Atrophin-1全长基因稳定转染和瞬时转染真核细胞系的表达分析

目的建立正常(CAG重复次数为19次)和异常(CAG重复次数为82和66次)atrophin-1[齿状核红核苍白球路易体萎缩(DRPLA)致病基因]全长基因真核表达体系,通过观察细胞形态及蛋白质表达水平和分布情况,比较稳定转染和瞬时转染两种细胞表达体系,为进一步研究建立良好的细胞模型。方法基于已构建的GFP-atrophin-1-Q19/Flp-In TREx293和GFP-atrophin-1-082/Flp-In TREx293两种稳定转染细胞株,利用四环素调控系统Tet-on诱导表达。正置荧光显微镜观察正常与异常atrophin-1融合蛋白在细胞中的表达定位,电子显微镜观察细胞超微结构的改变;脂质体介导法将重组真核表达质粒GFP-atrophin-1-Q19和GFP-atrophin-1-Q66瞬时转染293T和SH-SY5Y细胞,HE染色、倒置荧光显微镜和电子显微镜分别观察细胞形态结构、蛋白质表达定位和细胞超微结构,western blotting检测atrophin-1融合蛋白表达水平。结果在稳定转染体系中,绿色荧光信号大多位于细胞核内,异常蛋白质呈不均匀分布且有少数聚集现象;电子显微镜观察异常细胞胞核结构欠完整,核膜皱缩,核内结构紊乱。在瞬时转染293T细胞体系中,HE染色可见异常细胞核内形成粉紫色嗜酸性小体;大量atrophin-1融合蛋白在核内呈点状聚集现象;电子显微镜观察异常细胞核膜皱缩严重,核内大量电子致密物沉积且核仁消失;western blotting检测均表达atrophin-1融合蛋白。在转染的SH-SY5Y细胞体系中,蛋白质表达定位及western blotttng检测结果与转染293T细胞基本一致。结论包含异常扩增多聚谷氨酰胺链的atrophin-1融合蛋白在真核表达体系中可产生明显的核内聚集现象,并引起细胞形态改变,此为研究DRPLA核内包涵体的形成及其在发病中的作用,以及进一步的干预治疗奠定了理论基础。

Analysis of the expression of normal and mutant full-length atrophin-1 gene in stable and transient transfected eukaryotic cell line

Objective To establish eukaryotic expression system of full-length gene in normal [CAG repeats(Q19)]and abnormal[CAG repeats(Q82 and Q66)]atrophin - 1[the pathogenic gene of dentatorubral-pallidoluysian atrophy(DRPLA)].To compare the expression system of stable and transient transfected cell line by observing the cellular appearance and atrophin-1 fusion protein expression and its distribution for further research on the establishment of a fine cell model.Methods Based on the 2 established stable transfected green-fluoresent protein(GFP)-atrophin-1 - Q 19/Flp- In TREx293 and GFP-atrophin - 1 - Q82/Flp - In TREx293 cell lines,Tet - on system was used to induce the expression,which contained normal and abnormal atrophin - 1 full - length gene and GFP fusion proteins,respectively. Meanwhile.GFP - atrophin - 1 - Q19 and GFP - atrophin - 1 - Q66 recombinant plasmids were transiently transfected into 293T cells and SH-SY5Y cells respectively with Lipofectamine 2000.The expression and localization of normal and abnormal atrophin - 1 fusion proteins in the cells were detected by light microscope with HE staining and fluorescence microscope;the ultrastructure of the cells were observed by electron microscope;the expression of GFP - atrophin - 1 fusion proteins were detected by Western blotting.Results In stable transfected system,the green fluorescence signals were detected in the nucleus,and abnormal proteins were not uniformly distributed while some were aggregated.Electron microscope showed shrinkaged nuclear membrane and abnormal intranuclear structure in abnormal cells.In transient transfection of 293T,Q66 protein aggregated in the nucleus which was detected as acidophilic bodies by HE staining-light microscope and atrophin-1 fusion protein as punctuate pattern by fluorescence microscope.Under the observation of electron microscope,great quantity of electron - dense substance accumulation,distinct shrinkage nuclear membrane and nucleus disappearance were seen in abnormal cells.The expression of GFP - atrophin - 1 fusion proteins were detected by Western blotting.The location of protein expression and Western blotting results in transient transfected SH-SY5Y cells system were essentially similar to those in transfected 293T cells.Conclusion In eukaryotic cell expression system,atrophin - 1 with amplified polyglutamine chain may induce obvious aberrant aggregation in nucleus,and alteration of cellular appearance.The establishment of atrophin-1 expression eukaryotic cell model will provide the foundation for the research of pathogenesis of DRPLA,and therotic basis for further investigation of clincial intervention.