突变型APP基因转染对神经细胞尼古丁受体的影响

目的 探讨阿尔茨海默病(AD)发病机制中的重要物质淀粉样蛋白前体蛋白(APP)高表达对神经型尼古丁受体(nAChR)的影响.方法 将含有瑞典家族性AD双突变的APP670/671基因(APP_(SWE))转入神经母细胞瘤(SH-SY5Y)细胞和原代培养的大鼠神经细胞,得到体外构建的高表达APP_(SWE)细胞模型;用即时荧光定量PCR和Western blot方法分别测定APP mRNA、可溶性APP和总APP蛋白表达水平以及nAChR mRNA和蛋白表达水平;用放射性配体~3H]epibatidine进行nAChR-配体结合试验.结果 转染APP_(SWE)质粒的SH-SY5Y细胞和原代培养的神经细胞中APP mRNA和总APP蛋白表达水平显著升高,在SH-SY5Y细胞的培养液中检测到αAPP分泌量下降;SH-SY5Y细胞转染组α7 nAChR蛋白和mRNA表达水平显著升高,而α3 nAChR蛋白和mRNA表达水平明显下降,并且~3H]epibatidine配体结合位点明显减少;原代培养的神经细胞转染组α4 nAChR蛋白水平明显下降,但其mRNA水平无明显变化.结论 APP蛋白高表达使体外培养神经细胞中nAChR发生改变,其中α7 nAChR表达水平升高,而含α3和α4亚单位的nAChR表达水平降低,表明APP代谢紊乱可对nAChR产生一定的影响,在AD的发病机制中有重要作用.

Influence of APP_(SWE) transfection on nicotinic receptors in cultured neuronal cells

Objective To investigate the influence of APP_(SWF) on the expression of neuronal acetylcholine receptors ( nAChRs) and its relationship with Alzheimer's disease ( AD). Methods APP_(SWE) , carried the Swedish family AD double mutants, were transfected into SH-SY5Y cells and primary cultured neurons from rat brains to build a cellular model of AD. The mRNA levels of APP and nAChRs, and the protein levels of total APP, aAPPs and nAChRs in the cultured cells were measured using real-time PCR and Western blot, respectively. The numbers of α3 nAChR were determined by receptor-~3H] epibatidine binding assay. Results Increased expressions of Swedish 670/671 APP at mRNA and protein levels, and down-regulation of αAPPs were observed in both of the cultured neuronal cells transfected with APP_(SWE). A significant increase of α7 nAChR expression at protein and mRNA levels was detected in the APP_(SWE) transfected SH-SY5Y cells. On the other hand, after transfection with APP_(SWE), the expressions of α3 nAChR at protein and mRNA levels in SH-SY5Y cells, and α4 nAChR at mRNA level in primary cultured neurons were inhibited. In addition, the numbers of receptor bining sites were deceased in SH-SY5Y cells overexpressing with APP_(SWE). Conclusion Overexpression of APP_(SWE) can decrease αAPPs and modify nAChRs by increasing expression of Α7 nAChR and decreasing α3 and α4 nAChRs, which might play an important role in the pathogenesis of AD.