CN116077680B - 一种神经元铁沉积的靶向治疗制剂及应用 - Google Patents
一种神经元铁沉积的靶向治疗制剂及应用 Download PDFInfo
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Abstract
本发明公开了一种神经元铁沉积的靶向治疗制剂,制剂以生物磁编码基因mms6为基础,新型血脑屏障型AAV为载体,以神经元特异性转录因子syn为靶向转录条件的复合体生物制剂。本发明采用上述的一种神经元铁沉积的靶向治疗制剂及应用,使得Fe2+铁超载得到改善,铁死亡相关酶NOX2表达显著降低,线粒体损伤减轻,认知功能得到改善。
Description
技术领域
本发明涉及阿尔兹海默病治疗药物技术领域,尤其是涉及一种神经元铁沉积的靶向治疗制剂及应用。
背景技术
阿尔兹海默病(Alzheimer's disease,AD)是一种以记忆力缺失、认知功能障碍为主的疾病,是中老年人群最常见的神经退行性疾病。传统的药物是针对于Aβ淀粉样蛋白沉积、Tau蛋白异常磷酸化进行研发,均未获得重大突破,故仍需要探索新的治疗方向。
近年,研究发现铁超载是AD发病的重要病因,铁的转运、转出、转化等代谢过程障碍均会导致细胞内游离铁蓄积,即铁超载。中枢神经有成熟的铁转运机制,铁转运途径主要分为两种:转铁蛋白途径(经典途径,包括转铁蛋白、二价铁转运蛋白、血色素蛋白等)和非转铁蛋白途径如APP蛋白、Tau蛋白、朊病毒相关蛋白(PrionProtein,PRNP)等。其中PRNP具有铁还原酶活性,将Fe3+转变为Fe2+,促进细胞对铁摄取,PRNP表达增加会促进铁超载和神经纤维缠结的发生,加速AD病理发生。
其中,铁是细胞内大多数化学自由基产生的来源,过量的游离铁超载(主要是Fe2+)可触发铁依赖性的细胞内脂质体活性氧大量释放导致铁死亡。研究发现与AD密切相关的脑区-海马CA1/3区神经元对铁超载更敏感,接受高铁饮食的小鼠的海马组织铁浓度是皮质、小脑等部位组织铁浓度的1.5倍,更容易发生铁沉积。进一步证实,铁超载是导致海马神经元死亡、海马萎缩的重要原因,其铁含量与认知功能密切相关,这些证据均表明铁超载在AD致病环节的关键性作用。
并且,与AD发病机制关系最密切的APP蛋白、Tau蛋白等均参与正常铁稳态调控,铁沉积与Aβ蛋白沉积、Tau蛋白异常磷酸化、神经纤维缠结等病理改变密切相关。游离铁可以促进Aβ转运到细胞内,而且可以加速Aβ沉积,采用患者脑脊液模拟发生Aβ病理性改变所需要的时间,发现脑脊液中铁蛋白浓度越高,沉积速度越快。
另外,铁沉积是独立于Tau蛋白异常磷酸化、Aβ蛋白沉积的神经退行性病变的另一重要特征,其在一定程度上反映了疾病的发展阶段。研究淀粉样变、铁沉积程度对认知功能下降速度的影响,发现淀粉样变程度对认知功能下降的速度影响不显著,而铁沉积程度对痴呆患者认知功能下降速度影响较大,铁沉积程度越高,认知功能速度下降越快。
目前,用于治疗铁超载的药物有限,主要为去铁类螯合剂如去铁酮(Deferiprone,DFP)等,螯合剂不具有靶向性,可导致多种严重并发症,使铁超载治疗难以进入临床转化。并且,铁是所有细胞元素必需的微量元素,与血红蛋白、线粒体代谢等功能密切相关,系统性地使用铁螯合剂治疗可能会干扰生理状态下的铁代谢。临床研究发现,长期使用铁螯合剂会导致严重贫血、中性粒细胞减少败血症、神经功能退化等严重并发症。严重的副作用将大大限制去铁类螯合剂临床转化,而靶向治疗可能是解决这一问题的关键。
基因治疗是靶向治疗的重要手段,而与铁转运相关蛋白均为大分子功能蛋白,过表达会存在致病风险,能够编码具有螯合亚铁离子的小分子蛋白的基因是比较理想的候选基因。
发明内容
本发明的目的是提供一种神经元铁沉积的靶向治疗制剂及应用,采用mms6作为靶向治疗的关键基因,从其有效性进行研究,为mms6在铁超载靶向治疗的应用提供理论依据。
为实现上述目的,本发明提供了一种神经元铁沉积的靶向治疗制剂,制剂以生物磁编码基因mms6为基础,新型血脑屏障型AAV为载体,以神经元特异性转录因子syn为靶向转录条件的复合体生物制剂。
一种神经元铁沉积的靶向治疗制剂在制备治疗铁沉积特征的神经系统疾病药物中的应用。
优选的,疾病包括但不限于阿尔茨海默病、帕金森病、神经元铁沉积疾病。
因此,本发明采用上述一种神经元铁沉积的靶向治疗制剂及应用,由于铁超载在AD脑内存在时空差异性,采用生物磁编码基因mms6-BBB-AAV对关键亚群进行精准治疗,为阿尔兹海默病铁超载治疗提供了新的思路和方向。
具体来说,通过新型血脑屏障型AAV病毒(BBB-AAV)载体携带生物磁编码基因mms6和神经元特异性转录因子syn将mms6基因转染到AD小鼠脑内神经元,如图1,BBB-AAV病毒作为载体,通过尾静脉注射入小鼠体内1个月后进行切片染色,发现全脑神经元高效表达目的基因。简而言之,通过mms6基因嫁接技术方法改善神经元铁超载的技术方法,改善脑内神经元铁超载,进而改善AD小鼠海马Fe2+铁超载、线粒体损伤,进而改善海马神经元铁死亡,促进认知功能恢复。
下面通过附图和实施例,对本发明的技术方案做进一步的详细描述。
附图说明
图1是BBB-AAV靶向治疗流程图;
图2是pHBAAV-hsyn-MCS-T2A-ZsGreen载体图谱;
图3是支原体检测PCR胶图;
图4是滴度检测标准曲线图;
图5是mms6-syn-GFP-BBB-AAV靶向治疗改善AD小鼠认知功能示意图;
图6是mms6-BBB-AAV靶向治疗改善AD铁超载示意图。
具体实施方式
以下通过附图和实施例对本发明的技术方案作进一步说明。
除非另外定义,本发明使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的主旨或基本特征的情况下,能够以其它的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内,不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其它实施方式。这些其它实施方式也涵盖在本发明的保护范围内。
还应当理解,以上所述的具体实施例仅用于解释本发明,本发明的保护范围并不限于此,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,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明/发明的保护范围之内。
本公开使用的所有术语(包括技术术语或者科学术语)与本公开所属领域的普通技术人员理解的含义相同,除非另外特别定义。还应当理解,在诸如通用词典中定义的术语应当被理解为具有与它们在相关技术的上下文中的含义相一致的含义,而不应用理想化或极度形式化的意义来解释,除非本文有明确地这样定义。
对于相关领域普通技术人员已知的技术、方法和设备可能不作为详细讨论,但在适当情况下,所述技术、方法和设备应当被视为说明书的一部分。
本发明说明书中引用的现有技术文献所公开的内容整体均通过引用并入本发明中,并且因此是本发明公开内容的一部分。
实施例一
制备过表达腺相关病毒载体
(1)材料信息
如图2所示,载体为pHBAAV-hsyn-MCS-T2A-ZsGreen。
目的基因序列为(Seq ID NO:1):
ATGCCTGCTCAGATCGCCAACGGCGTGATATGTCCTCCTGGTGCTCCTGCCGGAACAAAAGCCGCTGCTGCCATGGGCGAGATGGAAAGAGAAGGCGCCGCTGCTAAAGCCGGCGCTGCAAAAACAGGCGCTGCCAAGACTGGAACCGTGGCCAAAACAGGCATTGCCGCCAAAACTGGCGTGGCCACAGCTGTTGCTGCTCCAGCTGCTCCTGCTAATGTGGCCGCTGCTCAAGGCGCTGGCACAAAAGTTGCTCTCGGAGCCGGAAAAGCTGCCGCTGGCGCTAAAGTTGTCGGCGGCACAATCTGGACAGGCAAAGGACTCGGACTCGGCCTTGGTCTTGGACTCGGAGCTTGGGGACCTATCATCCTGGGAGTTGTTGGAGCTGGCGCCGTGTACGCCTACATGAAGTCCAGAGATATCGAGAGCGCCCAGAGCGACGAGGAAGTGGAACTTAGGGACGCTCTGGCTTAA。
生物磁编码基因(Magnetite biomineralizationprotein gene 6,mms6)是具有趋磁的细菌,拥有抗氧化和生物利用游离铁合成磁小体(Fe3O4或者Fe3S4晶体)的能力,环境中的高活性氧可以触发磁小体的合成。而mms6是其中关键的磁小体矿化基因,它可以编码长度约62个氨基酸序列的小分子蛋白,羧基端对Fe2+有较强的螯合能力,而且它与趋磁细菌清除细胞内过量的活性氧密切相关。
(2)操作步骤如下:
S1、根据如表1中顺序依次加入每种试剂构建载体酶切体系,轻轻吸打混匀,置于37℃水浴锅中反应1-2h。酶切结束之后进行琼脂糖凝胶电泳,回收目的片段。
表1载体酶切体系
试剂 | 体积(μL) |
载体DNA(1ug/uL) | 1 |
10*buffer | 4 |
DdH2O | 32 |
BamHI | 1.5 |
BamHI | 1.5 |
total | 40 |
S2、获取目的片段
a、设计引物
AAV-mms6-Bam/Bam-F:agcgcagtcgagaaggtaccggatccgccaccatgcctgctcagat(SeqID NO:2);
AAV-mms6-Bam/Bam-R:ccctcactagtgctagcggatcctttgtcgtcatcatccttatag(SeqID NO:3)。
b、PCR扩增目的片段
配制如表2所示体系,轻轻混匀,置于PCR仪中进行反应;
表2目的片段PCR扩增体系
PCR程序:
S3、目的片段与载体连接
于冰水浴中配制HB infusionTM一步克隆连接体系,如果不慎将液体粘在管壁,可通过短暂离心使其沉入管底。连接反应液在50℃反应30min后,置于冰上5min,立即转化。
表3连接反应体系
组分名称 | 体积(μL) |
目的基因片段 | X(≥100ng) |
线性化载体 | Y(≥50ng) |
2×HB infusionTM Master mix | 10 |
ddH2O | Z(=10-X-Y) |
总体积 | 20 |
S4、转化
DH5α感受态细胞从-80℃冰箱拿出来之后,要立刻放到冰上融化,感受态分装过程操作轻柔,减轻对其机械破坏。
待感受态融化后,以每管50μL的体积分装(对于质粒转化,20μL就足够了),分装之后以不超过感受态体积1/10的量加入连接产物(目前加5μL连接产物),冰上放置20-30min;
42℃热激90s(这个时间要非常严格),热激完之后立刻插入冰上冰育2-3min;
在超净台中,加入500μL LB培养基(注意一定是无抗的LB培养基),轻柔的上下颠倒3-5次;37℃、230rpm震荡培养45-60min;
将菌液涂到相应抗性的固体平板上,涂布均匀,然后将板子倒放37℃恒温箱培养12-16h。
S5、菌液PCR鉴定
表4菌液PCR鉴定体系
组分名称 | 体积(μL) |
2xHieff PCR Master MIX(Dye) | 5 |
引物1 | 0.5 |
引物2 | 0.5 |
菌液 | 2 |
ddH2O | 2 |
总体积 | 10 |
表5菌液PCR鉴定程序
S6、测序
将筛选出来的阳性克隆,选择两个克隆进行测序。测序结果(Seq ID NO:4)如下:
CGGGCACGCGCTGTCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGTCGAGAAGGTACCGGATCCGCCACCATGCCTGCTCAGATCGCCAACGGCGTGATATGTCCTCCTGGTGCTCCTGCCGGAACAAAAGCCGCTGCTGCCATGGGCGAGATGGAAAGAGAAGGCGCCGCTGCTAAAGCCGGCGCTGCAAAAACAGGCGCTGCCAAGACTGGAACCGTGGCCAAAACAGGCATTGCCGCCAAAACTGGCGTGGCCACAGCTGTTGCTGCTCCAGCTGCTCCTGCTAATGTGGCCGCTGCTCAAGGCGCTGGCACAAAAGTTGCTCTCGGAGCCGGAAAAGCTGCCGCTGGCGCTAAAGTTGTCGGCGGCACAATCTGGACAGGCAAAGGACTCGGACTCGGCCTTGGTCTTGGACTCGGAGCTTGGGGACCTATCATCCTGGGAGTTGTTGGAGCTGGCGCCGTGTACGCCTACATGAAGTCCAGAGATATCGAGAGCGCCCAGAGCGACGAGGAAGTGGAACTTAGGGACGCTCTGGCTGGATCCTCGGTACCAAGCTTAAGTGACTACAAGGATGACGATGACAAGGATTACAAAGACGACGATGATAAGGACTATAAGGATGATGACGACAAAGGATCCGCTAGCACTAGTGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTACCGGTGCCACCATGGCCCAGTCCAAGCACGGCCTGACCAAGGAGATGACCATGAAGTACCGCATGGAGGGCTGCGTGGACGGCCACAAGTTCGTGATCACCGGCGAGGGCATCGGCTACCCCTTCAAGGGCAAGCAGGCCATCAACCTGTGCGTGGGTGGGGGGGCGGCCTCTTGCCTCTTTCGCCGAGGACATCTTGTCCGCCGTCTTCATGTACGGGCAACCGCGTGTTCACCGAGTACCCCCAGGGACATCGTCGACTACTTCAAGAA
测序结果与目的序列一致,该目的质粒构建成功。
S7、质粒抽提
测序成功之后,根据项目要求安排菌液扩增,进行质粒抽提纯化,质粒抽提的方案按照抽提试剂盒的说明书为准。抽提的质粒需要QC验证合格之后用于转染细胞。
质粒QC的原则是浓度大于200ng/uL,260-280在1.8-2.0之间。
实施例二
腺相关病毒包装与质量检测
腺相关病毒(adeno-associated virus,AAV),是微小病毒科依赖病毒属,病毒颗粒大小约为20~26nm,完整的生活周期需要辅助病毒参与。腺相关病毒编码两个末端的反向重复序列(ITR)中的cap和rep基因,其中cap基因编码病毒衣壳蛋白,rep基因参与病毒的复制和整合。AAV有多种血清型,不同血清型对不同组织的亲和力不同,适用于各种体内感染实验。其中,新型血脑屏障型AAV病毒(BBB-AAV)为突变株,其特殊性在于静脉注射后可以高效透过血脑屏障,使脑内神经细胞表达目的基因。
AAV包装系统内有三种质粒,包括可以插入外源基因的穿梭质粒、编码rep和cap蛋白编码基因的pAAV-RC、替代腺相关病毒所依赖的腺病毒的pHelper质粒。三种质粒共转染工具细胞AAV-293后,可以形成携带有外源插入基因的AAV病毒颗粒。
一、腺相关病毒包装
(1)将AAV-293细胞传代到100mm平皿中用于转染。操作完毕后置于37℃、5%CO2、95%相对湿度的培养箱中。
(2)转染
细胞观察:确认细胞密度达到约80~90%的汇合率即可进行转染。
脂质体转染:Opti MEM需在37℃水浴中预热,LipofiterTM转染试剂需恢复至室温方可使用,使用前需摇匀。
表6转染100mm平皿所需的转染复合物成分表
成分名称 | 含量 |
pAAV-RC质粒 | 10μg |
pHelper质粒 | 20μg |
穿梭质粒 | 10μg |
LipofiterTM | 120 |
(3)换液:转染后6h更换含10%胎牛血清FBS的新鲜完全培养基。
(4)细胞收集:转染后72h,将含AAV颗粒的细胞用细胞刮轻轻刮下,收集于15mL离心管中,150×g离心3min收集细胞,去除培养上清,用PBS洗一次,最后再用300μLPBS重悬细胞。
(5)细胞破碎:准备37℃恒温水浴锅和液氮,将装有细胞的离心管在液氮及37℃水浴反复冻融三次。4℃,2000×g,5min,去除细胞碎片,收集含AAV颗粒的裂解上清。
二、腺相关病毒纯化
全能核酸酶处理:每1mL病毒粗提物中加入0.1μL Benonase酶,37℃水浴1h,除去病毒液中的细胞基因组及残留的质粒DNA。600×g,4℃,离心10min,取上清。
柱纯化(根据Biomiga腺相关病毒纯化试剂盒V1469-01纯化):
将柱纯化得到的4mLAAV病毒样品液体,加入到超滤管中,1400×g离心30min,得到约1mLAAV。收集最终得到的纯化后的病毒,于-80℃保存。
三、腺相关病毒质量检测
(1)无菌检测
检测方法:取10uL病毒加入96孔板的Hela细胞进行验证,培养24h后显微镜镜检:
QC标准:培养基需澄清透明,细胞间隙无明显颗粒,无任何细菌及真菌污染。
(2)支原体检测
检测方法:取10uL病毒,96℃水浴15min后于超净台中配置PCR反应体系。PCR反应后电泳确定是否含有支原体污染。
QC标准:PCR胶图无明显条带,图3中,若500bp左右位置有条带,跑道1、2、3、6表示样品有支原体污染,跑道4、5没有条带表示没有支原体污染。
(3)滴度检测
使用DNase I及蛋白酶K消化AAV病毒样品。
表7DNase I酶反应体系表
37℃,水浴1h左右;100℃,10min处理;加入2μL蛋白酶K,55℃,水浴1h;100℃,10min处理,离心。
稀释标准品质粒,设置标准品的拷贝梯度为105、106、107、108、109、1010。配置QPCR反应体系,每个样品和标准品设计3个复孔。
表8QPCR体系表
试剂名称 | 体积 |
2×SYBR Green mix | 4.8μL |
Forward primer | 0.4μL |
Reverse primer | 0.4μL |
AAV基因组 | 1μL |
水 | 10μL |
表9QPCR反应程序表
三、数据分析(以HBAAV2/9-Hspa9 shRNA2-GFP滴度测度为例)
(1)Roche LC96实时荧光定量PCR仪所测Ct值数据如下:
如图4,以每组AAV标准品的Ct均值为纵坐标Y,其对应的拷贝数的对数为横坐标X,做标准曲线,得出标准曲线的函数公式及R平方值。
(2)待测样品滴度计算。将待测AAV样品Ct均值,代入公式,计算所加入AAV模板拷贝数X,再换算成滴度。
换算公式为:AAV病毒滴度=10x×40000(稀释倍数)vg/mL
HBAAV2/9-Hspa9 shRNA2-GFP滴度=1.2×1012vg/mL
对照病毒HBAAV2/9-GFP滴度=1.6×1012vg/mL
滴度结果为:
实施例三
铁超载靶向治疗的有效性测试
一、实验方法
AD模型小鼠铁超载治疗:将12月龄APP/PS1小鼠按照假治疗和生物磁靶向治疗随机双盲分配至分为mms6组和假治疗组并编号,两组在治疗前均进行行为学评估(水迷宫实验、筑巢实验);mms6组尾静脉注射100μlmms6-GFP-BBB-AAV(10*14VG/mL),每日腹腔注射生理盐水;假治疗组每日腹腔注射生理盐水,并尾静脉注射空载对照病毒100μlGFP-BBB-AAV(10*14VG/mL)。分别在1个月后、3个月后分别评估两组的认知功能、铁超载改善水平及病理改变。
行为学:研究采用水迷宫和筑巢实验评估两组治疗AD小鼠认知功能。
图5中,AD-G:AD小鼠对照组,使用syn-GFP-BBB-AAV尾静脉注射治疗;AD-M:AD小鼠试验组,mms6-syn-GFP-BBB-AAV尾静脉注射治疗。***表示P值<0.001。A为水迷宫测试流程;B:平台可见期mms6组与对照组无明显差异。C:平台隐藏期,mms6组学习能力较对照好,寻找平台的距离明显降低;D:无平台期,mms6组小鼠寻找平台区域潜伏期较对照组显著减少;E.筑巢行为学评分示意图;F:筑巢试验评分mms6组优于对照组。
铁超载水平变化研究:分别取两组小鼠海马、前额叶皮质、纹状体层面石蜡切片进行荧光染色、免疫组化染色、Aβ1-42用于对比两组经过不同治疗后的变化及病理损伤变化;并在人AD细胞模型(SH-SY5Y诱导的神经元孵育5μMAβ)中进一步实验。
图6中,*表示P值<0.05;***<0.001。ADM:AD动物模型或细胞模型mms6治疗组;ADG:AD动物模型或细胞模型治疗对照组。A.Fe(II)/Syn/DAPI荧光染色;B.mms6组海马Fe2+荧光强度较对照组显著降低;C.NOX2的免疫组化染色;D.mms6组NOX2的曲线下面积(AU)较对照组显著下降;E.采用流式细胞FL2通道检测Fe2+探针荧光强度,结果mms6显著降低铁超载条件下SH-SY5Y细胞内Fe2+水平;F.AD小鼠海马内大量的Aβ淀粉样斑块,斑块内还有大量的铁颗粒沉积;线粒体出现固缩;经mms6靶向治疗后,淀粉样斑块减少,铁颗粒反而蓄积在溶酶体内,此外,线粒体损伤较小,未见显著的固缩和膜结构破坏。
综上所述,使用BBB-AAV作为载体,Syn为特异性转录因子,mms6-syn-BBB靶向治疗工具治疗24月龄APP/PS1小鼠,水迷宫和筑巢实验结果表明mms6治疗组较对照组认知功能显著改善。
采用mms6-syn-AAV-BBB靶向治疗24月龄AD小鼠的海马Fe2+铁超载得到改善,铁死亡相关酶NOX2表达显著降低,线粒体损伤减轻,由此可见,mms6靶向治疗使海马神经元铁死亡改善。Mms6改善铁死亡功能进一步在人AD细胞模型(SH-SY5Y诱导的神经元孵育5μMAβ)中验证,表达组与非表达组同时给予铁超载诱导条件下,发现表达mms6显著降低细胞内Fe2+水平。
因此,本发明采用上述一种神经元铁沉积的靶向治疗制剂及应用,避免了传统上使用小分子铁螯合剂治疗铁超载相关疾病,但不具有靶向性,容易引起严重的并发症的问题,将铁超载靶向治疗为特色,利用时空转录组、iPSCs等新技术筛选AD小鼠脑内铁超载易感的细胞亚群,首次采用生物磁编码基因mms6-BBB-AAV对关键亚群进行精准治疗,指导Fe2+生物转化的功能嫁接到AD铁超载的治疗中,为阿尔兹海默病铁超载治疗提供了一个新的思路和方向。
最后应说明的是:以上实施例仅用以说明本发明的技术方案而非对其进行限制,尽管参照较佳实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对本发明的技术方案进行修改或者等同替换,而这些修改或者等同替换亦不能使修改后的技术方案脱离本发明技术方案的精神和范围。
Claims (1)
1.一种神经元铁超载的靶向治疗制剂在制备治疗铁超载特征的神经系统疾病药物中的应用,其特征在于:所述制剂是以新型血脑屏障型AAV为载体,携带生物磁编码基因mms6,携带神经元特异性启动子hsyn的复合体生物制剂;所述生物磁编码基因mms6的核苷酸序列如Seq ID NO:1所示;所述铁超载特征的神经系统疾病为阿尔茨海默病。
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