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小鼠睾丸生殖细胞瘤易感基因Dnd1编码的蛋白是一个在进化中保守的RNA结合蛋白.为探讨小鼠Dnd1的蛋白亚细胞定位和对细胞增殖的影响及其机制,利用生物信息学技术,采用组合的亚细胞定位分析软件对Dnd1进行真核生物亚细胞定位预测;利用融合绿色荧光蛋白(greenfluorescent protein,GFP)定位的方法,通过构建pEGFP-Dnd1重组质粒,将重组质粒pEGFP-Dnd1转染HeLa细胞和GC-1细胞,在荧光显微镜下观察Dnd1的蛋白亚细胞定位;用MTT法和流式细胞技术测定Dnd1过表达对HeLa细胞的增殖能力的影响和细胞周期的改变;在HeLa细胞系中检测Dnd1对AP-1转录活性的影响.结果表明:①生物信息学预测Dnd1主要在细胞核表达,在细胞质中也有少量表达;荧光显微镜下观察发现,Dnd1蛋白主要定位在细胞核,在细胞质中也有少量分布;②Dnd1基因在HeLa细胞系中的过表达抑制细胞增殖和诱导细胞周期G1期阻滞;③Dnd1抑制AP-1的转录活性,从而抑制AP-1介导的转录是Dnd1抑制细胞增殖的可能机制.本研究初步明确了Dnd1的蛋白亚细胞定位及其对HeLa细胞的生长抑制作用,这为进一步研究Dnd1基因的功能建立基础.
Mouse testicular germ cell tumor susceptibility gene Dnd1 encoded protein is a evolutionarily conserved RNA binding protein.To explore the mouse Dnd1 protein subcellular localization and cell proliferation and its mechanism, the use of bioinformatics technology, The combined subcellular localization analysis software was used to predict the subcellular localization of Dnd1 in eukaryotes. The recombinant plasmid pEGFP-Dnd1 was transfected by the fusion of green fluorescent protein (GFP) and the construction of pEGFP-Dnd1 recombinant plasmid HeLa cells and GC-1 cells were observed under fluorescent microscope Dnd1 protein subcellular localization; using MTT assay and flow cytometry Dnd1 overexpression HeLa cell proliferation and cell cycle changes in the HeLa cell line The results showed that: (1) It was predicted by bioinformatics that Dnd1 was mainly expressed in the nucleus and also in the cytoplasm. The results of fluorescence microscopy showed that Dnd1 mainly located in the nucleus and also in the cytoplasm A small amount of distribution; ②Dnd1 gene over-expression in HeLa cell line inhibited cell proliferation and induced cell cycle G1 arrest; ③Dnd1 The transcriptional activity of AP-1 and thus the inhibition of AP-1-mediated transcription are the possible mechanisms by which Dnd1 inhibits cell proliferation.In this study, we initially identified the protein subcellular localization of Dnd1 and its inhibitory effect on HeLa cell proliferation, To study the function of Dnd1 gene to establish the foundation.