重组腺病毒IκBαM在人肝癌细胞HepG2中的表达及对NF-κB活性的抑制

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。

The expression of Recombinant adenovirus IkappaBalphaM in human hepatocarcinoma HepG2 and it's inhibitive effect to the activity of NF-kappaB

OBJECTIVE: To investigate the expression of recombinant IkappaBalphaM (AdIkappaBalphaM) in human hepatocarcinoma cell line HepG(2) and the inhibiting effect to NF-kappaB. METHODS: To test the virus titer in 293 cells and the infective efficiency virus titer in HepG(2) cells with GFP and limited dilution method, then to assay the IkappaBalphaM expression of recombinant adenovirus and the alteration after induction of TNF-alpha in 293 cells and HepG(2) by Western blotting, furthermore, to observe NF-kappaB activity in HepG(2) before and after treatment of TNF-alpha by EMSA. RESULTS: The titer of AdIkappaBalphaM is 2 x 10(8) pfu/L, MOI equals to 20. AdIkappaBalphaM could be expressed stably and efficiently in HepG(2) and will not degrade by induction of TNF-alpha; but IkappaBalpha in the uninfection cell as well as AdIkappaBalpha control was increased at first and then decreased. EMSA demonstrated that the infected cells showed no activation of NF-kappaB before and after the treatment of TNF-alpha, but the cells of uninfected and infected with AdIkappaBalpha appeared excessive activation of NF-kappaB. CONCLUSION: AdIkappaBalphaM could be amplified in 293 cell and effectively infect to target cells HepG(2), and could be expressed stably in cells, and wouldn't be degrade with treatment of TNF-alpha, also it can effectually inhibit the excessive activation of NF-kappaB in HepG(2). The result of our research indicates the theoretical value of IkappaBalphaM as a super inhibitor, inhibition activity of NF-kappaB with IkappaBalphaM super-suppressor aided with routine anti-tumor therapy would become an effective method.