中文摘要：

目的构建携带人alpha-突触核蛋白真核重组质粒,检测其在人神经母细胞瘤SK-N-SH细胞中的表达及对细胞存活率的影响。方法 PCR扩增alpha-突触核蛋白基因,产物纯化后与pcDNA3.1（＋）载体进行连接反应,构建pcDNA3.1（＋）/alpha-突触核蛋白真核重组质粒。Lipofectamine法转染人神经母细胞瘤SK-N-SH细胞,RT-PCR方法检测SK-N-SH细胞中alpha-突触核蛋白mRNA的表达,四甲基偶氮唑盐（MTT）法检测其对细胞存活率的影响。结果酶切鉴定及基因测序证明人alpha-突触核蛋白基因已被完整、正确地插入到pcDNA3.1（＋）载体中。RT-PCR结果显示该表达载体转染SK-N-SH细胞后,细胞内alpha-突触核蛋白mRNA的表达水平明显增高,MTT方法检测结果显示该表达载体转染并未引起细胞存活率的改变。结论 alpha-突触核蛋白真核重组质粒构建成功,并可在SK-N-SH细胞内表达,且对细胞的存活率不产生任何影响,从而为进一步研究alpha-突触核蛋白在帕金森病发病机制中的作用奠定了基础。

英文摘要：

Objective To construct the eukaryotic expression plasmid of human alpha-synuclein gene and detect its expression in human SK-N-SH neuroblastoma cells and its effect on cell viability.Methods PCR was performed to amplify the entire fragment of human alpha-synuclein gene,and the amplified gene was inserted into the eukaryotic expression vector pcDNA3.1（＋）to form the new construct donated pcDNA3.1（＋）/alpha-synuclein after purification.Liposome-mediated gene transfection method was used to transfect the human SK-N-SH cells.RT-PCR was used to detect the transient expression of alpha-synuclein gene.MTT assay was used to detect the changes in cell viability.Results Recombinant contained the correct and entire nucleotide sequence of human alpha-synuclein gene was found to be successfully constructed via restriction enzyme digestion and sequencing methods.Levels of alpha-synuclein mRNA were obviously increased in SK-N-SH cells with reconstructive plasmid transfection.However,no significant changes in cell viability were observed in these transfected cells.Conclusion The eukaryotic expression plasmid of human alpha-synuclein gene is successfully constructed and expressed in SK-N-SH cells.The cell viability is not affected.All these results establish a foundation for a further study of the role of alpha-synuclein in the pathogenesy of Parkinson disease.