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目的 检测重组腺病毒IκBαM (AdIκBαM)在肝癌细胞HepG2 中的表达及TNF α诱导下IκBαM变化情况 ,并观察此超级抑制物对NF κB活性的抑制作用。方法 利用GFP及有限稀释法测定病毒滴度和感染靶细胞的效率 ,Western印迹法检测 2 93细胞和HepG2 中重组腺病毒介导IκBαM的表达及TNF α诱导下IκBαM变化情况 ,EMSA观测感染AdIκBαM前后经TNF α处理的HepG2 细胞核中NF κB活性水平的变化。结果 扩增AdIκBαM的滴度为 2× 10 8pfu/ml,MOI为 2 0 ;AdIκBαM在HepG2 中能稳定高效表达且不因TNF α的诱导而降解 ,未感染细胞基础水平的IκBα及转入的AdIκBα对照则随诱导时间延长而呈现先逐渐降低后升高的趋势。EMSA显示 ,感染AdIκBαM的细胞在处理前后均无NF κB活化迹象 ,而未感染及感染AdIκBα的细胞经TNF α诱导后则有NF κB过度活化情况。结论 AdIκBαM能高效扩增并有效感染靶细胞HepG2 ,能在HepG2 细胞中稳定高水平表达 ,且不会被TNF α诱导而降解 ,能稳定有效的抑制HepG2 细胞中NF κB的过度活化 ,上述结果初步表明 ,通过IκBαM超级抑制物抑制NF κB的活性 ,再辅以常规的抗肿瘤治疗 ,有望成为一种十分有效的肿瘤基因治疗方法。
Objective To detect the expression of IκBαM (AdIκBαM) in hepatocellular carcinoma cell HepG2 and the change of IκBαM induced by TNFα, and to observe the inhibitory effect of this superinhibitor on NF κB activity. Methods The viral titers and the efficiency of target cells were determined by GFP assay and limited dilution method. The expression of IκBαM in 293 cells and HepG2 cells was detected by Western blotting and the changes of IκBαM induced by TNFα were detected by EMSA. Before and after infection with AdIκBαM Changes of NFκB activity in HepG2 cells treated with TNFα. Results The titer of amplified AdIκBαM was 2 × 10 8 pfu / ml and the MOI was 20. AdIκBαM was stably and efficiently expressed in HepG2 and was not degraded by TNFα induction. The level of IκBα in uninfected cells and the transfected AdIκBα Control with the induction of time showed a downward trend and then increased. EMSA showed that cells infected with AdIκBαM showed no sign of NFκB activation before and after treatment, whereas NFκB over-activation was induced by TNFα in cells that were not infected and infected with AdIκBα. Conclusions AdIκBαM can efficiently and effectively infect target HepG2 cells, and can stably and highly expressed in HepG2 cells without being degraded by TNFα, which can stably and effectively inhibit the over-activation of NF κB in HepG2 cells It is indicated that the inhibition of NF κB activity by IκBαM superinfection combined with conventional anti-tumor therapy is expected to become a very effective method of oncology gene therapy.