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

目的构建Raf激酶抑制蛋白（RKIP）小干扰RNA（siRNA）重组质粒pGPHl-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pGPHl-GFP-RKIP，电穿孔转染至儿童白血病细胞CCRF—CEM，以Westernblot法检测细胞RKIP、磷酸化细胞外调节蛋白激酶（P．ERK）、磷酸化核因子KB抑制蛋白（p-IKB）；以不同浓度9-硝基喜树碱（9NC）处理上述细胞，Westernblotting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pG．PHl-GFP-RKIP；pGPHl-GFP-RKIP和pGPHl-GFP的转染效率分别为90．04％、91．32％；与pGPHI．GFP转染的细胞比较，pGPHl-GFP-RKIP转染的细胞RKIP表达下调，p-ERK、P—IKB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。

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

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.