Parkin基因过表达质粒和细胞模型的构建

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(P0.01)。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。

Construction of Parkin Overexpression Plasmid and the Cell Model

ABSTRACT 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 trans- fected 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). Conclusion: Using lipo- some transduction technology, the Parkin overexpression plasmid was successfully transfected into SH-SY5Y cells by lipo3000, the mR- NA and protein expression of Parkin was significantly increased. A cell model of Parkin gene overexpression was successfully estab- lished.