SH-SY5Y神经细胞中尼古丁受体α3亚单位基因表达抑制与APP代谢的关系

Correlation between inhibited gene expression of nicotinic acetylcholine receptor α3 subunit in SH-SY5Y cells and APP metabolism

目的用RNA干扰技术抑制神经型尼古丁受体(nAChR)α3亚单位基因表达并观察对神经细胞淀粉样蛋白前体蛋白(APP)代谢的影响。方法设计并体外合成α3nAChR的特异性编码siRNA序列的寡核苷酸,退火后克隆至pSliencer3.1-H1neo质粒中,构建重组质粒α3nAChR pSilencer3.1-H1neo。将α3nAChR pSilencer3.1-H1neo转染SH-SY5Y细胞,用含G418的培养液筛选,挑选阳性克隆后采用实时荧光定量PCR和蛋白质印迹方法检测转染细胞中α3nAChR mRNA及蛋白表达水平的变化;并测定分泌型APP、总APP蛋白表达的变化。结果成功构建α3nAChR 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,α3nAChR mRNA及蛋白表达量均降低(抑制率分别为98%和66%);分泌型APP表达下降,总APP水平比较表达水平无显著性差异。结论α3nAChR siRNA表达质粒能特异性抑制α3nAChR基因表达,α3nAChR可能通过增加α-分泌酶对APP的切割发挥神经保护作用。

Objective To investigate the influence of inhibited gene expression of α3 nicotinic acetylcholine receptor （nAChR） induced by RNA interference on the metabolism of （-amyloid precursor peptide （APP）. Methods The siRNA coding oligonucleotide sequences targeting α3 nAChR were designed and synthesized. The annealed product was cloned into pSliencer 3. 1 -HI neo vector. The recombinant α3 nAChR pSilencer 3. 1-H1 neo vector was transfected into the SH-SYSY 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. The levels of the （ -form of secreted APP （ （APPs） and total APP were also determined by Western blotting. Results Compared with control, the expression levels of mRNA and protein in the SH-SYSY cells transfected with the recombinant α3 nAChR pSilencer 3. 1-HI neo vector were decreased by the inhibitory efficiency with 98% and 66% respectively. The protein level of the αAPPs was reduced and no significant change of total APP was observed. Conclusions The inhibited gene expression of α3 nAChR resulted from the recombinant α3 nAChR siRNA can decrease cleavage of APP by （-secretase, which suggest that α3 nACbR may play a significant neuroprotective role in the pathogenesis of Alzheimer＇ s disease.