【摘要】 目的研究核磷蛋白A型突变体基因转染对人髓性白血病细胞系体外增殖的影响。方法构建含核磷蛋白A型突变基因(NPM-mA)的真核表达质粒pEGFPC1-NPM-mA,选择NPM-mA阴性、抑癌基因p14ARF有无缺失的两株人髓性白血病细胞KG-1a和K562作为靶细胞,将pEGFPC1-NPM-mA重组质粒和pEGFPC1空质粒分别转染白血病细胞,以未转染组为对照。用RT-PCR和免疫组化来分析转染细胞目的基因mRNA和蛋白质的表达。采用台盼蓝拒染法计数活细胞,绘制转染前后生长曲线,观察细胞体外生长的改变。结果转染后的两种白血病细胞株表达NPM-mA mRNA,胞浆内NPM-mA蛋白阳性,符合NPM突变蛋白胞质移位的特点。细胞生长曲线显示,转染NPM-mA质粒的KG-1a细胞较空质粒转染和未转染组细胞生长速度明显加快,而转染NPM-mA质粒的K562细胞则较对照组细胞生长明显受抑制(P<0.05)。结论NPM-mA基因稳定转染可影响白血病细胞的增殖活性,在无ARF缺失时NPM-mA突变体可促进白血病细胞体外生长。更多还原

【Abstract】 Objective To investigate the effect of type A mutant of nucleophosmin(NPM-mA)on the proliferation of human myelocytic leukemia cell lines in vitro.Methods The eukaryotic expression plasmid pEGFPC1-NPM-mA containing NPM-mA gene was constructed.Two NPM-mA-negtive leukemic cell lines(KG-1a and K562)were chosen as research objects,which expressed anti-oncogene p14 alternative reading frame(ARF)or not respectively.After stably transfection of the two cells with pEGFPC1-NPM-mA and empty plasmid pEGFPC1,RT-PCR and immunohistochemistry were used to detect the expression of mRNA and protein of NPM-mA respectively.The untransfected group was served as control.The proliferation effect was assessed by viable cell count(trypan blue)and cell growth curve.Results The expression of NPM-mA mRNA was detectable in transfected cells.NPM-mA protein was located in cytoplasm.The growth curve showed that after transfection of pEGFPC1-NPM-mA,KG-1a cells displayed high growth rate compared with empty plasmid transfected or untransfected group,however the growth of pEGFPC1-NPM-mA-transfected K562 cells were significantly suppressed compared with control groups(P<0.05).Conclusion The expression of NPM-mA may have effect on the proliferation of leukemia cells,and even if absence of ARF NPM-mA can promote the growth of leukemic cells in vitro.更多还原