APP695^K595N／M596L突变转基因小鼠的建立和病理表型动态分析

目的建立APP695^K595N／M596L（Swedish突变）转基因小鼠和评价痴呆表型的发生和发展过程。方法将APP695^K595N／M596L突变基因插入到小鼠朊蛋白（mouse prion protein）启动子下游，构建转基因表达载体，通过显微注射法建立APP695^K595N／M596L突变转基因C57BL／6J小鼠。PCR鉴定转基因小鼠的基因表型，Western blotting检测APP突变基因表达。Thioflavin-S染色检测不同年龄转基因小鼠大脑病理改变。Morris水迷宫动态观察小鼠行为学改变。结果建立了人APP695^K595N／M596L转基因小鼠，Thioflavin-S染色显示转基因小鼠9月龄时在脑海马区可检测到老年斑形成，并且在11、12月龄时明显增多。Morris水迷宫结果发现与同月龄野生型小鼠相比，该转基因小鼠5月龄开始出现学习记忆能力缺陷，7、9、11月行为学结果证实转基因小鼠的学习记忆能力缺陷随年龄增加而日趋严重（P＜0．05）。结论建立了人APP695^K595N／M596L转基因小鼠，并能再现人类阿尔茨海默症的行为学及神经病理学特征，为阿尔茨海默病发病机制研究和药物研发提供了有价值的动物模型。

Establishment of APP695^swediSh Transgenic Mouse Model and Analysis of the Development of Pathological Phenotypes

Objective To generate a transgenic mouse line expressing human APP695^K595N/M596L （Swedish mutation） and establish a transgenic Alzheimer disease model. Methods The transgenic plasmid was constructed by inserting the mutated APP695^K595N/M596L gene into the downstream of mouse prion protein promoter. The transgenic mice were produced by microinjection and the genotype was detected by PCR. The gene expression levels were determined by Western blotting. The senile plaques were detected by thioflavin-S staining and visualized directly by fluorescence microscopy. The behavioral changes was examined by Morris water maze test. Results Transgenic C57BL/6J mice were generated with the expression of the APP695^K595N/M596L in the brain tissue. The transgenic mice showed significant learning and memory impairments in the Morris water maze at 5 months of age and the extent of the impairments was developed at 7, 9 and 11 months of age, comparing with that of age-matched wild type mice （P 〈 0. 05）. Senile plaques were visualized in the CA1 area of hippocampus at 9 and increased at 12 months of age. Conclusions The transgenic mice show a progressive deficits of learning and memory and a progressive formation of senile plaques in the hippocampus, suggesting that this APP transgenic mouse is an useful animal model of Alzheimer disease.