SH-SY5Y神经细胞中α7神经型尼古丁受体基因沉默对CaM、CaMKⅡ蛋白水平的影响  

Influence of α7 neural nicotinic receptor gene silencing on the protein levels of CaM and CaMK Ⅱ in SH-SY5Y cells

作　　者：张淑丽[1] 官志忠[1,2] 肖雁[1] 吴昌学[1] 齐晓岚[1] 

机构地区：[1]贵州医科大学分子生物学重点实验室,贵州贵阳550004 [2]贵州医科大学病理学教研室,贵州贵阳550004

出　　处：《中风与神经疾病杂志》2016年第3期207-210,共4页Journal of Apoplexy and Nervous Diseases

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)

摘　　要：目的构建稳定的α7 n AChR沉默的神经母细胞瘤细胞(SH-SY5Y)细胞,研究α7神经型尼古丁受体(n AChR)基因沉默对钙调蛋白(Ca M)、钙调素依赖性蛋白激酶Ⅱ(Ca MKⅡ)水平的影响,了解α7 n AChR神经保护作用及其与阿尔茨海默病(AD)发病机制的关系。方法将α7 n AChR shRNA重组质转染到SH-SY5Y,用含嘌呤霉素的培养液筛选,挑选阳性克隆后采用实时荧光定量PCR和蛋白质印迹方法(Western-blot)检测细胞中α7n AChR mRNA及蛋白表达水平的变化;Western-blot方法测定Ca M、Ca MKⅡ蛋白表达水平。结果获得稳定转染α7 n AChR shRNA重组质粒的细胞克隆株,与对照组相比,α7 n AChR mRNA及蛋白表达量分别减少了95%和80%。Ca M、Ca MKⅡ蛋白表达量分别减少了48.5%和35%。结论成功构建了α7 n AChR mRNA沉默的SH-SY5Y细胞细胞株,α7 n AChR沉默降低了Ca M、Ca MKⅡ的蛋白水平,可能影响信号通路转导,这可能与阿尔茨海默病(AD)的发病有一定的关系。Objective Investigating that the influence of inhibited gene expression of α7 nicotinic acetylcholine receptor（ n AChR） induced by RNA interference on the protein levels of calmodulin（ Ca M） and calmodulin dependent protein kinase Ⅱ（ Ca MK Ⅱ）,and to understand the neuroprotective mechanism of α7 n AChR and its function in the pathogenesis of Alzheimer＇s disease（ AD）. Method The recombinant α7 n AChR shRNA were transfected into SH-SY5 Ycells,the stable clones were screened with puromycin,and used Real-time PCR and Western blot monitoring the levels of α7 n AChR mRNA and protein,respectively. The protein levels of the Ca M and Ca MK Ⅱ were also determined by Western blot. Results We got the cell clone strains with stable transfection of α7 n AChR shRNA recombinant plasmid,and as compared with controls,the expression levels of α7 n AChR mRNA and protein in such cells were decreased by the inhibitory efficiency with 95% and 80%,respectively. The protein levels of Ca M and Ca MK Ⅱ were decreased by 48. 5% and 35% respectively. Conclusion The results indicated that the inhibited gene expression of α7 n AChR could decrease the level of Ca M,Ca MK Ⅱ,which may affect the signal transduction pathway,suggesting that α7 n AChR may play a crucial neuroprotective role in Alzheimer disease＇s pathogenesis.

关 键 词：RNA干扰 α7神经型尼古丁受体 CAM CaMKⅡ SH-SY5Y细胞 

分 类 号：Q51[生物学—生物化学] R749.1[医药卫生—神经病学与精神病学]