pGPH1-GFP-RKIP的构建及其对白血病细胞株CCRF-CEM化疗敏感性的影响  

Establishment of pGPH1-GFP-RKIP plasmid vector and its effect on sensitivity to chemotherapy of leukemia cell line CCRF-CEM

作　　者：李青 多力坤[2] 

机构地区：[1]武警兵团指挥部医院,乌鲁木齐830063 [2]新疆医科大学第一附属医院

出　　处：《山东医药》2014年第15期20-23,共4页Shandong Medical Journal

摘　　要：目的构建Raf激酶抑制蛋白(RKIP)小干扰RNA(siRNA)重组质粒pGPH1-绿色荧光蛋白(GFP)-RKIP,观察其对白血病细胞株CCRF-CEM化疗敏感性的影响。方法构建RKIP的siRNA重组质粒pGPH1-GFPRKIP,电穿孔转染至儿童白血病细胞CCRF-CEM,以Western blot法检测细胞RKIP、磷酸化细胞外调节蛋白激酶(pERK)、磷酸化核因子κB抑制蛋白(p-IκB);以不同浓度9-硝基喜树碱(9NC)处理上述细胞,Western blotting法检测细胞RKIP,流式细胞仪法检测细胞caspase-3活性(计算细胞凋亡率),同上检测RKIP表达。结果成功构建pGPH1-GFP-RKIP;pGPH1-GFP-RKIP和pGPH1-GFP的转染效率分别为90.04%、91.32%;与pGPH1-GFP转染的细胞比较,pGPH1-GFP-RKIP转染的细胞RKIP表达下调,p-ERK、p-IκB表达上调(P均<0.05);9NC处理后CCRF-CEM细胞RKIP表达上调、细胞凋亡率下降(P均<0.05)。结论成功构建了RKIP的siRNA质组质粒,RKIP表达变化可能与白血病细胞化疗敏感性有关。Objective To establish plasmid vectors pGPH-green fluorescent protein （GFP）-Raf kinase inhibitor pro- tein （RKIP） expressing RKIP small interfering RNA （siRNA） and to observe the effect of RKIP RNA interference on sen- sitivity to chemotherapy of pediatric leukemia ceils CCRF-CEM. Methods Plasmid RKIP interference vectors pGPH-GFP- RKIP were established and transfected into CCRF-CEM cells by using electroporation technique. RKIP, phosphorylated ex- tracellular regulated protein kinase （p-ERK） and phosphorylated nuclear factor ~B inhibitor （p-IKB） levels in the trans- fected cells were detected by using Western blotting. 9-nitrocamptothecin （9NC） at various concentrations were used to in- duce CCRF-CEM cells apoptosis, and Western blotting was used to detect the RKIP expression, caspase-3 activities （ apop- tosis rates） were detected by using flow cytometry. Results The pGPH1-GFP-RKIP was successfully established. Trans- fection rates of pGPH1-GFP-RKIP and pGPH1-GFP plasmids were 90.04% and 91.32% , respectively. The expression levels of RKIP were effectively down-regulated and both p-ERR and p-IKB levels were up-regulated in CCRF-CEM cells transfected with pGPH-GFP-RKIP plasmids as compared with the ceils transfected with pGPH1-GFP plasmids （ all P 〈 O. 05）. RKIP levels in the CCRF-CEM cells exposed to 9NC were up-regulated and the CCRF-CEM cells transfected with pGPH-GFP-RKIP had down-regulated apoptosis rates （ all P 〈 O. 05 ）. Conclusions RKIP siRNA plasmid vectors are successfully established. RKIP expression changes may be associated with the sensitivity to chemotherapy of leukemia cells.

关 键 词：RAF激酶抑制蛋白 RNA干扰 儿童白血病 化疗敏感性 

分 类 号：R733.7[医药卫生—肿瘤]