摘要目的 探讨α7神经型尼古丁受体(nAChR)表达改变与淀粉样蛋白前体蛋白(APP)代谢、细胞存活率及脂质过氧化水平的关系,以了解α7 nAChR的神经保护作用,以及该受体水平与阿尔茨海默病发病的关系.方法 设计并合成α7 nAChR基因特异性小分子干扰RNA(siRNA),转染SH-SY5Y细胞;用20μmoL/L DMXB处理细胞;培养48 h后收集细胞,分别应用逆转录聚合酶链反应(RT-PCR)和Western blot方法检测转染细胞及DMXB处理后的细胞αnAChR mRNA及蛋白表达水平的变化;并用1μmol/L β-淀粉样肽25-35(Aβ25-35)处理细胞,测定分泌型APP、总APP蛋白表达的变化;比色法测定脂质过氧化产物含量;MTT方法测定细胞活力.结果 转染siRNA后,与对照组相比,α7 nAChR mRNA及蛋白表达量降低(抑制率分别为80%和69%)、脂质过氧化产物丙二醛含量明显增加、分泌型APP表达下降、细胞活力明显低于对照组,且能增强Aβ的细胞毒性作用;用DMXB处理细胞后能使α7nAChR的表达增加23%、增加分泌型APP的表达、并能对抗A13引起的细胞活力下降及脂质过氧化水平的增强.结论 α7nAChR可能通过增强α-分泌酶对APP的切割、增强细胞抗氧化能力及对抗AB的神经毒性作用来发挥神经保护作用,其表达减少与阿尔茨海默病的发病机制有密切的关系.

abstractsObjectives To investigate the neuroprotective function of α7 nicotinic receptor (nAChR)and its roles in the pathogenesis of Alzheimer's disease(AD).Method Specific RNA interference to α7 nAChR mRNA expression Was performed by gene specific small interference RNA(siRNA).SH-SY5Y cells were transfected with the siRNA or treated with 20 μmoL/L 3-[2,4-dimethoxybenzylidene] anabaseine(DMXB),an α7 nAChR agonist.After 48 hrs culture,levels of α7nAChR mRNA and protein were monitored by RT-PCR and Western blotting,respectively.In the second experiment,SH-SY5Y cells treated with siRNA or DMXB were exposed to 1μmoL/L Aβ25-35,followed by protein analysis of α-form of secreted β-amyloid precursor peptide(αAPPs),and total APP was assayed by Western blotting.In addition,lipid peroxidation and MTT[3-(4,5-dimethyhhiazol-2-y1)-2,5-diphenyhetrazolium bromide]reduction were measured by spectrophotometry.Result In RNA interference group.as compared with controls,α7 nAChR mRNA and protein levels were decreased with inhibitory efficiency by 80%and 69%.respectively,along with a decrease in protein levels of αAPP and reduction of MTT.However the product of lipid peroxidation was increased.There was an enhanced gene inhibition of α7nAChR by Aβ.While cells treated with DMXB.the α7 nAChR protein was increased by 23%as compared with that of the control.along with decrease of αAPP and ERK 1/2 at the protein level.The enhanced expression of α7 nAChR reduced the neurotoxic effects resulted from Aβ.Conclusion The findings indicate that α7 nAChR may play a significant neuroprotective role by enhancing cleavage of APP,improving antioxidant defenses and limiting the toxicity of Aβ.which has been implied in the pathogenesis of AD.