Park Soojin, Huh Jae-Won, Eom Taekil, Na Naeun, Lee Youngjeon, Kim Ju-Sung, Kim Sun-Uk, Shim Insop, Lee Sang-Rae, Kim Ekyune
Graduate School of Medicine, Osaka University, Suita, Osaka 5650871 Japan.
National Primate Research Center (NPRC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Deajeon 34141, Republic of Korea.
J Microbiol Biotechnol. 2017 Nov 28;27(11):2044-2051. doi: 10.4014/jmb.1707.07003.
The main pathological hallmark of Alzheimer's disease is the deposition of amyloid-beta (Aβ) peptides in the brain. Aβ has been widely used to mimic several aspects of Alzheimer's disease. However, several characteristics of amyloid-induced Alzheimer's disease pathology are not well established, especially in mice. The present study aimed to develop a new Alzheimer's disease model by investigating how Aβ can be effectively aggregated using prokaryotes and eukaryotes. To express the Aβ42 complex in HEK293 cells, we cloned the Aβ42 region in a tandem repeat and incorporated the resulting construct into a eukaryotic expression vector. Following transfection into HEK293 cells via lipofection, cell viability assay and western blotting analysis revealed that exogenous Aβ42 can induce cell death and apoptosis. In addition, recombinant His-tagged Aβ42 was successfully expressed in BL21 (DE3) and not only readily formed Aβ complexes, but also inhibited the proliferation of SH-SY5Y cells and . For in vivo testing, recombinant His-tagged Aβ42 solution (3 μg/μl in 1× PBS containing 1 mM Ni²⁺) was injected stereotaxically into the left and right lateral ventricles of the brains of C57BL/6J mice ( = 8). Control mice were injected with 1× PBS containing 1 mM Ni²⁺ following the same procedure. Ten days after the sample injection, the Morris water maze test confirmed that exogenous Aβ caused an increase in memory loss. These findings demonstrated that Ni²⁺ is capable of complexing the 50-kDa amyloid and that intracerebroventricular injection of Aβ42 can lead to cognitive impairment, thereby providing improved Alzheimer's disease models.
阿尔茨海默病的主要病理标志是β-淀粉样蛋白（Aβ）肽在大脑中的沉积。Aβ已被广泛用于模拟阿尔茨海默病的多个方面。然而，淀粉样蛋白诱导的阿尔茨海默病病理学的几个特征尚未完全明确，尤其是在小鼠中。本研究旨在通过研究如何利用原核生物和真核生物有效地聚集Aβ来开发一种新的阿尔茨海默病模型。为了在HEK293细胞中表达Aβ42复合物，我们将Aβ42区域克隆成串联重复序列，并将所得构建体整合到真核表达载体中。通过脂质体转染将其转染到HEK293细胞后，细胞活力测定和蛋白质印迹分析表明，外源性Aβ42可诱导细胞死亡和凋亡。此外，重组His标签的Aβ42在BL21（DE3）中成功表达，不仅易于形成Aβ复合物，还抑制了SH-SY5Y细胞的增殖。为了进行体内测试，将重组His标签的Aβ42溶液（在含有1 mM Ni²⁺的1×PBS中为3 μg/μl）立体定向注射到C57BL/6J小鼠（n = 8）大脑的左右侧脑室中。按照相同程序给对照小鼠注射含有1 mM Ni²⁺的1×PBS。样本注射十天后，莫里斯水迷宫试验证实外源性Aβ导致记忆丧失增加。这些发现表明Ni²⁺能够与50 kDa的淀粉样蛋白复合，脑室内注射Aβ42可导致认知障碍，从而提供了改进的阿尔茨海默病模型。
