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目的构建稳定表达人核仁磷酸蛋白1(Nucleophosmin 1,NPM1)A型突变蛋白(NPM1-mA)的白血病髓性细胞系,并进行鉴定。方法利用xfectTM转染试剂将含人NPM1-mA基因的重组质粒pEGFP-C1-NPM1-mA分别转染THP-1和K562细胞系,G418筛选阳性克隆,建立稳定表达NPM1-mA的白血病细胞株THP-1-mA和K562-mA。采用RT-PCR和Western blot检测细胞NPM1-mA mRNA和蛋白水平的表达,细胞免疫化学法检测NPM1-mA蛋白的亚细胞定位,细胞生长曲线观察细胞体外增殖能力的改变。结果构建的白血病细胞株THP-1-mA和K562-mA的RT-PCR产物均可见446 bp的NPM1-mA基因条带;NPM1-mA蛋白的表达量明显升高;NPM1-mA蛋白存在于构建的2株白血病细胞胞浆中;与空载体转染组和未转染组细胞相比,转染NPM1-mA基因后,THP-1细胞体外增殖能力增强,K562细胞体外增殖能力减弱。结论已成功构建了稳定表达NPM1-mA的两株白血病细胞株THP-1-mA和K562-mA,为进一步研究NPM1基因突变对白血病细胞生物学特性的影响提供了良好的细胞模型。
Objective To construct and identify leukemic myeloid cell lines stably expressing nucleophosmin 1 (NPM1) A mutant protein (NPM1-mA). Methods The recombinant plasmid pEGFP-C1-NPM1-mA containing human NPM1-mA gene was transfected into THP-1 and K562 cell lines by xfectTM transfection reagent. Positive clones were screened by G418 to establish THP-expressing leukemia cell lines stably expressing NPM1-mA -1-mA and K562-mA. The expression of NPM1-mA mRNA and protein was detected by RT-PCR and Western blot. The subcellular localization of NPM1-mA protein was detected by immunocytochemistry. The proliferation of cells was observed by cell growth curve. RESULTS: The 446 bp NPM1-mA gene band was found in the RT-PCR products of the leukemia cell lines THP-1-mA and K562-mA. The NPM1-mA protein expression was significantly increased. NPM1- The proliferation of THP-1 cells was enhanced in vitro, and the proliferation of K562 cells in vitro was weaker than that of the untransfected cells. Conclusions Two leukemia cell lines, THP-1-mA and K562-mA, stably expressing NPM1-mA have been successfully constructed, which provides a good cell model for further studying the influence of NPM1 gene mutation on the biological characteristics of leukemia cells.