RNA干扰抑制α3神经型尼古丁受体的表达对SH-SY5Y神经细胞抗氧化能力的影响

目的 探讨通过RNA干扰技术抑制α3神经型尼古丁受体(nAChR)基因表达后该受体对神经细胞抗氧化能力的影响,来了解α3 nAChR的神经保护作用.方法 设计并体外合成α3 nAChR的特异性编码siRNA序列的寡核苷酸,退火后克隆至pSilencer 3.1-H1 nco质粒中,构建重组质粒α3 nAChR pSilencer 3.1-H1 neo.将α3 nAChR pSilencer 3.1-H1 neo转染SH-SYSY细胞,用含G418的培养液筛选,挑选阳性克隆后采用即时荧光定量PCR和Western blot方法检测转染细胞中α3 nAChR mRNA及蛋白表达水平的变化.用1 μmo1/Lβ淀粉样肽1-42(Aβ1-42)处理转染细胞后,用四甲基偶氮唑盐(MTT)方法测定细胞活力;用比色法测定其脂质过氧化产物含量、超氧化物歧化酶活性、谷胱甘肽过氧化物酶活性.结果 成功构建α3 nAChR siRNA表达质粒,转染后经G418筛选获得稳定转染重组质粒的细胞克隆株,与对照组相比,α3 nAChR mRNA及蛋白表达量均降低(抑制率分别为98%和66%);经转染处理的细胞活力明显低于对照组,细胞中脂质过氧化产物丙二醛含量明显增加,超氧化物歧化酶活性、谷胱甘肽过氧化物酶活性不同程度的降低;抑制α3 nAChR基因表达后能增强Aβ的神经细胞毒性作用.结论 成功构建的α3 nAChR siRNA表达质粒能特异性抑制α3 nAChR基因表达,α3 nAChR基因表达抑制后可引起氧化应激水平升高,并增加Aβ的神经毒性,表明α3 nAChR具有一定的抗氧化能力和神经保护作用.

Influence of inhibited gene expression of α3 nicotinic acetyicholine receptor by RNA Interference on anti-oxidation in SH-SY5Y cells

Objectives To investigate the neuroprotective function of 0.3 nicotinic scetylcholine receptor (nAChR) by inhibiting the gene expression in human neuroblastoma (SH-SY5Y) cells using small interference RNA(siRNA). Methods The siRNA coding oligonucleofide sequences targeting a3 nAChR were designed and synthesized. The annealed product was cloned into pSilencer 3.1-HI neo vector. The recombinant α3 nAChR pSilencer 3.1-H1 neo vector was transfected into the SH-SY5Y cells. The stable clones were screened by G418 medium, and the levels of α3 nAChR mRNA and protein were monitored by using real-time PCR and Western blotting,respectively. After the SH-SY5Y cells with siRNA treatment were exposed to 1 μmol/L Aβ1-42, MTT 3-(4,5-dimethylthiasoi-2-yl)-2,5-diphenyltetrazolium bromide], SOD, GSH-px and the lipid peroxidation were measured by spectrophotometry. Results Compared with the controls, the expression levels of mRNA and protein in the stable SH-SY5Y clone cells transfected with the recombinant 0.3 nAChR pSilencer 3.1-H1 neo vector were decreased with inhibitory efficiency of 98% and 66%, respectively, the MTr reduction decreased; the product of lipid peroxidation was increased and the activities of SOD and GSH-px were decreased. Biologically, the gane expression inhibition of α3 nAChR enhanced the toxicity induced by Aβ in SH-SY5Y cells.Conclusions The expression inhibition ofα3 nAChR as a result of recombinant α3 nAChR siRNA can induce oxidative stress and improve the toxicity of Aβ on SH-SY5Y cells,indicating that α3 nAChR may play a sinaificant neuroprotective role in the pathogenesis of Alzheimer disease.