PcDNA3.1-rhGM-CSF表达质粒的构建及其对K562细胞生长与分化的影响作用  

Construction of the eukaryotic expressing vector of PcDNA3.1 rhGM-CSF and its effect on the proliferation and differentiation of K562 cells

作　　者：朱红[1,2] 杨晓红[3] 王亚平[1] 唐恩洁[3] 

机构地区：[1]重庆医科大学基础医学院,重庆400042 [2]川北医学院,南充637000 [3]川北医学院医学微生物学与免疫学教研室,南充637000

出　　处：《中国免疫学杂志》2010年第6期494-497,502,共5页Chinese Journal of Immunology

基　　金：四川省科技厅项目资助(2009SZ0260)

摘　　要：目的:观察GM-CSF对髓系白血病细胞(K562)生长、分化的影响作用。方法:采用PCR、T-A克隆及基因定向克隆技术,将rhGM-CSF基因插入PcDNA3.1(+)空载体中,构建了PcDNA3.1-rhGM-CSF真核表达载体。经限制性内切酶消化和DNA测序鉴定确认,转染人K562髓系白血病细胞、72小时后采用细胞形态学检测、MTT试验、免疫组化技术观察和分析PcD-NA3.1-rhGM-CSF在K562细胞中的表达及其影响该细胞生长与分化的作用。结果:①成功构建PcDNA3.1-rhGM-CSF真核细胞表达载体;②重组质粒PcDNA3.1-rhGM-CSF能在K562细胞中表达,并诱导K562细胞向单核细胞系、巨噬细胞系分化;③K562细胞增殖受到明显抑制。结论:成功构建了PcDNA3.1-rhGM-CSF真核细胞表达载体;转染K562细胞能向单核细胞系、巨噬细胞系分化。Objective：To construct eukaryotic expressing vector of PcDNA3.1-rhGM-CSF and to investigate its effects of suppressing growth and inducing differentiation in K562 cells.Methods：The eukaryotic expressing vector PcDNA3.1-hGM-CSF was constructed by means of PCR and T-A clone techniques as well as directional cloning techniques.It was affirmed by the restriction map and DNA sequence analysis,and then transfected into K562 cells.It was observed that the target gene of the recombinant vector was expressed and exerted in K562 cells 72 h later,RT-PCR was used to affirm the expression of rhGM-CSF in K562 cells;Cell morphological,cell proliferation assay and immunohistochemistry were used to observe the effect of PcDNA3.1-rhGM-CSF on the growth and differentiation of K562 cells.Results：①The recombinant eukaryotic expressing vector PcDNA3.1-GM-CSF was constructed successfully;②Recombinant GM-CSF was expressed in the transfected K562 cells and the transfected K562 cells could differentiate into monocyte and macrophage cells;③ The proliferation of K562 cells was suppressed.Conclusion：The recombinant eukaryotic expression vector PcDNA3.1-rhGM-CSF was constructed successfully;Transfected K562 cell could differentiate into monocyte and macrophage cell.

关 键 词：粒细胞-巨噬细胞集落刺激因子 重组表达载体 细胞分化 K562 

分 类 号：R733.7[医药卫生—肿瘤]