siRNA特异性抑制HEL细胞evi1基因的实验研究

本研究探讨小干扰RNA（small interfering RNA,siRNA）沉默人红白细胞白血病细胞（HEL）的evi1基因后对细胞evi1基因表达的抑制作用及细胞生物学特征变化及其作用机制。体外合成3条evi1基因特异性的siRNA（siRNA-1、siRNA-2、siRNA-3）并转染HEL细胞,并设对照。用四甲基偶氮唑蓝法评价细胞增殖抑制情况,半定量逆转录聚合酶链反应（semiquantitative RT-PCR）检测evi1基因mRNA的表达,台盼蓝染色试验测定细胞活性的变化,流式细胞术检测细胞周期变化和凋亡率。结果显示,细胞转染24、48、72小时后,以siRNA-1作用最强,转染后48小时抑制作用最明显。siRNA浓度为120nmol/L时,抑制率达到最大。转染48小时后siRNA-1对HEL细胞的增殖抑制率为（72.22±2.80）%,evi1-mRNA相对表达率为（27.31±1.11）%,HEL细胞活率为（26.05±2.49）%,与其他各组相比有显著性差异（p〈0.001）。siRNA可使细胞周期阻滞在G0/G1期,S期细胞明显减少,凋亡率明显增高（p〈0.01）。结论：evi1基因特异性siRNA可抑制HEL细胞增殖,使evi1-mRNA表达水平降低,细胞活性下降,HEL细胞周期阻滞在G0/G1期,抑制细胞的有丝分裂,促进细胞凋亡。

Inhibition of evi1 Expression by siRNA in HEL Cell Line

The aim of study was to investigate the inhibitory effect of small interfering RNA on evi1 gene expression and biological characteristics in HEL cells and its mechanism. 3 siRNA（siRNA-1,siRNA-2,siRNA-3） specific for evi1 gene were synthesized and transfected into HEL cells in vitro. Experiments were divided into test and control groups. MTT method was used to assay the inhibitory effect of siRNA on cell proliferation; semiquantitative RT-PCR was used to detect the expression of evi1 gene mRNA; the cell viability was determined by trypan blue dye test; the change of cell cycle and apoptosis of cells were analyzed by flow cytometry. The results showed that siRNA-1 had stongest effect,and inhibitory effect was most obvious at 48 hours after trasfection. When the concentration of siRNA raised to 120 nmol/L,the inhibitory rate reached to the peak. The inhibitory rate of siRNA-1 on proliferation of HEL cells,relative expression level of evi1 mRNA and cell viability at 48 hours after transfection were 72.22±2.80%,27.31±1.11% and 26.05±2.49%,which had significant difference from other groups （p0.001）. The siRNA resulted in arrest of cell cycle at G0/G1 phase,the cell amount at S phase obviously decreased,the apoptotic rate of HEL cells obviously increased （p0.01）. It is concluded that the siRNA specific for evi1 gene can suppress the proliferation of HEL cells,reduce the expression of evi1 mRNA,decrease the cell viability,arrest the cell cycle at G0/G1 phase,suppress cell mitosis,and promote cell apoptosis.