Parkin基因过表达质粒和细胞模型的构建

Construction of Parkin Overexpression Plasmid and the Cell Model

目的:构建Parkin基因过表达质粒并转染SH-SY5Y细胞,为进一步研究帕金森病的发病机制及中药的作用环节奠定基础。方法:首先构建Parkin基因过表达质粒,采用脂质体转导技术,将Parkin过表达质粒应用Lipo3000转染SH-SY5Y细胞。荧光显微镜观察细胞绿色荧光蛋白的表达;RT-PCR检测其Parkin mRNA的表达;Western Blot技术检测其Parkin蛋白的表达。结果:转染组可观测到较多的绿色荧光蛋白表达;Parkin过表达细胞Parkin mRNA和蛋白的表达均显著提高(P<0.01)。结论:通过脂质体转导技术,应用Lipo3000可将Parkin过表达质粒成功转染入SH-SY5Y细胞,转染的基因和蛋白表达均较高,提示此法可成功构建Parkin基因过表达的细胞模型。

Objective： Construct Parkin overexpression plasmid and transfect into human neuroblastoma SH-SY5Y cells, with the aim of laying a foundation for studying the pathogenesis of Parkinson＇s disease and the role of traditional Chinese medicine. Methods：Firstly, construct Parkin overexpression plasmid. Using liposome transduction technology, the Parkin overexpression plasmid was transfected into SH-SY5Y cells by lipo3000. The green fluorescent protein was observed by fluorescence microscopy; RT-PCR technique was used to detect the expression of Parkin at mRNA level; Western-blot technique was used to detect the expression of Parkin at protein level.Results： More expression of enhanced green fluorescent protein（EGFP） was observed in the transfected group; the mRNA and protein expression of Parkin was significantly increased after transfection with Parkin overexpression plasmid（P〈0.01）. Conclusions： Using liposome transduction technology, the Parkin overexpression plasmid was successfully transfected into SH-SY5Y cells by lipo3000, the mRNA and protein expression of Parkin was significantly increased. A cell model of Parkin gene overexpression was successfully established.