表达APPswe_(695)基因细胞系的构建及Aβ分泌水平的分析

Preparation of the Cells Transfected with Human APPswe_(695) Gene and the Determination of Aβ Production

为探究β-淀粉样前体蛋白(amyloid-βprecursor protein, APP)在阿尔茨海默病(Alzheimer’s disease, AD)发病过程中的作用及构建应用于AD发病机理研究的实验细胞模型,该研究构建了过表达APP695瑞典型突变体(APPswe_(695))的SH-SY5Y细胞系(APPswe_(695)细胞)并分析了该细胞系中Aβ的分泌水平。采用慢病毒介导转染方法,将APPswe_(695)表达质粒转染至SH-SY5Y细胞,抗性药物筛选阳性转染细胞。分别采用RT-PCR、Western blot技术验证APPswe_(695) mRNA、APP蛋白的表达, ELISA分析Aβ1-40和Aβ1-42的分泌水平。结果显示,转染慢病毒包装的APPswe_(695)质粒后,细胞的APPswe_(695)mRNA表达呈现阳性;与野生型细胞和转染质粒空白细胞相比,转染APPswe_(695)基因细胞表达APP695的瑞典型突变蛋白(APPswe_(695))。APPswe_(695)具有与内源性APP770相同的细胞分布。转染APPswe_(695)基因后,细胞内分泌Aβ水平增加(P<0.05)而细胞外液中Aβ含量并没有显著变化。由此说明, APPswe_(695)细胞能够表达被转染的APPswe_(695)基因, APPswe_(695)细胞倾向于产生更多的胞内而不是胞外Aβ,APPswe_(695)细胞可应用于阿尔茨海默病发病机理及药物治疗的研究。

Alzheimer’s disease(AD) is a neurodegenerative disorder,and amyloid-β(Aβ) precursor protein(APP) is related to AD development.In this study,we developed a cell line that stably expressed APP gene with Swedish mutation(APPswe695 cells) and the production of amyloid-β was investigated.The expression plasmid with expressing APPswe695 gene was transfected into SH-SY5 Y cells mediated by lentiviral vector.The gene expression of APPswe695 was confirmed at the levels of transcription and translation.The location of APP was analyzed by the method of immunocytochemistry.Aβ peptide,a product derived from APP by β-cleavage,was detected by the method of enzyme-linked immunosorbent assay(ELISA).The results indicated that APPswe695 cells could transcribe the mRNA and translate the protein of the transfected APPswe695 gene.The Swedish mutant protein of APP695(APPswe695) had the same cellular distribution as endogenous APP770.Interestingly,APPswe cells are in-clined to generate more intracellular but not extracellular Aβ.In general,these results indicated that APPswe695 cells can express the transfected APPswe695 gene and that APPswe695 is prone to generate more intracellular but not extracellular Aβ.This study implied that the APPswe695 cells can be used to investigate the pathogenesis and drug therapy of Alzheimer’s disease.