人淀粉样蛋白前体695基因及人烟碱乙酰胆碱受体α4β2亚单位基因共表达细胞模型的构建

目的为满足寻找可能影响淀粉样β蛋白(Aβ)在阿尔茨海默病过程中的药物实验的需求,建立共表达人淀粉样蛋白前体(APP)695基因和烟碱乙酰胆碱受体(nAChR)α4β2亚单位基因的细胞模型。方法用脂质体转染方法把APP695基因转染进已稳定转染nAChRα4β2亚单位基因的SH-EP1细胞。用500mg.L-1G418加压筛选,并挑选细胞单克隆。用RT-PCR和Western蛋白印迹法对转染后的细胞单克隆进行鉴定,挑取成功转染并高表达APP695的细胞进行克隆。膜片钳检测所挑细胞克隆的α4β2受体功能。结果挑出成功稳定转染人APP695基因及人nAChRα4β2亚单位基因的共表达细胞克隆株。膜片钳方法检测到此细胞克隆株上nAChRα4β2存在活性。结论成功制备了共表达人APP695基因及人nAChRα4β2亚单位基因的细胞模型,为探讨神经元nAChRα4β2亚型对Aβ加工影响的药物实验提供了条件。

Construction of coexpression cell model of human amyloid protein recursor 695 gene and human nicotinic acetylcholinergicα4β2 subunit genes

AIM For the need of cell model in drugs research that may have effect on amyloid β-protein in Alzheimer′s disease processing, the cells coexpressed with human amyloid protein precursor (APP) 695 gene and human nicotinic acetylcholine receptor(nAChR) α4β2 subunit genes were constructed. METHODS Liposome was used to transfect human APP 695 gene into SH-EP1 cells which stably expressed nAChR α4β2 genes. Neomycin (500 mg·L^-1) was used to ensure stable expression of APP 695, and limiting dilution assay was used to obtain single transfected cell clones. RT-PCR and Western blot were used to verify the clones. The highly expressed cell clones were selected, and the activity of nAChR α4β2 was justified by patch clamp. RESULTS Cell clone with stable coexpression of APP 695 gene and nAChR α4β2 genes was constructed successfully, and the activity of α4β2 receptors in the cell clone was confirmed by patch clamp. CONCLUSION Coexpression cell model of human APP 695 and nAChR α4β2 genes was constructed success fully, which may provide good method for pharmaceutical experiments on effects of nAChR α4β2 on amyloid processing.