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PLK1mRNA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PLK1mRNA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。
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 cell cycle arrest at G2/M, and increase intracellular ADM accumulation, so that enhance cell sensitivity to ADM.
出处
《中国实验血液学杂志》
CAS
CSCD
2006年第2期241-246,共6页
Journal of Experimental Hematology
