BCR/ABL特异性siRNA真核表达载体构建及其对K562细胞的影响

Construction of Eukaryotic Expression Vector of siRNA Specific for BCR/ABL Fusion Gene and Its Effects on K562 Cells

目的:构建特异性BCR/ABL的siRNA真核细胞表达载体并探讨其对K562细胞BCR/ABL的干扰和生长增殖的影响。方法:根据GenBank数据库提供的BCR/BAL基因核苷酸序列,按照Tuschl设计原则,选择设计双链小干扰RNA(small interfering RNA,siRNA),再转化为能表达其小发卡结构RNA(Small hairpin RNAs,shRNA)的DNA序列,并与pTER质粒定向连接,构建受控于人RNA聚合酶Ⅲ启动子H1的真核表达载体pTER117和pTER363,经限制性内切酶酶切和DNA测序进行鉴定;该载体带有zeocin药物抗性和受四环素(tet)调控的开关基因,通过Lipofectamine 2000转染K562细胞,并筛选出阳性K562细胞克隆。采用TaqMan real-time quantitative RTPCR(RQ-PCR)和Western blot技术检测BCR/ABL mRNA和P210蛋白表达;台盼蓝染色法观察细胞的生长增殖,流式细胞术(FCM)检测细胞的凋亡。结果:构建BCR/ABL融合基因siRNA真核表达载体pTER117和pTER363,经限制性内切酶酶切和DNA测序证实与设计完全一致。用筛选出重组载体转染阳性K562细胞克隆,经过四环素诱导基因表达后,细胞的生长增殖明显受到抑制;pTER117和pTER363诱导K562细胞48 h和72 h后,其凋亡率分别为34.4%、31.8%和58.1%;54.6%;BCR/ABL的mRNA表达明显下调,分别为诱导前的10%、18%和8.5%、16%;P210蛋白表达下降明显,几乎检测不到表达。结论:BCR/ABL融合基因siRNA真核细胞表达载体构建成功,并有效干扰K562细胞BCR/ABL的表达,抑制细胞生长,诱导细胞调亡。

Objective：To construct eukaryotic expression vector of siRNA specific for BCR/ABL and to investigate the effect of recombinant plasmid on BCR/ABL and P210 protein expression in K562 cells.Methods：siRNA（small interfering RNA） was designed according to the Tuschl＇s principle of Ai- based medicine,and was converted into cDNA coding expression of shRNA（small hairpin RNAs） of siRNA for BCR/ABL fusion gene.The cDNA was synthesized and inserted into plasmid pTER.The pTER117 and pTER363 of recombinant plasmid being eukaryotic expression vector was controlled by the H1 promoter of RNA polymerase Ⅲ,and identified by the restriction map and the sequence analysis.The recombinant plasmid did not only have the screening resisting antibiotics,its expression but also are induced by tetracycline（tet）.After steadily transfection into K562 cells by Lipofectamine,their positive mono- cell clones being resistant to Zeocin were isolated.TaqMan real- time quantitative RT-PCR（RQ-PCR） and Western blot respectively detected expression of BCR/ABL mRNA and P210 protein.Trypaum blue dying was used to analyze the proliferation of K562 cells.Cell apoptosis was observed by flow cytometer.Results：the recombinant plasmid was steadily transfected into K562 cells by Lipofectamine 2000,Their positive mono-cell clones being resistant to Zeocin were isolated.The proliferation of K562 cells were remarkably inhibited by the recombinant plasmid induced gene expression by tetracycline.Tetracycline induced its expression for 48 h and 72 h.pTER117,pTER363 decreased the mRNA level of BCR/ABL 90%,82%and 91.5%,84%,respectively,P210 protein were almost measured in K562 cells.FCM analysis showed that the recombinant plasmid induced apoptosis in K562 cells,the apoptosis rate were respectively 34.4%,58.1%in K562 cells treated by pTER117 for 48 h and 72 h,apoptosis rate were 31.8%,54.6%by pTER363,but the control groups did not show these effects on K562 cells.Conclusion：The siRNA eukaryotic expression vector against BCR/ABL mRNA has been successfully conctructed,and effectively inhibits the expression of BCR/ABL in K562 cells,inhibite cell growth and induce cell apoptosis.