SH-SY5Y神经细胞中α7神经型尼古丁受体基因过表达对钙调蛋白表达的影响

发布时间：2018-10-11 14:59

【摘要】：目的构建稳定的α7神经型尼古丁受体(α7 nAChR)过表达的神经母细胞瘤细胞SH-SY5Y,研究α7 nAChR过表达对钙调蛋白(CaM)表达水平的影响。方法设计α7 nAChR引物并扩增α7 nAChR特异性编码的核苷酸序列,退火后克隆至pc DNA 3.1质粒,构建α7 nAChR-pcDNA 3.1重组质粒。将α7 nAChR-pcDNA 3.1重组质粒转染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(Realtime)PCR和蛋白质印迹方法检测转染细胞中α7 nAChR mRNA及蛋白表达水平的变化,蛋白质印迹方法检测CaM表达水平的变化。结果构建的α7 nAChR mRNA过表达质粒成功转入SH-SY5Y细胞后,经G418筛选获得稳定转染细胞克隆株,与对照组相比,α7 nAChR mRNA及蛋白表达量分别增加了533%和110%,CaM表达量增加了43%。结论成功构建了α7 nAChR mRNA过表达的SH-SY5Y细胞株,α7 nAChR表达上调增加了CaM表达水平,这可能与阿尔茨海默病的发病有一定的关系。
[Abstract]:Objective to construct a stable 伪 7 neuronal nicotine receptor (伪 7 nAChR) overexpressed neuroblastoma cell line SH-SY5Y, to study the effect of 伪 7 nAChR overexpression on the expression of calmodulin (CaM). Methods 伪 7 nAChR primers were designed and the nucleotide sequences specifically encoded by 伪 7 nAChR were amplified and cloned into pc DNA 3.1 plasmid after annealing. The recombinant plasmid 伪 7 nAChR-pcDNA 3.1 was constructed. The recombinant plasmid 伪 7 nAChR-pcDNA 3.1 was transfected into SH-SY5Y cells. The positive clones were selected and the expression levels of 伪 7 nAChR mRNA and protein in the transfected cells were detected by real-time fluorescent quantitative (Realtime) PCR and Western blotting. The expression of CaM was detected by Western blot. Results the 伪 7 nAChR mRNA overexpression plasmid was successfully transferred into SH-SY5Y cells, and stable transfected cell lines were obtained by G418 screening. Compared with the control group, the expression of 伪 7 nAChR mRNA and protein increased by 533% and 110%, respectively. Conclusion A SH-SY5Y cell line with overexpression of 伪 7 nAChR mRNA was successfully constructed, and the expression of 伪 7 nAChR increased CaM expression, which may be related to the pathogenesis of Alzheimer's disease.
【作者单位】：贵州医科大学分子生物学重点实验室;贵州医科大学病理学教研室;
【基金】：国家自然科学基金资助项目(81360178) 教育部“长江学者和创新团队发展计划资助”(IRT13058) 贵州省科技厅重大专项(黔科合重大专项字2014〔6008〕);贵州省科技厅项目(201344,黔科合SY字〔2013〕3020)
【分类号】：R749.16

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