反义RelA下调白血病耐药细胞株HL-60/E6 bcl-x_L mRNA

Antisense Oligodeoxynucleotide Directed to NF-κB-RelA Down-Regulates bcl-x_L mRNA in Drug-Resistant Leukemia Cell Line HL-60/E6

为了探讨NF κB对白血病耐药细胞株HL 6 0 E6bcl x基因转录的影响 ,首先在体外采用间歇小剂量表阿霉素 (6ng ml)作用于HL 6 0细胞的方法 ,建立了性能稳定的广谱耐药细胞株HL 6 0 E6。然后用RT PCR方法检测 5 μmol L硫代修饰反义RelA作用于HL 6 0 E6细胞后bcl x基因mRNA水平的变化。同时应用免疫荧光技术和流式细胞术分别对HL 6 0 E6细胞中NF κB RelA的功能状态及脂质体作为寡核苷酸载体的转染效率进行评估。结果表明 ,在HL 6 0 E6细胞中RelA表达于胞核 ,NF κB处于持续活化状态。脂质体介导的寡核苷酸 (ODN)的转染效率明显高于裸ODN组 ,在 4 ,6和12小时时有显著差异 (P <0 .0 1)。反义RelA作用于HL 6 0 E6细胞后 ,bcl xL mRNA水平下降呈时间依赖性 ,8小时抑制达到最高峰 ,15小时后逐渐恢复 ,而对照组bcl xL mRNA水平变化不明显。分析结果认为 ,NF κB对bcl x有转录调节作用 ,反义技术封闭RelA亚基可以减弱NF κB对bcl xL 基因的转录激活 ,进而推测NF κB可能是白血病细胞耐药的一个重要因子。

In order to explore the effect of nuclear factor-kappa B(NF-κB) on bcl-x gene transcrtiption in drug-resistant leukemia cell line HL-60/E6, first of all, drug-resistant subline HL-60/E6 was derived by intermittently exposing HL-60 cells to 6 ng/ml epirubicin, and then bcl-x L mRNA levels were detected by RT-PCR after exposing HL-60/E6 cells to 5 μmol/L AS-PS-ODN-RelA at different times. Morever, indirect immuno-fluorescence and FCM were used to demonstrate the location of NF-κB-RelA in HL-60/E6 cells and the efficiency of liposome-mediated ODN transfection. The results showed that RelA kept active and located at the nuclei of HL-60/E6 cells, and the efficiency of liposome-mediated ODN transfection was significantly higher than that of null ODN (P<0.01 in 4, 6 and 12 h). Exposure of HL-60/E6 cells to 5 μmol/L AS-PS-ODN-RelA led to a maximal 40% decline of bcl-x L mRNA levels. No significant change of bcl-x L mRNA level occurred in control group. It was concluded that NF-κB was involved in regulating bcl-x transcription. Suppressing NF-κB-RelA by antisense technology may down-regulate level of bcl-x L mRNA.