药学学报

Year	Animal	Injection site	Dose (pmol/ mouse or rat)	Duration of modeling and behavioral test
2008^[29]	C57BL/6 mice (12 w)	Lateral ventricle	500	-	-	4 day	-	7-14 day	-	-
2010^[12]	C57BL/6 mice (7-8 w)	Lateral ventricle	7.5 (two injections)	1 day (recovered after 10 days)	-	-	-	-	-	-
2010^[30]	C57BL/6 mice (20 w)	Lateral ventricle	222	-	-	-	1 day	-	-	-
2012^[31]	Swiss mice (12 w)	Lateral ventricle	10	1 day	-	-		-	1, 8 day	1 day
2013^[32]	C57BL/6J mice (12 w)	Lateral ventricle	50	-	-	4 day	-	-	-	-
2013^[33]	Swiss mice (12 w)	Lateral ventricle	10, 50	1, 7, 14 day	-	-	-	-	-	-
2016^[34]	ICR mice (7 w)	Lateral ventricle	500	-	-	3 day	-	-	-	-
2017^[35]	Swiss mice (8-12 w)	Lateral ventricle	10	1 day (novel object) 8 day (object location)	-	-	-	-	-	-
2011^[36]	ICR mice (8 w)	Granule cell layer of hippocampus	30	6 day	8 day	-	-	-	-	-
2011^[37]	ICR mice (27-30 g)	Hilus of hippocampus	30	-	8 day	6 day	-	-	-	-
2015^[38]	C57BL/6 mice (12 w)	Hippocampus	50	-	-	6 day (recovered after 30 days)	-	-	-	-
2012^[39]	Wistar rats (12-48 w, 200-250 g)	Lateral ventricle	600/day (30-days infusion)	-	-	-	-	32-36 day	-	-
2012^[40]	SD rats (200-300 g)	Lateral ventricle	144/day (30-days infusion)	-	-	-	-	31-35 day	-	-
2014^[41]	SD rats (220-280 g)	Lateral ventricle	5000	-	-	-	-	2-10 day	-	-
2015^[42]	SD rats (250 g)	Lateral ventricle	10.8	2 h	-	-	-	-	-	-
2015^[43]	Wistar rats (280-300 g)	Lateral ventricle	5000	-	-	-	-	10-16 day	-	-
2016^[44]	Wistar rats (280-300 g)	Lateral ventricle	5000	-	-	-	-	10-16 day	-	-
2016^[45]	Wistar rats (24 w)	Lateral ventricle	5100	-	-	-	-	8-12, 19 day	-	-
2016^[46]	SD rats (250-350 g)	Lateral ventricle	400/2 day (16-days infusion)	-	-	-	-	8-16 day	-	-
2017^[47]	Charles River- Harlan rats (250-300 g)	Lateral ventricle	375, 1 125 and 3 000	-	-	-	-	8-12 day	-	-
2013^[48]	Wistar rats (24 w, 220-250 g)	CA1 molecular layer of hippocampus	149.53	-	-	-	-	8-11 day	-	-

Year	Animal	Injection site	Dose (pmol/ mouse or rat)	Duration of modeling and behavioral test
2008^[29]	C57BL/6 mice (12 w)	Lateral ventricle	500	-	-	4 day	-	7-14 day	-	-
2010^[12]	C57BL/6 mice (7-8 w)	Lateral ventricle	7.5 (two injections)	1 day (recovered after 10 days)	-	-	-	-	-	-
2010^[30]	C57BL/6 mice (20 w)	Lateral ventricle	222	-	-	-	1 day	-	-	-
2012^[31]	Swiss mice (12 w)	Lateral ventricle	10	1 day	-	-		-	1, 8 day	1 day
2013^[32]	C57BL/6J mice (12 w)	Lateral ventricle	50	-	-	4 day	-	-	-	-
2013^[33]	Swiss mice (12 w)	Lateral ventricle	10, 50	1, 7, 14 day	-	-	-	-	-	-
2016^[34]	ICR mice (7 w)	Lateral ventricle	500	-	-	3 day	-	-	-	-
2017^[35]	Swiss mice (8-12 w)	Lateral ventricle	10	1 day (novel object) 8 day (object location)	-	-	-	-	-	-
2011^[36]	ICR mice (8 w)	Granule cell layer of hippocampus	30	6 day	8 day	-	-	-	-	-
2011^[37]	ICR mice (27-30 g)	Hilus of hippocampus	30	-	8 day	6 day	-	-	-	-
2015^[38]	C57BL/6 mice (12 w)	Hippocampus	50	-	-	6 day (recovered after 30 days)	-	-	-	-
2012^[39]	Wistar rats (12-48 w, 200-250 g)	Lateral ventricle	600/day (30-days infusion)	-	-	-	-	32-36 day	-	-
2012^[40]	SD rats (200-300 g)	Lateral ventricle	144/day (30-days infusion)	-	-	-	-	31-35 day	-	-
2014^[41]	SD rats (220-280 g)	Lateral ventricle	5000	-	-	-	-	2-10 day	-	-
2015^[42]	SD rats (250 g)	Lateral ventricle	10.8	2 h	-	-	-	-	-	-
2015^[43]	Wistar rats (280-300 g)	Lateral ventricle	5000	-	-	-	-	10-16 day	-	-
2016^[44]	Wistar rats (280-300 g)	Lateral ventricle	5000	-	-	-	-	10-16 day	-	-
2016^[45]	Wistar rats (24 w)	Lateral ventricle	5100	-	-	-	-	8-12, 19 day	-	-
2016^[46]	SD rats (250-350 g)	Lateral ventricle	400/2 day (16-days infusion)	-	-	-	-	8-16 day	-	-
2017^[47]	Charles River- Harlan rats (250-300 g)	Lateral ventricle	375, 1 125 and 3 000	-	-	-	-	8-12 day	-	-
2013^[48]	Wistar rats (24 w, 220-250 g)	CA1 molecular layer of hippocampus	149.53	-	-	-	-	8-11 day	-	-

β-淀粉样蛋白寡聚体脑内注射动物模型在阿尔茨海默病研究中的应用

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王丽莎 , 刘新民 , 陶雪 , 张梦荻 , 周云丰 , 常琪 ^*

药学学报 | 综述 2018,53(7): 1060-1067

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药学学报 | 综述 2018, 53(7): 1060-1067

β-淀粉样蛋白寡聚体脑内注射动物模型在阿尔茨海默病研究中的应用

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王丽莎, 刘新民, 陶雪, 张梦荻, 周云丰, 常琪^*

作者信息

中国医学科学院、北京协和医学院药用植物研究所, 北京 100193

通讯作者:

* 常琪, Tel:86-10-57833468, E-mail:qchang@implad.ac.cn

Application of the animal model of intracerebral injection of amyloid-β oligomers to the study of Alzheimer's disease

Li-sha WANG, Xin-min LIU, Xue TAO, Meng-di ZHANG, Yun-feng ZHOU, Qi CHANG^*

Affiliations

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China

出版时间: 2018-07-12 doi: 10.16438/j.0513-4870.2018-0088

文章导航

摘要

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脑内β-淀粉样蛋白(amyloid-β peptide, Aβ)的异常沉积在阿尔茨海默病(Alzheimer's disease, AD)的发病进程中起重要作用。Aβ寡聚体脑内注射动物模型不仅为深入探索Aβ在AD中的作用机制提供了方法, 而且也可用来筛选靶向于Aβ寡聚体的候选药物。迄今为止, 已有大量文献报道利用该动物模型进行抗AD药物的活性评价和作用机制研究。本文拟对脑内注射可溶性Aβ寡聚体的研究进展进行总结, 主要包括实验动物、Aβ的种类及其寡聚体的体外制备方法、注射部位和剂量、造模时长和行为学变化, 以及该动物模型相关的病理学机制等, 为该模型的合理应用提供参考。

关键词

阿尔茨海默病 / β-淀粉样蛋白 / 寡聚体 / 脑内注射 / 动物模型

Abstract

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Progressive accumulation of the amyloid-β peptide (Aβ) in the brain plays a central role in the pathogenesis of Alzheimer's disease (AD).The animal model of intracerebral injection of Aβ oligomers not only provides a method for further exploring the mechanism of Aβ in AD, but also can be used to screen drug candidates targeting Aβ oligomers.This animal model has been widely used in the study of anti-AD drugs and mechanism of AD.In this paper, we summarize the research progress in the animal model of intracerebral injection of soluble Aβ oligomers, including experimental animals, the types of Aβ, the preparation of Aβ oligomers in vitro, injection sites and doses, the duration of modeling, animal behavioral changes, and the pathological mechanisms relating to this animal model, which will contribute to the application of the animal model to various conditions.

Key words

Alzheimer's disease / amyloid-β peptide / oligomer / intracerebral injection / animal model

引用本文

王丽莎, 刘新民, 陶雪, 张梦荻, 周云丰, 常琪. β-淀粉样蛋白寡聚体脑内注射动物模型在阿尔茨海默病研究中的应用. 药学学报, 2018 , 53 (7) : 1060 -1067 . DOI: 10.16438/j.0513-4870.2018-0088

Li-sha WANG, Xin-min LIU, Xue TAO, Meng-di ZHANG, Yun-feng ZHOU, Qi CHANG. Application of the animal model of intracerebral injection of amyloid-β oligomers to the study of Alzheimer's disease[J]. Acta Pharmaceutica Sinica, 2018 , 53 (7) : 1060 -1067 . DOI: 10.16438/j.0513-4870.2018-0088

正文

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阿尔茨海默病(Alzheimer’s disease, AD)是一种多发生于老年人的中枢神经系统退行性疾病, 临床主要表现为记忆力减退、认知功能障碍、运动障碍及人格改变等。随着人类寿命的延长和人口老龄化的加剧, AD的发病率呈快速增长趋势。到2015年, 全世界有4.68千万名AD患者, 医疗花费约为8.18千亿美元, 到2018年其费用将会突破万亿美元^[1], 这给个人、家庭和社会带来了沉重的经济和精神负担。

AD的发病机制复杂, 至今尚不明确。AD患者脑内含有大量纤维化淀粉样蛋白沉积^[2]。在近几十年的研究中, 淀粉样蛋白级联假说占主导地位。该假说认为遗传或其他因素引起β-淀粉样蛋白(amyloid-β peptide, Aβ)聚集形成纤维化沉积, 进而形成β淀粉样斑块, 随后tau蛋白过度磷酸化形成神经纤维缠结, 并产生突触和神经元功能损伤, 最终导致认知功能障碍。支持这一假设的最有力证据来自于家族性AD的许多基因突变^{[3, 4]}。这些突变使Aβ总量增加, 提高Aβ_1-42与Aβ_1-40的比值^[4], 也可产生更易形成淀粉样聚集的突变Aβ^[5]。早期研究认为, Aβ纤维化聚集体, 而非单体, 与AD的淀粉样斑块类似, 具有神经毒性^[6]。但AD患者的痴呆程度与脑内纤维化斑块密度的相关性较差^[7]。淀粉样前体蛋白(amyloid precursor protein, APP)转基因小鼠在淀粉样斑块形成之前就出现了学习记忆障碍和长时程增强(long-term potentiation, LTP)的抑制^{[8, 9]}。Mucke等^[9]观察到突触损伤与淀粉样蛋白沉积无关, 但与可溶性提取物中的Aβ免疫反应性水平相关。之后的研究进一步表明Aβ毒性与可溶性寡聚体有关, 而不是纤维化聚集体^[10-12]。Gong等^[13]已使用特异性抗体在AD患者脑中检测到这种寡聚体, 它与体外聚集生成的Aβ寡聚体一致。在APP cDNA转染的中国仓鼠卵巢细胞培养液中^[14]和Tg2576转基因小鼠的脑提取物中也观察到相似的Aβ寡聚体^[15]。在细胞和动物模型中, Aβ寡聚体能够产生与AD相关的病理改变, 包括tau蛋白磷酸化、氧化应激和突触损伤等^[16-18]。APP转基因小鼠的突触损伤发生在淀粉样斑块核团周围^[19], 这可能是因为淀粉样斑块作为Aβ寡聚体的惰性储存器, 在脂质存在下易分解, 局部产生Aβ寡聚体^[20]。Aβ纤维化聚集体的形成需要较高水平的Aβ, 而Aβ寡聚体在体内的形成只需要较低水平的Aβ, 这意味着体内首先产生Aβ寡聚体这一毒性形式, 其在AD早期阶段起重要作用^[21]。

基于Aβ假说, AD动物模型的发展主要集中于脑内Aβ的异常过量。APP或早老素基因的突变可引起Aβ失衡升高, 最终导致家族性AD^[22], 在此基础上建立的转基因小鼠(Tg2576、PDAPP、APP/PS1和APP23)模型被广泛应用, 它们能够较好地模拟人类AD病理变化进程。然而, 转基因小鼠不但价格较高, 还需要数月才能形成Aβ斑块, 甚至更长时间才能显示出Aβ诱导的突触或行为异常^[23]。而Aβ注射模型克服了转基因小鼠模型的上述缺点, 不仅可以模拟AD行为异常, 还可以表现家族性和散发性AD的病理特征。这一模型的最大优点在于其可控性, 如Aβ的聚集形态(单体、寡聚物、纤维化或混合物)、种类(Aβ_1-40、Aβ_1-42和Aβ_25-35等)、注射时间点、注射部位和造模时长等。因此, 在研究由Aβ寡聚体诱导的神经毒性或炎症时、或研究尚未形成淀粉样斑块的AD病理学时、或评价作用于AD早期阶段的候选药药效时, 建议采用可溶性Aβ寡聚体进行脑内注射。

本文集中总结了脑内注射可溶性Aβ寡聚体的研究, 主要包括实验动物、Aβ种类、Aβ寡聚体的体外制备、注射部位和剂量、造模时长和行为学检测及与Aβ寡聚体相关的病理学机制, 并对此模型的应用进行了展望。

1 实验动物

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文献报道用于脑内注射Aβ的模型动物主要有非人灵长类动物和啮齿类动物, 其中啮齿类动物最为常用^{[24, 25]}。各个月龄的鼠都有使用, 有选用2个月左右的成年鼠, 也有选用9个月甚至16个月龄的老年鼠, 以更好模拟老年人AD的病理情况^[26]。研究大多数只采用雄性鼠, 因为雌性鼠体内波动的雌激素具有神经保护作用^[27]。Liu等^[28]研究表明, 侧脑室注射Aβ_25-35后, 雄性昆明小鼠在水迷宫实验中学习记忆水平的降低程度明显大于雌性昆明小鼠。除了昆明小鼠外, 较多研究还采用C57BL/6J、ICR和Swiss等小鼠以及SD和Wistar等大鼠(表 1)^{[12, 29-48]}。

2 Aβ种类

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较多研究报道, 脑内注射Aβ_1-40、Aβ_1-42和Aβ_25-35等可产生神经毒性, 引起认知功能障碍和LTP的抑制。Aβ含有36~43个氨基酸残基, 是由APP通过水解酶水解而得到。Aβ_1-40和Aβ_1-42是AD患者脑内Aβ的两个主要成分, 前者的含量高于后者。而Aβ_1-42在AD患者脑组织中比在同龄正常人群中更丰富^[49], 且被认为是引起Aβ_1-40聚集的“种子”^[50]。细胞转染实验中, 能够引起AD的APP和早老素基因突变可以使Aβ_1-42增多^[51]。研究表明Aβ_1-40和Aβ_1-42的肽链组成基本相同, 但Aβ_1-42在C-末端多了个二肽片段(异亮氨酸-丙氨酸), 肽链更长、更疏水, 自身的聚合反应更快^[52], 毒性更大^[53]。Aβ_25-35片段能够迅速聚集, 产生神经毒性^[54]。但这种合成的Aβ片段不存在于AD患者脑内, 不能完全代表天然Aβ, 尤其是它不含有与共同沉积在神经炎斑块中的载脂蛋白ApoE、α1-抗胰凝乳蛋白酶、肝素硫酸蛋白聚糖等分子发生相互作用的结构域。目前尚不清楚Aβ_25-35是否可以模拟AD中病理生理过程以及蛋白质-蛋白质或蛋白质-脂质的相互作用^[55]。

与人类的Aβ相比, 啮齿类动物的Aβ在脑中不能形成纤维化沉积, 且较少形成β-折叠结构^{[56, 57]}。这是由于人与啮齿类动物Aβ_1-42之间的一级氨基酸序列不同。啮齿动物Aβ_1-42含有人肽序列的3个氨基酸变异, 即精氨酸5、酪氨酸10和组氨酸13分别被甘氨酸、苯丙氨酸和精氨酸所取代^[58]。由于氨基酸变异发生在与Cu²⁺结合的结构域内, 啮齿类动物的Aβ缺乏还原Cu²⁺的能力, 因此, 推测其不具有与人Aβ_1-42相同的毒性。而Boyd-Kimball等^[59]研究表明:虽然啮齿类动物Aβ_1-42的氨基酸变异发生在人Aβ的Cu²⁺结合和还原结构域内, 但不足以阻止啮齿类动物Aβ_1-42引起氧化损伤和形态学改变, 尽管其氧化损伤略低于人Aβ_1-42。

3 Aβ寡聚体的体外制备

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Aβ有多种聚集形态, 包括寡聚体(二聚体、三聚体和四聚体等)、原纤维和纤维化聚集体等。Aβ的种类和浓度、孵育时间和温度、介质的pH和离子强度等都影响Aβ的聚集^[60]。Aβ通过以下两种途径聚集:一种是成核依赖性途径, 是由Aβ单体通过非共价键结合形成核或“种子”, 然后逐渐聚集形成可溶性原纤维中间体, 最终形成不溶性纤维状聚集体; 另一种是可溶性寡聚体形成途径, 随孵育时间的延长寡聚体逐渐变大^[61]。

脑内注射的Aβ多采用双蒸水、PBS或生理盐水直接溶解。引用次数较多的是Maurice等^[62]所采用的方法:将Aβ_25-35和Aβ_1-28溶解于双蒸水中(1 mg·mL^-1), 置于-20 ℃保存。使用前将其在37 ℃孵育4天。用光镜观察“老化”的Aβ_25-35, 发现其包含两种不溶性沉积:双折射纤维状和球状聚集体。在2003年Stine等^[60]的研究中, Aβ冻干粉直接溶于双蒸水中, 原子力显微镜(atomic force microscopy, AFM)显示其包含寡聚体、纤维状和球状聚集体等各种形态。这些形态结构主要依赖于最初Aβ冻干粉中聚集体的形态, 它们能进一步聚集, 影响最终溶液内Aβ的形态和生物活性。Aβ冻干粉直接溶解也可能形成无定型聚集体, 影响Aβ正常聚集。由于最初选用的Aβ冻干粉状态不可控, 虽然用水溶液溶解孵育条件一致, 但不能保证最终形成的溶液中Aβ聚集状态一致, 因此会导致研究结果存在差异。

采用冰冷的1, 1, 1, 3, 3, 3-六氟-2-丙醇(1, 1, 1, 3, 3, 3- hexafluoro-2-propanol, HFIP)溶解Aβ冻干粉(1 mmol·L^-1), 可去除其中预先存在的不同聚集态结构, 使Aβ单体化, 经挥干HFIP后形成无色透明薄膜状肽, 它可在-80 ℃下保存6个月。使用前将薄膜状肽用无水二甲基亚砜超声复溶(5 mmol·L^-1), 这样可以形成均一可控的Aβ单体溶液。然后采用PBS或细胞培养基(不含酚红的Ham’s F-12, BioSource)稀释(100 μmol·L^-1), 于4 ℃孵育12~24 h即可形成寡聚体。若采用10 mmol·L^-1 HCl稀释并于37 ℃孵育24 h则形成纤维状聚集体。溶液中Aβ的形态可以采用AFM和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳免疫印迹法进行表征^[60]。在HFIP挥干的过程中, 需要注意Aβ的稳定性, 温度偏高会引起Aβ降解, 使其变成棕色, 不能形成无色透明的薄膜状肽。因此, 需要密切关注这一过程中的温度, 使其维持在室温^[63]。值得注意的是, 在整个制备过程中应选用聚丙烯塑料制品, 如离心管、吸头等, 因为聚丙烯塑料蛋白结合率低, 不影响溶液中不同Aβ形态的比例^[64]。

4 注射部位和剂量

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AD病变发生在特定脑区, 尤其在海马和皮层部位^[65]。Aβ寡聚体诱导神经元死亡具有区域特异性, 它能够选择性杀伤海马CA1区和内嗅皮层的神经元, 而对小脑的神经元无毒性作用^[66], 这与其在不同脑区的分布特性和受体表达差异有关^[21]。较多的研究报道是将Aβ注射于侧脑室或海马(CA1区和齿状回等)等部位(表 1)。Jean等^[26]认为, 由于针头插入脑内会引起注射部位组织损伤, 导致额外的细胞死亡和胶质增生, 因此, 在进行脑片检测时必须远离注射处进行分析。如要研究Aβ对海马依赖性行为的影响, 则应在海马附近进行注射, 而不是直接注射进入海马, Aβ可通过扩散进入海马内产生影响。

可以根据AD患者脑内的实际浓度选择造模所需的Aβ剂量。AD患者脑内皮层中可溶性Aβ的平均浓度为1.5 μg·g^-1, 而正常人脑中的为2 ng·g^-1[67]。Epelbaum等^[38]的体内实验显示, Aβ用量采用与AD患者脑内可溶性Aβ相同的数量级, < 1 μg·g^-1[68], 即每只小鼠注射50 pmol, 相当于0.225 μg Aβ扩散于约500 mg的小鼠脑中达到0.45 μg·g^-1的浓度。其他研究也有采用高于此范围的剂量(表 1)。

5 造模时长和行为学检测

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表 1总结了近10年来Aβ寡聚体脑内注射动物模型的造模时长和行为学检测方法。关于小鼠的研究报道中, 应用较多的是物体认知实验, 以检测动物短期学习记忆水平。总结这些文献发现, 14天内模型组小鼠和对照组比较, 其学习记忆水平显著降低。但2010年Balducci等^[12]研究表明, 在造模后第10天的检测中模型组和对照组的学习记忆水平没有统计学差异。推测原因可能包括: Aβ寡聚体的制备方法与其他研究的不完全一致, 采用碱性溶液溶解Aβ后再用PBS稀释孵育, 而不是“Aβ寡聚体的体外制备”中总结的常规方法, 可能包含不同或不同比例的寡聚体形态; 在熟悉期和测试期前2 h分别给予两次较低剂量的Aβ寡聚体(7.5 pmol)而不是熟悉期前单次注射稍多剂量的Aβ寡聚体(10和50 pmol)。在海马部位注射Aβ后的第8天, 避暗实验表现出明显差异, 表明Aβ寡聚体也可影响动物的长期记忆水平^{[36, 37]}。Y迷宫实验中, 造模后6天内动物表现出空间工作能力的降低, 而30天后恢复正常^[38]。这可能是由于小胶质细胞和星形胶质细胞对脑内Aβ的清除作用^{[69, 70]}。在C57小鼠侧脑室中一次性注射500 pmol Aβ寡聚体可以在第7~14天的水迷宫实验中表现出空间学习记忆能力的损伤^[29]。Ledo等^[31]的研究发现, 造模后小鼠不仅表现出学习记忆水平的下降, 还表现出AD的另一症状——抑郁。在悬尾和强迫游泳实验中, 造模后第1天和第8天都可以显著增加小鼠的不动时间; 造模后第1天也显著降低模型小鼠对糖水的偏爱程度。关于大鼠的研究报道中, 应用较多的是水迷宫实验。多数研究选在造模后1周开始检测水迷宫^{[43, 45, 47, 48]}, 也有研究采用每天注射一定量Aβ寡聚体, 连续30天之后再开始进行检测^{[39, 40]}。Rammes等^[42]研究表明, 注射Aβ寡聚体2 h后可以显著降低大鼠在新物体识别中的短期学习记忆水平。

6 相关病理学机制

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Aβ寡聚体引发的细胞损伤几乎包含AD病理学的所有重要方面, 如突触损伤、tau蛋白异常磷酸化、胆碱能丢失、氧化应激、胰岛素抵抗、神经炎症和选择性神经元死亡等^[71]。大量研究表明, 可溶性Aβ寡聚体在AD中诱导突触丢失。Aβ诱导的突触功能障碍依赖于对N-甲基-D-天冬氨酸受体(N-methyl-D- aspartate receptors, NMDARs)的过度刺激, 导致氧化还原异常激活以及细胞质内Ca²⁺水平升高, 这又进一步触发后续反应, 涉及磷酸tau蛋白、caspases、Cdk5/dynamin相关蛋白1、钙调神经磷酸酶/PP2B、PP2A、Gsk-3β、Fyn、cofilin和CaMKⅡ等, 并引起AMPA受体和NMDARs的内吞作用。这些信号通路的异常改变导致线粒体功能障碍和生物能量损失, 随后引起突触功能障碍和损伤, 抑制LTP, 降低认知水平。也有研究指出Aβ至少部分能够通过抑制谷氨酸摄取或促进胶质细胞释放谷氨酸来异常升高突触后谷氨酸水平。随后突触后NMDARs被过度刺激, 通过上述途径导致突触功能障碍。与此一致的是, NMDARs拮抗剂(美金刚和NitroMemantine)可以部分改善Aβ的突触毒性^[72]。

7 结语

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尽管一些Aβ抗体(礼来的solanezumab、强生的bapineuzumab和罗氏的gantenerumab)在最近的AD Ⅲ期临床试验中失败^[73], 但作用于调节Aβ的候选药物和新型临床试验策略仍有希望^[74]。Solanezumab靶向于可溶性Aβ单体, bapineuzumab和gantenerumab靶向于β淀粉样斑块。体内Aβ具有多种不同形态, 对应的抗体需要包含不同结合表位, 因此目前Aβ抗体临床试验的失败不能表明靶向于Aβ寡聚体等形态的抗体药物没有希望。另外, AD药物的研发也越来越趋向于其早期治疗。如礼来正在进行的solanezumab Ⅲ期EXPEDITION-PRO试验(NCT02760602), 预计将在2021年完成^[75]。总之, Aβ寡聚体脑内注射动物模型为这些临床研究奠定基础, 不仅能深入探索Aβ在体内的作用机制, 而且可以筛选靶向Aβ寡聚体的候选药物, 为阻止AD病理进程和逆转早期AD功能障碍提供方法^[21]。

基金

收起

国家自然科学基金资助项目(81773930)
国家重大创新药创制专项(2017ZX09301029)
中国医学科学院创新工程项目(2016-I2M-1-012)

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2018年第53卷第7期

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doi: 10.16438/j.0513-4870.2018-0088

接收时间：2018-01-24
首发时间：2026-01-15
出版时间：2018-07-12

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收稿日期：2018-01-24
修回日期：2018-03-14

基金

国家自然科学基金资助项目(81773930)

国家重大创新药创制专项(2017ZX09301029)

中国医学科学院创新工程项目(2016-I2M-1-012)

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中国医学科学院、北京协和医学院药用植物研究所, 北京 100193

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* 常琪, Tel:86-10-57833468, E-mail:qchang@implad.ac.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Year	Animal	Injection site	Dose (pmol/ mouse or rat)	Duration of modeling and behavioral test
2008^[29]	C57BL/6 mice (12 w)	Lateral ventricle	500	-	-	4 day	-	7-14 day	-	-
2010^[12]	C57BL/6 mice (7-8 w)	Lateral ventricle	7.5 (two injections)	1 day (recovered after 10 days)	-	-	-	-	-	-
2010^[30]	C57BL/6 mice (20 w)	Lateral ventricle	222	-	-	-	1 day	-	-	-
2012^[31]	Swiss mice (12 w)	Lateral ventricle	10	1 day	-	-		-	1, 8 day	1 day
2013^[32]	C57BL/6J mice (12 w)	Lateral ventricle	50	-	-	4 day	-	-	-	-
2013^[33]	Swiss mice (12 w)	Lateral ventricle	10, 50	1, 7, 14 day	-	-	-	-	-	-
2016^[34]	ICR mice (7 w)	Lateral ventricle	500	-	-	3 day	-	-	-	-
2017^[35]	Swiss mice (8-12 w)	Lateral ventricle	10	1 day (novel object) 8 day (object location)	-	-	-	-	-	-
2011^[36]	ICR mice (8 w)	Granule cell layer of hippocampus	30	6 day	8 day	-	-	-	-	-
2011^[37]	ICR mice (27-30 g)	Hilus of hippocampus	30	-	8 day	6 day	-	-	-	-
2015^[38]	C57BL/6 mice (12 w)	Hippocampus	50	-	-	6 day (recovered after 30 days)	-	-	-	-
2012^[39]	Wistar rats (12-48 w, 200-250 g)	Lateral ventricle	600/day (30-days infusion)	-	-	-	-	32-36 day	-	-
2012^[40]	SD rats (200-300 g)	Lateral ventricle	144/day (30-days infusion)	-	-	-	-	31-35 day	-	-
2014^[41]	SD rats (220-280 g)	Lateral ventricle	5000	-	-	-	-	2-10 day	-	-
2015^[42]	SD rats (250 g)	Lateral ventricle	10.8	2 h	-	-	-	-	-	-
2015^[43]	Wistar rats (280-300 g)	Lateral ventricle	5000	-	-	-	-	10-16 day	-	-
2016^[44]	Wistar rats (280-300 g)	Lateral ventricle	5000	-	-	-	-	10-16 day	-	-
2016^[45]	Wistar rats (24 w)	Lateral ventricle	5100	-	-	-	-	8-12, 19 day	-	-
2016^[46]	SD rats (250-350 g)	Lateral ventricle	400/2 day (16-days infusion)	-	-	-	-	8-16 day	-	-
2017^[47]	Charles River- Harlan rats (250-300 g)	Lateral ventricle	375, 1 125 and 3 000	-	-	-	-	8-12 day	-	-
2013^[48]	Wistar rats (24 w, 220-250 g)	CA1 molecular layer of hippocampus	149.53	-	-	-	-	8-11 day	-	-

Year

Animal

Injection site

Dose (pmol/
mouse
or rat)

Duration of modeling and behavioral test

Object
recognition

Passive avoidance

Y
maze

Fear
conditioning

Water
maze

Tail
suspension and forced swim

Sucrose preference

2008^[29]

C57BL/6 mice
(12 w)

Lateral ventricle

500

4 day

7-14 day

2010^[12]

C57BL/6 mice
(7-8 w)

Lateral ventricle

7.5 (two
injections)

1 day (recovered after 10 days)

2010^[30]

C57BL/6 mice
(20 w)

Lateral ventricle

222

1 day

2012^[31]

Swiss mice
(12 w)

Lateral ventricle

1 day

1, 8 day

1 day

2013^[32]

C57BL/6J mice
(12 w)

Lateral ventricle

4 day

2013^[33]

Swiss mice
(12 w)

Lateral ventricle

10, 50

1, 7, 14 day

2016^[34]

ICR mice
(7 w)

Lateral ventricle

500

3 day

2017^[35]

Swiss mice
(8-12 w)

Lateral ventricle

1 day (novel object) 8 day (object location)

2011^[36]

ICR mice
(8 w)

Granule cell layer
of hippocampus

6 day

8 day

2011^[37]

ICR mice
(27-30 g)

Hilus of
hippocampus

8 day

6 day

2015^[38]

C57BL/6 mice
(12 w)

Hippocampus

6 day (recovered after 30 days)

2012^[39]

Wistar rats
(12-48 w,
200-250 g)

Lateral ventricle

600/day (30-days
infusion)

32-36 day

2012^[40]

SD rats
(200-300 g)

Lateral ventricle

144/day (30-days
infusion)

31-35 day

2014^[41]

SD rats
(220-280 g)

Lateral ventricle

5000

2-10 day

2015^[42]

SD rats
(250 g)

Lateral ventricle

10.8

2 h

2015^[43]

Wistar rats
(280-300 g)

Lateral ventricle

5000

10-16 day

2016^[44]

Wistar rats
(280-300 g)

Lateral ventricle

5000

10-16 day

2016^[45]

Wistar rats
(24 w)

Lateral ventricle

5100

8-12, 19 day

2016^[46]

SD rats
(250-350 g)

Lateral ventricle

400/2 day (16-days
infusion)

8-16 day

2017^[47]

Charles River-
Harlan rats
(250-300 g)

Lateral ventricle

375, 1 125
and 3 000

8-12 day

2013^[48]

Wistar rats
(24 w,
220-250 g)

CA1 molecular
layer of
hippocampus

149.53

8-11 day