高表达人淀粉样前体蛋白降低SH-SY5Y细胞M胆碱受体结合  

Over-expressed human amyloid precursor protein depressed M cholinergic receptor binding in SH-SY5Y cells

作　　者：阮灿军[1] 王抒[1] 孙兰[1] 杜冠华[2] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所药理室,北京100005 [2]中国医学科学院中国协和医科大学药物研究所,北京100050

出　　处：《基础医学与临床》2007年第12期1334-1338,共5页Basic and Clinical Medicine

基　　金：北京市科技计划项目(H020220020220)

摘　　要：目的观察阿尔茨海默病致病相关基因淀粉样前体蛋白（APP）基因的高表达致高度生成，对人神经母细胞瘤SH-SYSY细胞的胆碱乙酰转移酶（CHAT）活性及M和N胆碱受体功能的影响，探讨β-淀粉样肽（Aβ）生成增加对细胞胆碱功能的作用。方法采用Lipofectamine2000试剂盒将携带野生型人APP基因的pCMV695质粒转染至人神经母细胞瘤SH-SYSY细胞中。以ELISA方法测定Aβ生成量。取稳定高表达Aβ细胞的克隆，用放射免疫法测定ChAT的活性；放射配基结合测定M、N乙酰胆碱受体结合。结果与未转染APP的SH-SYSY细胞相比，当Aβ生成量为对照的2—2．6倍时，SH-SYSY-APP细胞中未观察到明确的细胞毒性形态学改变；ChAT活性无明显变化，但细胞M受体结合降低18．5％-21．8％（P〈0．05）；N受体结合未出现明显变化。结论单纯Aβ产生增加就可通过影响M受体结合功能而引起脑内胆碱能功能障碍。Objective To observe the effect of over-expressed amyloid procursor protein （APP） gene on the cholinergic receptor binding and the ChAT activity in human neuroblastoma cell line SH-SYSY. Methods SH-SYSY cells were transfected with pCMV695 plasmid containing wild type human APP695 gene by Lipofectamine 2000 method. The expression of APP was detected by Western blot. Aβ contents were test by ELISA assay in over-expression SH-SYSY cell clones （SH-SYSY-APP）. The special binding of muscarinic and nicotinic cholinergic receptors in those Aβ-overproducing cell clones was determined by radio-ligand binding method. The cholinergic acetyl transferase （CHAT） activity was assayed by radiao-immunoassay. Results No evident morphologic changes of cytotoxicity were detected after transfection. When Aβ production was 2 - 2. 6 times as much as that of normal cells, muscarinic receptor binding was decreased from 18.5 % to 21.9 % （P 〈0. 05 ）. However, no alterations in nicotinic receptor binding and the ChAT activity were found. Conclusion Muscarinic receptor binding inhibition was shown before cytotoxicity, nicotinic receptor suggested that the increase of Aβ production could binding and ChAT activity changes in SH-SY5Y-APP. It is independently cause the cholinergic dysfunction in early stage of AD brain tissues by muscarinic receptor binding inhibition.

关 键 词：阿尔茨海默病 淀粉样前体蛋白 Β-淀粉样肽 胆碱能 受体 

分 类 号：R749.1[医药卫生—神经病学与精神病学] Q516[医药卫生—临床医学]