SH-SY5Y神经细胞APP高表达对胆碱酯酶活性影响

目的研究类阿尔茨海默病(AD)神经细胞模型中APP异常代谢下胆碱酯酶活性变化,探讨AD的发病机制。方法采用脂质体转染法将含有瑞典家族性AD双突变的淀粉样蛋白前体蛋白APP670/671基因(APPSWE)转染至神经母细胞瘤细胞(SH-SY5Y)中;利用实时荧光定量PCR方法和Western blotting的方法测定APPmRNA、可溶性APP(αAPPs)和总APP蛋白表达水平;采用改进的Ellman比色法测定细胞培养基中乙酰胆碱酯酶(Acetylcholinesterase,AChE)及丁酰胆碱酯酶(Butyrylcholinesterase,BuChE)活性。结果转染APPSWE质粒的SH-SY5Y细胞APP总mRNA和蛋白表达水平显著升高,αAPPs分泌量下降。转染组SH-SY5Y细胞与对照组相比,AChE和BuChE活性都升高。结论体外神经细胞实验证明APP蛋白过度表达能升高AChE和BuChE的活性,其机制可能与APP蛋白代谢紊乱使Aβ升高及下调αAPPs分泌有关。

Influence of APP_(SWE) overexpression on activity of cholinesterase in SH-SY5Y cells

Objective To investigate the influence of APP_(SWE) overexpression on activity of cholinesterase in the neuroblastoma SH-SY5Y cells and reveal the pathogenesis of Alzheimer's disease.Methods In the study,the plasmid carried the Swedish 670/671 APP double mutant gene(APP_(SWE))was transfected into SH-SY5Y cells to build cellular model of AD.The levels of total APP mRNA and protein,and αAPPs in the cell models were detected by Real-time PCR and Western blotting,respectively.The acetylcholinesterase(AChE)and butyrylcholinesterase(BuChE)activities in the cells were measured by improved Ellman's colorimetric method.Results The expressions of APP mRNA and protein were significantly increased in SH-SY5Y cells transfected with APP_(SWE).The down-regulation of αAPPs expression was detected in the condition medium of the cultured cells treated with APP_(SWE).On the other hand,elevation in the activities of AChE and BuChE were observed in the SH-SY5Y cells with APP_(SWE).Conclusion These results indicated that the overexpression of APP_(SWE) can up-regulate the activity of AChE and BuChE,which might relate with the down-regulation of αAPPs.