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

Influence of inhibited gene expression of α3 nicotinic acetylcholine receptor by RNA interference on anli-oxidalion in SH-SYSY cells

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 neo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-SY5Y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1μmol／L β-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。

Objectives To investigate the neuroprotective function of α3 nicotinic acetylcholine receptor （nAChR） by inhibiting the gene expression in human neuroblastoma （SH-SY5Y） cells using small interference RNA（siRNA）. Methods The siRNA coding oligonucleotide sequences targeting α3 nAChR were designed and synthesized. The annealed product was cloned into pSilencer 3.1-H1 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-dimethyhhiazol-2-yl） -2,5-diphenyhetrazolium 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 α3 nAChR pSilencer 3.1-H1 neo vector were decreased with inhibitory efficiency of 98% and 66% , respectively, the MTT reduction decreased; the product of lipid peroxidation was increased and the activities of SOD and GSH-px were decreased. Biologically, the gene expression inhibition of α3 nAChR enhanced the toxicity induced by Aβ in SH-SY5Y cells. Conclusions The expression inhibition of α3 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 significant neuroprotective role in the pathogenesis of Alzheimer disease.