Chk1基因沉默增强K562/A02细胞对阿霉素敏感性的实验研究  

Chk1 Gene Silence Enhancing the Drug Sensitivity of K562/A02 Cells to Adriamycin

作　　者：王海燕[1] 邹萍[1] 张敏[1] 游泳[1] 刘芳[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院血液科,武汉430022

出　　处：《华中科技大学学报（医学版）》2006年第2期173-176,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.30240022)

摘　　要：目的研究Chk1基因沉默对人红白血病耐药细胞株K562/A02(耐阿霉素)生长的影响,探讨Chk1在白血病细胞耐药中的作用。方法通过电穿孔转染法将特异性靶向Chk1的短发卡状RNA(shRNA)导入K562/A02细胞,应用RT-PCR、Weston-blot检测细胞内Chk1的表达,经阿霉素作用后,流式细胞术(FCM)检测其细胞周期分布及细胞凋亡率,MTT法检测细胞增殖。结果与对照组和空载体转染组相比,shRNA使细胞中Chk1 mRNA水平下降了66%,蛋白水平下降了60%。抑制Chk1的表达可解除阿霉素引起的G2/M期阻滞;使阿霉素诱导的细胞凋亡率由转染前的(5.67±0.78)%上升到(19.25±1.41)%;在阿霉素浓度为0.4 mg/L、4 mg/L时,细胞的增殖活性分别下降12%、20%。结论靶向Chk1 shRNA有效地抑制了Chk1的表达,阻断了细胞周期检测点信号传导通路,从而增强了K562/A02细胞对阿霉素的敏感性,有可能为临床上克服白血病的化疗耐药提供新的作用靶点及治疗途径。Objective To investigate the effect of Chk1 gene silence on the cell viability of human erythroleukemic cell line K562/A02 resistant to adriamycin, and explore the role of Chk1 in drug-resistance existed in leukemia cells. Methods The short hairpin RNA targeting at Chk1 gene was constructed and transfected into K562/A02 cells via electroporation. The mRNA and protein expression of Chk1 was detected by RT PCR and Western-blot respectively. Six h after treatment of K562/A02 cells with adriamycin, cell cycle and apoptotie rate were analyzed by flow cytometry. Cell viability was determined via MTT assay. Results The Chk1 expression at mRNA and protein levels in Chk1 shRNA-transfected group was reduced by about 66 % and 60 % respectively as compared with that in negative control and empty vector-transfected group （P〈0.05）. Inhibition of the Chk1 expression in Chk1 shRNA-transfected group significantly abrogated G2/M arrest induced by adriamycin, and the proportion of the cells in G2/M phase was （17.10±0.97） %, lower than that in other two groups （[50.96±1.25] %,[52.08 ±1.36] % respectively, P〈0.05）; and increased cell apoptotic rates from （5.67±0.78） % to （19. 25±1. 41） % （P〈： 0.05）. Treated with 0.4 mg/L or 4 mg/L of adriamycin, cells-transfected viability was decreased by 12 % or 20 % respectively （P〈0.05）. Conclusion shRNA targeting at Chkl gene can efficiently inhibit the Chk1 expression in K562/A02 cells, which enhanced the drug sensitivity of K562/A02 cells to adriamycin through blocking the signal transduction pathways of cell cycle checkpoint. So Chk1 gene silence may provide a new target and an effective way against drug resistance of leukemia cells.

关 键 词：Chk1基因 K562/A02细胞 RNA干扰 白血病细胞耐药 

分 类 号：R73[医药卫生—肿瘤]