论文部分内容阅读
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。
To investigate the effect of NF κB on the transcription of the HL-60 E6bclx gene in a leukemic drug-resistant cell line, we first established a stable and widely used method by intermittently injecting small doses of epirubicin (6 ng ml) into HL 60 cells in vitro. Spectrum-resistant cell line HL 6 0 E6. RT-PCR method was used to detect the change of bcl x mRNA level after 5 μmol L thio-modified antisense RelA acted on HL 60 E6 cells. Immunofluorescence and flow cytometry were used to evaluate the functional status of NF-κB RelA in HL 60 E6 cells and the transfection efficiency of liposomes as oligonucleotide carriers. The results showed that RelA was expressed in the nucleus in HL60 E6 cells, and NFκB was in a sustained activation state. The liposome-mediated oligonucleotide (ODN) transfection efficiency was significantly higher than that of the naked ODN group, with significant differences at 4, 6, and 12 hours (P < 0.01). After antisense RelA acted on HL60 E6 cells, the bclxL mRNA level decreased in a time-dependent manner. The 8-hour inhibition peaked and gradually recovered after 15 hours. However, the bclxL mRNA level in the control group did not change significantly. The analysis results suggest that NF κB has transcriptional regulation on bcl x , and that blocking the RelA subunit by antisense technology can attenuate the transcriptional activation of bcl xL gene by NF κB, and further speculate that NF κB may be an important factor for drug resistance in leukemia cells.