosttranslationalModification of Neurofibrillary Degeneration in Alzheimer’s Disease CurrentAlzheimer Research,2010,7,708-716)等位点可以在不同蛋白酶水解下发生截断。在阿尔茨海默氏症病人的脑脊液中存在Tau蛋白的截短片段。Park等用Aβ处理AD小鼠的原代培养神经元，激活神经元细胞中的Calpain水解Tau产生17kD的Tau蛋白片段(tau45-230)，并诱导原代培养神经元细胞的凋亡。耿俊华选用果蝇作为模式生物，依据Tau蛋白上潜在的Calpain水解位点将Tau蛋白截短成不同的截短片段Tau1-44、Tau45-441、Tau45-230和Tau231-441。(人源Tau蛋白截短片段转基因果蝇模型的建立，生命科学研究，第19卷第2期)。CN03816647.X公开了N端和C端双重截短tau分子，所述分子相对于含四重复的tau43而言截短至少前236个N端氨基酸和至少后45个C端氨基酸。又如Norbert Zilka公开了构建人截短tau蛋白151-391的转基因大鼠，但该转基因大鼠tau病理仅发生在脑干和小脑深部核团，而并未发生在AD病变相关的海马区和大脑皮层。
目前人们对tau蛋白截短体的功能研究很有限，几乎没有使用tau蛋白截短体构建阿尔茨海默病小鼠模型的研究，现有技术在阿尔茨海默病小鼠模型的构建上还一直未有突破。
本发明考察了tau蛋白、tau蛋白截短体tau1-150、tau392-441、tau151-391对野生型FVB和转基因FVB-Fgf14Tg(tetO-MAPT*P301L)4510Kha/JlwsJ小鼠的阿尔茨海默病模型的构建效果。结果显示tau蛋白、tau蛋白截短体1-150、tau392-441均不能在基因型FVB-Fgf14Tg(tetO-MAPT*P301L)4510Kha/JlwsJ雌性小鼠中诱导tau病理；tau151-391不能在FVB野生型小鼠中诱导tau病理。仅有采用基因型FVB-Fgf14Tg(tetO-MAPT*P301L)4510Kha/JlwsJ雌性小鼠，于小鼠4月龄时脑注射tau151-391或者表达人截短tau蛋白的载体，成功快速获得阿尔茨海默病小鼠模型(脑海马区注射病毒AAV9-Syn promoter-tau151-391，使海马区神经元在腺相关病毒AAV9载体介导下，表达人tau蛋白截断体151-391，诱导tau病理产生。该模型于小鼠4月龄时进行脑注射，6月龄可在脑中检测到诱导性tau病理，而应的雄性小鼠，同样注射该病毒，6月龄未能检测到tau病理)。本发明将tau病理模型的构建周期缩短至6个月，节省了50％以上的时间。且采用定点注射，只有海马区出现tau病理，其他脑区未见tau病理。
附图说明
图1.为本发明所述tau病理模型构建流程图。
图2为使用tau151-391蛋白构建阿尔茨海默病雌性小鼠模型与对照组的病理切片。(图2A和2B为生理盐水组，图2C和2D为AAV9-tau151-391病毒组，B图和D图分别为A图和C图的放大图，图中箭头指出处为AT8染色)。
图3为使用tau全长、tau1-150、tau392-441注射15815雌性鼠脑后的阴性病理切片。(图3A为AAV9-tau全长组，图3B为AAV9-tau1-150组，图3C为AAV9-tau392-441组)。
图4为使用tau151-391注射15815雄性和野生型FVB鼠脑后的阴性病理切片。(图4A为15815雄鼠组，图4B为野生型FVB组)。
具体实施方式
实施例1阿尔茨海默病小鼠模型的构建
分别构建整合人tau蛋白截短体tau151-391、tau1-150、tau392-441和tau蛋白的病毒载体。tau151-391蛋白氨基酸序列如SEQ ID NO:1，所示，DNA序列如SEQ ID NO:2所示；tau1-150蛋白氨基酸序列如SEQ ID NO:3，所示，DNA序列如SEQ ID NO:4所示；tau392-441蛋白氨基酸序列如SEQ ID NO:5，所示，DNA序列如SEQ ID NO:6所示；tau蛋白氨基酸序列如SEQ ID NO:7，所示，DNA序列如SEQ ID NO:8所示。
构建hSyn promoter-MCS-EGFP-3FLAG-SV40 PolyA病毒载体，整合入人tau蛋白全长及各截短体的相应DNA序列，包装腺相关病毒AAV9。
根据人tau蛋白截短体tau151-391、tau1-150、tau392-441和tau蛋白DNA序列为模板，设计引物如下：
tau151-391引物序列：
151-391F：5’-ccggaattcactgccacaccgcgggg-3’(SEQ ID NO:9)
151-391R：5’-cgcggatcctcactccgccccgtggtctg-3’(SEQ ID NO:10)
tau1-150引物序列
Tau1-150F：5’-ccggaattcatggctgagccccgcca-3’(SEQ ID NO:11)
Tau1-150R：5’-cgcggatcctcactccgccccgtggtctg-3’(SEQ ID NO:12)
tau392-441引物序列
tau392-441F：5’-ccggaattcatcgtgtacaagtcgccagtg-3’(SEQ ID NO:13)
tau392-441R：5’-cgcggatcctcacaaaccctgcttggc-3’(SEQ ID NO:14)
tau蛋白全长引物序列
Tau-F：同tau1-150F(SEQ ID NO:11)
Tau-R：同tau392-441R(SEQ ID NO:14)
1.载体构建
A.以人cDNA为模板扩增目的片段
PCR体系：
PCR反应条件：
向50μl PCR产物中加入10μl 6×DNA Loading buffer，1％琼脂糖凝胶进行电泳，120V，40min。分别切胶回收tau151-391、tau1-150、tau392-441和tau全长DNA的PCR产物片段，测定DNA浓度。
B.PCR片段与载体酶切连接
PCR产物酶切体系：
载体酶切体系：
37℃水浴4小时，向50μl酶切产物中加入10μl 6×DNA Loading buffer，1％琼脂糖凝胶进行电泳，120V，40min。分别切胶回收tau151-391、tau1-150、tau392-441和tau全长DNA的片段和载体片段，测定DNA浓度。
C.酶切产物连接：
16℃过夜。
D.连接产物转化至大肠杆菌中扩增，提取后测序鉴定，取正确载体进行质粒大量抽提，并调整各质粒浓度为1μg/μl。
E.AAV-293细胞转染
将AAV-293细胞种于T75细胞培养瓶，加入10mL含10％胎牛血清(FBS)的DMEM培养基。细胞生长至80％-90％密度时，进行转染。
转染后72小时收集细胞。
F.病毒收集和提纯
将包装好病毒颗粒的细胞连同上清一起收集到15mL离心管，1000rpm/min离心3分钟，分离细胞和上清，分别收集。细胞用1mL PBS重悬，反复冻融4次裂解细胞。10000rpm离心去除细胞碎片，取上清和细胞培养的上清混合，0.45μm滤器过滤杂质。加入1/2体积1MNaCl，10％PEG8000，混合均与后置于4度过夜。12000rpm离心2小时，去上清，将病毒沉淀以适量PBS重悬，0.22μm滤器过滤除菌。加入终浓度50U/ml Benzonase核酸酶，37℃孵育30分钟消化残留DNA。0.45μm滤器过滤得AAV病毒粗提物。
病毒粗提物中加入CsCl至密度1.41g/ml,175000g离心24小时，收取不同密度的样品，测定滴度后收集富含AAV颗粒的组分。重复该步骤一次。
将收集的AAV病毒颗粒装入100kDa透析袋，4℃透析脱盐过夜，分别获得纯化的病毒AAV9-tau151-391、AAV9-tau1-150、AAV9-tau392-441和AAV9-tau。2.小鼠脑注射AAV9-Syn promoter-tau151-391病毒
FVB-Fgf14Tg(tetO-MAPT*P301L)4510Kha/JlwsJ小鼠购于美国jackson实验室，编号为15815。随机分为以下7组：1.15815雌性小鼠注射生理盐水；2.15815雌性小鼠注射AAV9-tau151-391；3.15815雌性小鼠注射AAV9-tau全长；4.15815雌性小鼠注射AAV9-tau1-150；5.15815雌性小鼠注射AAV9-tau392-441；6.15815雄性小鼠注射AAV9-tau151-391；7.野生型FVB雌性小鼠注射AAV9-tau151-391；每组3只。
4月龄15815雌性小鼠麻醉后，以前囟为原点，向右1.8-2.0mm，后2.4-2.6mm，深1.66-1.70mm区域注射上述病毒3μL，注射速度1μL/min。注射完成后停针2min，让注射液被脑组织充分吸收。将小鼠置于保温垫上至苏醒后，转至正常饲养至6月龄，处死小鼠，进行病理检测。
实施例2鼠脑tau病理检测
将不同组注射病毒的15815小鼠大脑矢状位40μM厚组织切片进行PBS清洗，含0.5％Triton-X 100的PBS进行透膜处理，5％山羊血清封闭液封闭30min，针对磷酸化tau(tau病理)的特异性抗体mouse-AT8 1:1000 4度孵育过夜，PBS清洗3次，Alexa-555荧光标记的抗小鼠二抗室温避光孵育2hr，加入1:5000Hochest孵育15min，PBS清洗三次，贴片封片。荧光显微镜检测tau病理。结果如图2-4所示，结果显示15815雌性小鼠4月龄时进行脑注射AAV9-tau151-391，6月龄时用Ser202/Thr205位点磷酸化tau的特异性抗体AT8染色标记tau病理，低倍镜下可见海马区部分神经元显示明显的AT8染色，而皮层区域没有AT8染色神经元，说明病毒仅在海马区有效诱导tau病理的产生，同时不累及其他脑区(图2C)。放大图片后，40倍镜下可见AT8染色的磷酸化tau在神经元胞体和突触中大量堆积，信号明显增强，与文献报道的tau病理吻合，进一步说明该模型成功诱导tau病理的产生(图2D)。而对照组小鼠4月龄时脑注射生理盐水，6月龄时海马区和皮层AT8染色呈阴性，均未检测到tau病理(图2A、B)，说明15815雌性小鼠脑注射AAV9-tau151-391后产生的tau病理确由病毒注射引起，而非年龄增长而出现的自然现象。
15815雌性小鼠4月龄时脑注射AAV9-tau全长(图3A)，AAV9-tau1-150(图3B)或AAV9-tau392-441(图3C)，6月龄时海马区AT8抗体染色呈阴性，未检测到tau病理，说明只有tau151-391这一截短体才能在注射后2月内特异性诱发tau病理，而tau全长、tau1-150或tau392-441均无法达到这种效果。
15815雄性小鼠注射AAV9-tau151-391(图4A)，野生型FVB雌性小鼠注射AAV9-tau151-391(图4B)，6月龄时海马区AT8抗体染色呈阴性，均未能检测到tau病理，说明15815雌性小鼠对AAV9-tau151-391尤其敏感，是构建该tau病理模型的适宜小鼠品系。
实施例3.本发明所述小鼠tau病理模型的应用举例
(1)筛选可抑制tau病理的基因。
构建靶基因腺相关病毒载体，注射入4月龄15815小鼠海马区，使靶基因高表达。2周后，同一位置注射AAV9-tau151-391病毒，诱导tau病理产生。6月龄时，取鼠脑进行免疫荧光，比较注射靶基因病毒和对照病毒的鼠脑tau病理强度(tau病理特异性抗体mouse-AT8阳性细胞的数目)，判断靶基因是否影响tau病理产生，从而筛选出可有效抑制tau病理的基因。
(2)筛选可抑制tau病理的药物
4月龄15815小鼠海马区注射AAV9-tau151-391病毒，诱导tau病理产生。注射后第二天，通过腹腔注射或灌胃给小鼠施加相应浓度的药物，持续1个月。6月龄时，取鼠脑进行免疫荧光，比较给药组和对照组之间tau病理强度(tau病理特异性抗体mouse-AT8阳性细胞的数目)，判断药物是否影响tau病理传播，从而筛选出可有效抑制tau病理的药物。
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Ser Ala Ser Leu Ala Lys Gln Gly Leu
435 440
<210> 8
<211> 1326
<212> DNA
<213> 人(human)
<400> 8
atggctgagc cccgccagga gttcgaagtg atggaagatc acgctgggac gtacgggttg 60
ggggacagga aagatcaggg gggctacacc atgcaccaag accaagaggg tgacacggac 120
gctggcctga aagaatctcc cctgcagacc cccactgagg acggatctga ggaaccgggc 180
tctgaaacct ctgatgctaa gagcactcca acagcggaag atgtgacagc acccttagtg 240
gatgagggag ctcccggcaa gcaggctgcc gcgcagcccc acacggagat cccagaagga 300
accacagctg aagaagcagg cattggagac acccccagcc tggaagacga agctgctggt 360
cacgtgaccc aagctcgcat ggtcagtaaa agcaaagacg ggactggaag cgatgacaaa 420
aaagccaagg gggctgatgg taaaacgaag atcgccacac cgcggggagc agcccctcca 480
ggccagaagg gccaggccaa cgccaccagg attccagcaa aaaccccgcc cgctccaaag 540
acaccaccca gctctggtga acctccaaaa tcaggggatc gcagcggcta cagcagcccc 600
ggctccccag gcactcccgg cagccgctcc cgcaccccgt cccttccaac cccacccacc 660
cgggagccca agaaggtggc agtggtccgt actccaccca agtcgccgtc ttccgccaag 720
agccgcctgc agacagcccc cgtgcccatg ccagacctga agaatgtcaa gtccaagatc 780
ggctccactg agaacctgaa gcaccagccg ggaggcggga aggtgcagat aattaataag 840
aagctggatc ttagcaacgt ccagtccaag tgtggctcaa aggataatat caaacacgtc 900
ccgggaggcg gcagtgtgca aatagtctac aaaccagttg acctgagcaa ggtgacctcc 960
aagtgtggct cattaggcaa catccatcat aaaccaggag gtggccaggt ggaagtaaaa 1020
tctgagaagc ttgacttcaa ggacagagtc cagtcgaaga ttgggtccct ggacaatatc 1080
acccacgtcc ctggcggagg aaataaaaag attgaaaccc acaagctgac cttccgcgag 1140
aacgccaaag ccaagacaga ccacggggcg gagatcgtgt acaagtcgcc agtggtgtct 1200
ggggacacgt ctccacggca tctcagcaat gtctcctcca ccggcagcat cgacatggta 1260
gactcgcccc agctcgccac gctagctgac gaggtgtctg cctccctggc caagcagggt 1320
ttgtga 1326
<210> 9
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccggaattca ctgccacacc gcgggg 26
<210> 10
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cgcggatcct cactccgccc cgtggtctg 29
<210> 11
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ccggaattca tggctgagcc ccgcca 26
<210> 12
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cgcggatcct cactccgccc cgtggtctg 29
<210> 13
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ccggaattca tcgtgtacaa gtcgccagtg 30
<210> 14
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
cgcggatcct cacaaaccct gcttggc 27
Claims (5)
1.一种阿尔茨海默病小鼠模型的构建方法，其特征在于采用基因型 FVB-Fgf14T g(tetO-MAPT*P301L)4510Kha/JlwsJ 雌性小鼠，于小鼠4月龄时脑注射截短tau 蛋白或者表达截短tau蛋白的载体后饲养 2-4个月，所述截短tau蛋白氨基酸序 列如 SEQ ID NO:1 所示，脑注射位置为：以前囟为原点，向右1.8-2.0mm，后2.4-2.6mm，深1.66-1.70mm区域。
2.根据权利要求 1 所述的方法，其特征在于所述小鼠4月龄时脑注射截短 tau蛋白或者表达截短 tau 蛋白的载体后正常饮食饲养2个月。
3.根据权利要求 1 所述的方法，其特征在于所述表达截短tau蛋白的载体为腺病毒。
4.根据权利要求 1 所述的方法，其特征在于所述表达截短tau蛋白的载体为腺病毒AAV9 载体。
5.氨基酸序列如 SEQ ID NO:1 所示的截短 tau 蛋白在快速构建阿尔茨海默病小鼠模型中的应用，其特征在于采用基因型 FVB-Fgf14Tg(tetO-MAPT*P301L)4510 Kha/JlwsJ雌性小鼠造模。
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| CN113430230A (zh) * | 2021-06-24 | 2021-09-24 | 南通大学 | tau截断体蛋白在诱导tau病理聚集中的应用 |
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| CN115125244A (zh) * | 2022-05-14 | 2022-09-30 | 吉林大学 | 构建阿尔兹海默症的兔模型方法 |
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