α3神经型尼古丁受体基因上调对SH-SY5Y细胞突触素水平的影响

Effect of α3 neuronal nicotinic receptor gene up-regulated on the expression of SYP protein in SH-SY5Y cells

目的构建使α3nAChR基因上调的重组质粒α3nAChR-pcDNA3.1并转染至神经母细胞瘤细胞(SH-SY5Y),研究α3nAChR基因上调对细胞突触素(SYP)水平的影响。方法设计α3nAChR上下游引物,通过逆转录PCR的方法获取人α3nAChR特异核苷酸序列,并将其克隆到质粒载体pcDNA3.1上,构建重组质粒α3nAChR-pcDNA3.1;瞬时转染至SH-SY5Y细胞后,用Real-time法和蛋白免疫印迹法分别α3nAChR及蛋白水平,并检测上调细胞中SYPmRNA和蛋白水平的变化。结果成功构建了使α3nAChR基因上调的重组质粒α3nAChRpcDNA3.1;将α3nAChR-pcDNA3.1质粒转染到SH-SY5Y细胞后,与空载质粒组及正常对照组相比,α3nAChRmRNA及蛋白表达水平分别增加了422%和106%(P<0.05);SYPmRNA及蛋白表达水平分别增加了115%和43%(P<0.05)。结论α3nAChR表达的上调可使细胞SYP的表达增加,说明了α3nAChR与SYP密切相关,在AD的发生中可能起着重要作用。

Objective To study the effect of α3 nAChR gene up-regulated on the level of SYP protein in SH-SY5 Y cells. Methods Constructing recombinant plasmid（ α3 n AChR-pc DNA3. 1） by reverse transcription PCR; Detecting α3 n AChR、SYP mRNA and protein levels by real-time PCR and Western blotting respectively after transient transfection. Results Obtaining recombinant plasmid（ α3 n AChR-pc DNA3. 1） with increasing α3 n AChR successfully. As compared with controls,the expression levels of α3 n AChR mRNA and protein in such cells were increased by 422% and 106%,and the mRNA and protein level of SYP were increased by 115% and 43%,respectively. Conclusion Enhancing the level of α3 nAChR in SH-SY5 Y cells can increase the level of SYP protein. The result indicated that synaptic function is closely related to α3 nAChR in cells,suggesting that the α3 n AChR might play a neuroprotective role in connection with the pathogenesis of AD.