变异IkBα抑制肝癌细胞株SMMC—7721中NF—kB活性及细胞生长

目的了解变异I κBα(mI κBα)转染到肝癌细胞株SMMC-7721细胞中是否抑制NF-κB向核内转位活性及细胞的生长.方法电泳迁移率分析检测32p标记的寡核苷酸探针与NF-κB结合情况,westernblot检测核内NF-κB表达情况,细胞生长曲线分析和细胞增殖实验分析肝癌细胞生长情况.结果转染mI κB α质粒肝癌细胞在0、24、48、96 h未见核内蛋白与κ B探针结合转染,而转染对照PcDNA 3质粒肝癌细胞始终可见核内蛋白与κB探针强结合; Western blot结果也显示0、24、48、96 h未见核内NF-κB表达,而对照PcDNA 3质粒核内NF-κB高水平表达.细胞增殖实验分析发现转染mI κB α质粒肝癌细胞生长受到抑制,而转染对照P cDNA 3质粒肝癌细胞生长未受影响,第2天开始转染mI κB α质粒肝癌细胞与其它两种细胞比较差异有非常显著性,增殖效率值分别是5 092.63±541.41、7 851.87±72.76、8 240.88±603.26,t值分别是14.29、10.99,P＜0.01.结论转染mI κBα质粒肝癌细胞可以持续表达mI κBα,抑制NF-κB向核内转位,从而抑制肿瘤细胞的生长.

Nuclear factor kappa B activity and cell viability of SMMC-7721 inhibited by mutated inhibitor kappa Balpha

OBJECTIVE: To investigate the inhibition consequence of NF-kappaB activity and cell viability by transfecting mutated inhibitor kappa B alpha (mI(kappa)B(alpha)) into liver cancer cell line of SMMC-7721 cells. METHODS: The nucleic proteins of SMMC-7721 cells transfected with mI(kappa)B(alpha) plasmid and cells with empty pcDNA3 vector were used to determine not only the binding of the 32P-labelled kappaB probes by EMSA, but also the expression of NF-kappaB by western blot. Cell viability was also analyzed. RESULTS: NF-kappaB nuclear translocation was inhibited remarkably in SMMC-7721 cells transfected with mI(kappa)B(alpha) at 0, 24, 48 and 96 hours. Furthermore, NF-kappaB was not detected in the nucleic protein of mI(kappa)B(alpha) -transfected cells at the same intended time by western blot. Compared with that of control cells, the growth of SMMC-7721 cells transfected with mI(kappa)B(alpha) was suppressed evidently, especially on the second day, the cpm values of mI(kappa)B(alpha) -transfected cells, pcDNA3-transfected cells, and control cells were 5,092.63+/-541.41, 7,851.87+/-72.76, and 8,240.8+/-603.26 respectively (t = 14.29, P<0.01; t = 10.99, P<0.01). CONCLUSION: Stable expression of mI(kappa)B(alpha) in SMMC-7721 cells transfected with mI(kappa)B(alpha) plasmid inhibits NF-kappaB nuclear translocation, then suppresses the cell growth.