【摘要】 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.更多还原

【Abstract】 Dnd1 is an evolutionary conserved RNA binding protein（RBP） known as a mouse testicular germ cell tumor（TGCT） susceptible protein.To understand its subcellular localization in mammalian cells in connection with cell proliferation and cell cycle,we transfected the recombinant plasmid pEGFP-Dnd1 into HeLa or GC-1 cell lines and detected its subcellular localization by fluorescent microscopy.The results were compared with the predicted subcellular localization of Dnd1 using several softwares,such as Target P,Subloc V,and WOLFPSORT.The cell growth curve and cell cycle analysis of Dnd1-transfected HeLa cells were determined by MTT assay and flow cytometry,and the effect of Dnd1 on AP-1 transcription activity was also assayed.We found that EGFP-Dnd1 mainly located in the nuclear and little in the cytoplasma in both HeLa and GC-1 cells,which is consistent with the bioinformatic predictions.Dnd1 inhibited cell proliferation and led to cell cycle arrest at G₁/S phase in HeLa cells,with down-regulated transcription activity of AP-1.Our work may have important implication for further studies of Dnd1 function.更多还原