利用脑立体定位技术将人源三突变APP基因导入海马区构建阿尔茨海默病大鼠模型

doi:10.12300/j.issn.1674-5817.2025.030

实验动物与比较医学 ›› 2025, Vol. 45 ›› Issue (3): 269-278.DOI: 10.12300/j.issn.1674-5817.2025.030

利用脑立体定位技术将人源三突变APP基因导入海马区构建阿尔茨海默病大鼠模型

肖林林¹^,², 杨逸萱¹^,², 黎珊杉¹^,², 罗兰诗雨¹^,², 尹思威¹^,², 孙俊铭¹, 施维¹, 欧阳轶强¹()(), 李习艺³()()

^1.广西医科大学实验动物中心, 南宁 530021
^2.广西医科大学基础医学院, 南宁 530021
^3.广西医科大学公共卫生学院, 南宁 530021

收稿日期:2025-03-01 修回日期:2025-04-14 出版日期:2025-07-07 发布日期:2025-06-25
通讯作者: 李习艺（1974—），女，博士，教授，研究方向：营养学，人类疾病模型。E-mail： leeciyee@163.com。ORCID：0009-0006-0280-8451；
欧阳轶强（1977—），男，博士，高级实验师，研究方向：人类疾病模型。E-mail： ouyangyiqiang@stu.gxmu.edu.cn。ORCID： 0000-0003-2277-4018
作者简介:肖林林（1999—），女，硕士研究生，研究方向：阿尔茨海默病模型。E-mail：2721357235@qq.com
基金资助:
广西自然科学基金"组织特异性基因编辑技术制作树鼩肝癌模型的探索"(2023GXNSFDA026069)

Establishment of a Rat Model of Alzheimer's Disease by Introducing Human Triple Mutant APP Gene into Hippocampus via Brain Stereotactic Technology

XIAO Linlin¹^,², YANG Yixuan¹^,², LI Shanshan¹^,², LUO Lanshiyu¹^,², YIN Siwei¹^,², SUN Juming¹, SHI Wei¹, OUYANG Yiqiang¹()(), LI Xiyi³()()

^1.Laboratory Animal Center, Guangxi Medical University, Nanning 530021, China
^2.School of Basic Medical Sciences, Guangxi Medical University, Nanning 530021, China
^3.School of Public Health, Guangxi Medical University, Nanning 530021, China

Received:2025-03-01 Revised:2025-04-14 Published:2025-06-25 Online:2025-07-07
Contact: LI Xiyi (ORCID: 0009-0006-0280-8451), E-mail: leeciyee@163.com;
OUYANG Yiqiang g (ORCID: 0000-0003-2277-4018), E-mail: ouyangyiqiang@stu.gxmu.edu.cn

摘要/Abstract

摘要：

目的构建脑海马区表达人源三突变淀粉样前体蛋白（amyloid precursor protein，APP）的阿尔茨海默病（Alzheimer's disease，AD）大鼠模型，为疾病机制和药物研究奠定基础。方法将24只12周龄SPF级雌性SD大鼠随机分成空白对照组、空载病毒组和实验组，每组8只；其中，实验组通过脑立体定位在海马区注射携带人源三突变APP和NanoLuc萤光素酶基因的腺相关病毒（adeno-associated virus，AAV）进行造模。采用活体成像法观察各组大鼠脑内的病毒表达情况，新事物识别实验检测各组大鼠的认知记忆能力，实时荧光定量PCR检测APP基因表达水平，HE染色观察脑组织病理，采用尼氏染色观察海马区病变情况，用免疫组织化学检测β-淀粉样蛋白（amyloid β-protein，Aβ）沉积情况。结果活体成像显示实验组与空载病毒组大鼠脑部均检测到报告荧光。新事物识别实验发现实验组大鼠的认知记忆能力比对照组显著下降（P<0.01）。荧光定量PCR显示APP基因在实验组大鼠脑内表达水平显著增高（P<0.01）。重组AAV感染大鼠6个月后，脑组织HE染色和尼氏染色显示，实验组大鼠海马体CA1区神经元细胞和尼氏体数量减少而且排列混乱；免疫组织化学检测实验组大鼠海马CA1区及锥形细胞层可以看到明显的棕褐色沉淀，提示产生了Aβ沉积。结论通过脑立体定位技术联合AAV转入人源三突变APP基因成功构建的大鼠模型可体现出典型AD特征，该模型可为基于Aβ沉积的AD病理研究和药物治疗研究提供动物模型并奠定基础。

关键词: 阿尔茨海默病, 淀粉样前体蛋白, 人源三突变基因, 脑立体定位, 大鼠模型

Abstract:

Objective To establish a rat model of Alzheimer's disease (AD) expressing human triple mutant amyloid precursor protein (APP) in the hippocampus, and to provide a model for the study of disease mechanisms and drug development. Methods Twenty-four 12-week-old SPF-grade female SD rats were randomly divided into a blank control group, a virus control group and an experimental group, with eight rats in each group; among them, the experimental group received a stereotaxic injection of adeno-associated virus (AAV) carrying the human triple mutant APP and NanoLuc luciferase genes into the hippocampus. In vivo imaging was used to observe viral expression in the brains of rats in each group, the novel object recognition test was used to assess the recognition memory of the rats in each group, real-time fluorescent quantitative PCR was used to detect the expression level of the APP gene, HE staining was used to examine the brain histopathology, Nissl staining was used to assess the hippocampal lesions, and immunohistochemistry was used to detect the deposition of amyloid β-protein (Aβ). Results In vivo imaging showed that reporter fluorescence was detected in the brains of rats in both experimental and virus control groups. Fluorescence quantitative PCR showed that the expression level of the APP gene was significantly increased in the brains of rats in the experimental group (P<0.01). Novel object recognition test revealed that the recognition memory of rats in the experimental group was significantly reduced compared with that of the blank control group (P<0.01). Six months after recombinant AAV virus infection, HE staining and Nissl staining of brain tissues showed that the number of neurons and Nissl bodies in the CA1 region of the hippocampus in the experimental group was reduced and disorganized; immuno-histochemistry testing of the CA1 region of the hippocampus and the pyramidal cell layer of the experimental group revealed prominent brown deposits, indicating Aβ protein deposition. Conclusion The rat model successfully established by stereotaxic injection and AAV-mediated delivery of human triple mutant APP gene exhibits typical AD features, providing a valuable animal model for studying AD pathology and developing drug therapies targeting Aβ protein deposition.

Key words: Alzheimer's disease, Amyloid precursor protein, human triple mutant gene, Brain stereotaxis, Rat model

中图分类号:

R-332

肖林林,杨逸萱,黎珊杉,等. 利用脑立体定位技术将人源三突变APP基因导入海马区构建阿尔茨海默病大鼠模型[J]. 实验动物与比较医学, 2025, 45(3): 269-278. DOI: 10.12300/j.issn.1674-5817.2025.030.

XIAO Linlin,YANG Yixuan,LI Shanshan,et al. Establishment of a Rat Model of Alzheimer's Disease by Introducing Human Triple Mutant APP Gene into Hippocampus via Brain Stereotactic Technology[J]. Laboratory Animal and Comparative Medicine, 2025, 45(3): 269-278. DOI: 10.12300/j.issn.1674-5817.2025.030.

图/表 5

图1 重组腺相关病毒载体结构示意图注：A为空载病毒质粒结构图；B为携带人源三突变APP基因的质粒结构图。APPsla-L-A代表人源三突变APP基因，Nluc代表NanoLuc萤光素酶基因，SYN promoter是神经元特异性启动子。

Figure 1 Schematic diagram of recombinant adeno-associated viral vector structureNote：A， structure of the empty viral plasmid； B， structure of the plasmid carrying the human triple mutant APP gene， APPsla-L-A represents the human triple mutant APP gene， Nluc represents the NanoLuc luciferase gene， and SYN promoter is the neuron-specific promoter.

图2 注射病毒2周和6个月后大鼠活体成像情况注：BC、VC、Exp分别为空白对照组、空载病毒组、实验组（通过脑立体定位在海马区注射携带人源三突变APP和NanoLuc萤光素酶基因的腺相关病毒）。各组大鼠腹腔注射萤光素酶底物Furimazine后10 min观察荧光素报告酶活性。2周后实验组与空载病毒组头部区域可检测到荧光发光，6个月后实验组和空载病毒组的荧光强度都减弱，但仍然可以被检测到；空白对照组未检测到荧光。

Figure 2 In vivo imaging of rats two weeks and six months after virus injectionNote：BC， VC， Exp are blank control group， virus control group， and experimental group （adeno-associated virus carrying human triple mutant APP and NanoLuc luciferase genes was injected in the hippocampus by brain stereotaxic localization）， respectively. The luciferase reporter activity was observed 10 min after intraperitoneal injection of the luciferase substrate Furimazine into rats in each group. 2 weeks later， fluorescent luminescence could be detected in the head area of the experimental group and the virus control group， and 6 months later， the fluorescence intensity of both the experimental group and the virus control group was weakened， but it could be detected； the fluorescence was not detected in the blank control group.

图3 新物体识别实验检测大鼠认知记忆能力注：A展示大鼠探索两个相同物体；B为更换了新物体；C为以距离为统计指标的新物体识别实验结果；D为以时间为统计指标的新物体识别实验结果。RI：新事物识别指数。BC、VC、Exp分别为空白对照组、空载病毒组、实验组（通过脑立体定位在海马区注射携带人源三突变APP和NanoLuc萤光素酶基因的腺相关病毒），每组8只大鼠（n=8）；与空白对照组相比，**P<0.01；与空载病毒组相比，#P<0.05。

Figure 3 Novel object recognition test to assess recognition memory of ratsNote：A shows rats exploring two identical objects； B is a new object replaced； C is the result of the new object recognition experiment using distance as a statistical index； D is the result of the new object recognition experiment using time as a statistical index. RI：Recognition index.BC， VC， Exp are blank control group， virus control group and experimental group （adeno-associated virus carrying human triple mutant APP and NanoLuc luciferase genes was injected in the hippocampus by brain stereotaxic localization）， respectively， 8 rats per group （n=8）； **P<0.01， compared with blank control group； #P<0.05， compared with virus control group.

图4 大鼠海马体APP 基因表达及测序结果注：A，为实时荧光定量PCR检测结果［BC、VC、Exp分别为空白对照组、空载病毒组、实验组（通过脑立体定位在海马区注射携带人源三突变APP和NanoLuc萤光素酶基因的腺相关病毒），每组4只大鼠（n=4）；与空白对照组相比，**P<0.01；与空载病毒组相比，##P<0.01］。B为PCR产物测序比对结果，黄色高亮部分指示突变位点。

Figure 4 APP gene expression and sequencing results in rat hippocampusNote： A is the result of real-time fluorescence quantitative PCR. BC， VC， Exp are blank control group， virus control group and experimental group （adeno-associated virus carrying human triple mutant APP and NanoLuc luciferase genes was injected in the hippocampus by brain stereotaxic localization）， respectively. 4 rats in each group （n=4）； Compared with blank control group， **P<0.01； Compared with virus control group， ##P<0.01. B is the result of real-time fluorescence quantitative PCR product sequencing comparison， and the yellow highlighted part is the indicated mutation site.

图5 大鼠海马体组织的HE染色（A）、尼氏染色（B）和免疫组织化学染色（C）结果注：BC、VC、Exp分别为空白对照组、空载病毒组、实验组［通过脑立体定位在海马区注射携带人源三突变淀粉样前体蛋白（APP）和NanoLuc萤光素酶基因的腺相关病毒］。黑色箭头所指区域为皱缩坏死的细胞；红色箭头所指区域为尼氏体稀疏，染色浅淡的细胞；蓝色箭头所指区域为β-淀粉样蛋白沉积区域。

Figure 5 HE staining （A）， Nissl staining （B）， and immunohistochemical staining （C） results of rat hippocampal regionNote： BC， VC， Exp are blank control group， virus control group and experimental group （adeno-associated virus carrying human triple mutant APP and NanoLuc luciferase genes was injected in the hippocampus by brain stereotaxic localization）， respectively. The areas indicated by black arrows are crumpled necrotic cells. The areas indicated by red arrows are cells with sparse Nissl bodies and light staining. The area indicated by blue arrow is the area of amyloid β-protein deposition.

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利用脑立体定位技术将人源三突变APP基因导入海马区构建阿尔茨海默病大鼠模型

Establishment of a Rat Model of Alzheimer's Disease by Introducing Human Triple Mutant APP Gene into Hippocampus via Brain Stereotactic Technology

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