摘要
目的:研究靶向人白血病K562细胞内高表达的三磷酸腺苷结合盒转运体G2(ATP binding cassette transporter G2,ABCG2)基因的RNA干扰(RNA interference,RNAi)对K562细胞化疗敏感性的影响。方法:构建靶向ABCG2基因的RNAi重组腺病毒Ad-siABCG2,以其感染K562细胞后,采用RT-PCR、免疫细胞化学和Western印迹法检测K562细胞中ABCG2 mRNA和蛋白的表达,MTT法检测K562细胞对药物的敏感性变化,FCM检测细胞内多柔比星(adriamycin,ADM)和柔红霉素(dauno-rubicin,DNR)的平均荧光强度。结果:成功构建靶向ABCG2的RNAi重组腺病毒Ad-siABCG2,经感染K562细胞后可有效沉默ABCG2基因表达;MTT检测发现,感染Ad-siABCG2的K562细胞对ADM和DNR的敏感性是未感染时的2～6倍(P<0.01);FCM检测显示,感染Ad-siABCG2的K562细胞内DNR和ADM平均荧光强度(86.19和180.26)分别高于未感染的K562细胞(63.65和141.60),差异有统计学意义(P<0.05)。结论:重组腺病毒Ad-siABCG2沉默ABCG2基因表达可增加K562细胞对ADM和DNR的敏感性,表明ABCG2基因表达可导致K562细胞产生多药耐药;Ad-siABCG2对ABCG2介导的细胞多药耐药有逆转作用。
Objective：To investigate the sensitivity of human leukemia cell lines K562 to chemotherapy after ATP binding cassette transporter G2 （ABCG2） gene was silenced by RNA interference. Methods：The recombined adenovirus targeting ABCG2 （Ad-siABCG2） was constructed and infected K562 cells. The ABCG2 mRNA was detected with RT-PCR and protein expression was measured by immunocytochemistry and Western blotting before and after K562 cells were infected with Ad-siABCG2. The drug sensitivity of K562 cells was detected with MTT assay. The average fluorescence intensities of intracellular adriamycin （ADM） and daunorubicin （DNR） were determined by flow cytometry（FCM）.Results：The recombinant adenovirus Ad-siABCG2 targeting ABCG2 gene was constucted successfully. Transfection of Ad-siABCG2 effectively silenced the expression of ABCG2 gene. MTT assay showed that the drug sensitivity of infected K562 cells to DNR and ADM was raised 2 to 6 times as compared with uninfected cells （P〈0.01）. FCM analysis showed that the average fluorescence intensities of ADM and DNR were higher than uninfected cells （86.19 vs 63.65 and 180.26 vs 141.60）. The differences were significant （P〈0.05）. Conclusion：Silencing ABCG2 gene by the recombinant adenovirus Ad-siABCG2 increases the sensitivity of K562 cells to ADM and DNR of K562 cells,which indicate that overexpression of ABCG2 gene maybe cause multidrug resistance of leukemia K562 cells and Ad-siABCG2 can reverse the multidrug resistance induced by ABCG2 gene.
出处
《肿瘤》
CAS
CSCD
北大核心
2009年第10期929-933,共5页
Tumor
基金
国家自然科学基金资助项目(编号:30330590)
