摘要
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 a leukemia cell line for stable expression of human nucleophosmin 1 mutant of type A（NPM1-mA） gene.Methods THP-1 and K562 cell lines were transfected with recombinant plasmid pEGFP-C1-NPM1-mA carrying NPM1-mA gene with xfectTM transfection agent,based on which positive clones were screened with G418 for construction of leukemia cell strains THP-1-mA and K562-mA for stable expression of NPM1-mA.The expressions of NPM1-mA at mRNA and protein levels were determined by RT-PCR and Western blot respectively,and the subcellular location of NPM1-mA by immunocytochemical assay.The in vitro proliferation abilities of constructed leukemia cell strains were observed by cell growth curve.Results Both THP-1-mA and K562-mA cell strains contained NPM1-mA gene fragment at a length of 446 bp as proved by RT-PCR,in which the expression level of NPM1-mA protein increased significantly.The expressed NPM1-mA protein was located in the cytoplasm of the two strains.Compared with those transfected with empty vector and those untransfected,the in vitro proliferation level of THP-1 cell strain after transfection with NPM1-mA increased,while that of K562 cell strain decreased.Conclusion The leukemia cell strains THP-1-mA and K562-mA for stable expression of NPM1-mA were successfully constructed,which provided a good cell model for further study on the effect of NPM1 gene mutation on biological characters of leukemia cells.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第2期141-145,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金面上项目资助课题(30872418)
重庆市科委自然科学基金计划资助项目(CSTC
2010BB5363)
