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AD大鼠模型

doi:10.12300/j.issn.1674-5817.2025.030

摘要/Abstract

摘要：

目的构建海马脑区表达人源三突变淀粉样前体蛋白基因（Amyloid precursor protein，APP）的阿尔茨海默症（Alzheimer's disease，AD）大鼠模型，为疾病机制和药物研究提供模型。方法将24只SD大鼠随机分成空白对照组、空载病毒组、实验组，实验组脑立体定位海马区注射携带人源三突变APP和NanoLuc荧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病毒的表达情况，qPCR检测转入的APP的表达水平，对各组大鼠进行行为学实验检测其认知记忆能力，取大鼠脑组织进行HE染色，尼氏染色检测海马区病变情况，免疫组织化学检测Aβ蛋白沉积情况。结果大鼠的活体成像结果显示，除空白对照组外，实验组与空载病毒组均检测到荧光，表明携带NanoLuc荧光素酶基因标记的AAV成功在大鼠脑部表达；荧光定量PCR结果显示APP基因在实验组大鼠脑内表达水平显著增高（P<0.01）；新物体识别实验观察到，实验组大鼠认知记忆能力与空白对照组以及空载病毒组相比显著下降（P<0.01）；重组AAV病毒感染大鼠6个月后，脑组织HE染色和尼氏染色结果显示，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棕褐色沉淀，即产生了Aβ蛋白沉积。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蛋白沉积的AD病理研究和药物治疗研究提供动物模型和奠定基础。

关键词: 阿尔茨海默症, 人源三突变APP, 脑立体定位, 大鼠模型

Abstract:

Objective To construct a rat model of Alzheimer's disease (AD) in the hippocampal brain region expressing human triple mutant amyloid precursor protein (APP), and to provide a model for mechanistic study and preclinical research. Methods Twenty-four SD rats were randomly divided into negative control, virus control and experimental groups. The experimental group was prepared by stereotactic injection of AAV carrying the human triple mutant APP genes and the NanoLuc luciferase gene. In vivo imaging was used to observe the expression of the virus, qPCR was used to examine the expression level of the mutant APP, the behavioral experiments were conducted to detect the cognitive and memory ability, the pathological changes in the hippocampus were detected by HE staining and Nissl staining, and the deposition of Aβ protein was detected by immunohistochemistry. Results The in vivo imaging results showed that the AAV was successfully expressed in the rat brain; The qPCR results showed that the expression level of APP genes was significantly increased in the rat of experimental group (p<0.01); The recognition of new objects test observed that the cognitive and memory abilities of the experimental group of rats were significantly decreased compared with the blank control group or the empty virus group (p<0.01); Six months after recombinant AAV virus infection, the HE staining and Nissl staining of brain tissue showed that the number of neuronal cells and Nissl bodies in hippocampal CA1 area of the experimental group was reduced and disorganized, the deposition of Aβ protein in CA1 region and conical cell layer of hippocampus of experimental rats was detected by immunohistochemistry. Conclusion The rat model successfully constructed by brain stereotaxic injections of AAV expressing human triple mutant APP genes can reflect typical AD characteristics, providing a rat model for the AD pathological and pharmacological research based on Aβ protein deposition.

Key words: Alzheimer's disease, Human triple mutant APP, Brain stereotaxis, Rat model

中图分类号:

Q95-33

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AD大鼠模型[J]. 实验动物与比较医学.. DOI: 10.12300/j.issn.1674-5817.2025.030.

XIAO Linlin,YANG Yixuan,LI Shanshan,et al. A Rat Model of Alzheimer's Disease by Introducing Human Triple Mutant APP Gene into Hippocampus using Brain Stereotactic Technology[J]. Laboratory Animal and Comparative Medicine. DOI: 10.12300/j.issn.1674-5817.2025.030.

图/表 7

图1 重组腺相关病毒载体图注：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；载体图中APLsla-L-A代表人源三突变APP基因，Nluc代表NanoLuc荧光素酶基因，SYN promoter是神经元特异性启动子。

Figure 1 Diagram of adeno-associated viral vectorsNote：A， Structure of the empty viral plasmid； B， Structure of the plasmid carrying the human triple mutant APP gene； APLsla-L-A in the vector diagram represents the human triple mutant APP genes， Nluc represents the NanoLuc luciferase gene， and SYN promoter is the neuron-specific promoter.

图2 注射病毒两周和6个月后大鼠活体成像情况注：各组大鼠腹腔注射荧光素酶底物Furimazine后10 min观察荧光素报告酶活性。A为空白对照组两周后活体成像图；B为空载病毒组造模两周后活体成像；C为实验组造模两周后活体成像；D为空白对照组六个月后活体成像；E为空载病毒组造模六个月后活体成像；F为实验组造模六个月后活体成像。

Figure 2 In vivo imaging of rats two weeks and six months after virus injectionNote：Luciferase activity was observed 10 min after intraperitoneal injection of luciferase substrate Furimazine in each group of rats. A，In vivo imaging a rat in the blank control group； B， In vivo imaging a rat two weeks after injection of the empty virus； C，In vivo imaging a rat two weeks after injection of of AAV virus expressing the human triple mutant APP genes in the experimental group； D， In vivo imaging a rat survived for 6 months in the blank control group； E， In vivo imaging a rat six months after injection of the empty virus； F， In vivoimaging a rat six months after injection of the mutant APP genes in the experimental group.

图3 大鼠海马体APP 表达检测结果注：A为qPCR检测结果，与空白对照组相比，**P<0.01；与空载病毒组相比，##P<0.01，n=4；B为qPCR产物测序比对结果，黄色高亮部分是突变位点。

Figure 3 Results of APP expression detection in rat hippocampusNote：A， qPCR results，compared with blank control group， **P<0.01； compared with empty virus group，##P<0.01； B， sequencing comparison results of qPCR products， yellow highlights are mutation sites.

图4 新物体识别实验结果注：A为大鼠探索两个相同物体的探索行为图；B为大鼠对新旧物体的探索行为图；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；NC，VC，Exp分别为空白对照组、空载病毒组、实验组；与空白对照组相比，**P<0.01；与空载病毒组相比，#P<0.05，n=8。

Figure 4 Experimental results of new object recognitionNote：A， Exploratory behavior graphs of rats exploring two identical objects； B， Exploratory behavior graphs of rats exploring new and old objects； C， Results of new object recognition experiments using distance as a statistical index； D， Results of new object recognition experiments using time as a statistical index； NC， VC， and Exp are blank control group， empty virus group， and experimental group， respectively； compared with the blank control group，**P<0.01； compared with the airborne virus group， #P<0.05， n=8 .

图5 大鼠海马区HE染色结果注：NC，VC，Exp分别为空白对照组，空载病毒组，实验组HE染色结果。标尺在图中标出。箭头所指区域为皱缩坏死的细胞。

Figure 5 HE staining results of rat hippocampal regionNote：NC， VC， Exp are the HE staining results of the blank control group， the empty virus group， and the experimental group， respectively. Scale bars are shown. The areas indicated by arrows are crumpled necrotic cells.

图6 大鼠海马区尼氏体染色结果注：NC，VC，Exp分别为空白对照组，空载病毒组，实验组尼氏染色结果。标尺在图中标出。箭头所指区域为尼氏体稀疏，染色浅淡的细胞。

Figure 6 Staining results of Nystrom's body in the hippocampus of the ratNote：NC， VC， Exp are the Nissl staining results of the blank control group， the empty virus group， and the experimental group， respectively. Scale bars are shown. Areas indicated by arrows are cells with sparse nidus and light staining.

图7 大鼠海马区免疫组织化学结果注：NC，VC，Exp分别为空白对照组，空载病毒组，实验组免疫结果。标尺在图中标出。箭头所指区域为Aβ蛋白沉积区域。

Figure 7 Immunohistochemical results of rat hippocampal regionNote：NC， VC， Exp are the immunization results of blank control group， empty virus group， and experimental group， respectively. Scale bars are shown. The area indicated by the arrow is the area of Aβ protein deposition.

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