PLK1基因沉默对K562-A02细胞周期、增殖及耐药性影响.docVIP

PLK1基因沉默对K562/A02细胞周期、增殖及耐药性影响 　 作者：刘林，张敏，邹萍，田蕾，刘芳 【摘要】 为了探讨小干扰RNA（siRNA）对K562/A02细胞Polo样激酶1（PLK1）基因的抑制作用及其对细胞周期、细胞增殖和耐药性的影响，将构建的针对PLK1 mRNA的siRNA真核质粒，导入K562/A02细胞，用RT-PCR和Western-blot方法分析PLK1基因在转染前后的表达差异；台盼蓝拒染试验检测细胞存活率；流式细胞术（FACS）检测细胞周期和细胞内阿霉素（ADM）的积累量；MTT试验检测细胞对ADM的药物敏感性。结果显示，与对照组相比，转染24和48小时后，siRNA-PLK1质粒转染组的PLK1 mRNA下降（34.7±2.1）%和（56.6±1.5）%，蛋白水平下降（49.9±3.2）%和（62.1±1.7）%；转染24和48小时后，细胞存活率下降了30%和59%；转染48小时后，G2/M期细胞比例提高了2.77倍，同时细胞内ADM积累量显著提高；对ADM药物敏感性的相对率为73.8%。结论： PLK1基因沉默能有效抑制K562/A02细胞生长，使细胞周期停滞在G2/M期，同时使得细胞内ADM积累量提高，对ADM敏感性增强。 【关键词】 PLK1基因沉默 　　Effect of PLK1 Gene Silence on Cell Cycle，Proliferation and Drug Resistance in K562/A02 Cells 　　Abstract The study was purposed to investigate the effect of small interference RNA (siRNA) targeting Polo-like kinase 1 (PLK1) gene on cell cycle progression，proliferation and drug resistance in K562/A02 cells.siRNA plasmid vector specifically targeting PLK1 gene with enhanced green fluorescence protein (EGFP) was transfected into K562/A02 cells.Expressions of PLK1 mRNA and protein were assayed by RT-PCR and Western-blot; cell proliferation was evaluated by direct cell counting after trypan blue staining.Cell cycle and intracellular adriamycin（ADM）accumulation was determined by flow cytometry; 50% inhibition concentration (IC50) of ADM on K562/A02 cells was determined by MTT method.The results showed that，as compared with control cells，siRNA plasmid reduced PLK1 mRNA expression by（34.7±2.1）% for 24 hours and by（56.6±1.5）% for 48 hours，PLK1 protein significantly decreased simultaneously by (49.9±3.2)% and by (62.1±1.7)%.After being transfected for 24 and 48 hours，the rate of survival cells decreased by 30% and 59% respectively.Forty-eight hours after transfection，the ratio of K562/A02 cells at G2/M increased by 2.77-fold，at the same time，intracellular ADM accumulation increased and the relative efficiency of K562/A02 cells to ADM was 73.8%.It is concluded that PLK1 gene silence can inhibit K562/A02 cell proliferation，induce c