mIκBα基因转染肝癌细胞HepG2的放射增敏作用

Enhancement of HepG2 cell radiosensitivity by mutant IκBα gene transfection

目的探讨mIκBα基因是否增加肝癌细胞的放射敏感性。方法实验分3组:亲本细胞对照组、HepG2细胞转染Adv组、转染mIκBα基因的HepG2细胞-AdmIκBα组。在6GyX射线照射前后分别测定3组细胞下列指标:Westernblot检测肝癌细胞浆内IκBα值;电泳迁移率法测定肝癌细胞核内NF-κB活性;TUNEL染色法测定肝癌细胞凋亡指数。2Gy放射线照射后的肝癌细胞存活分数和用多靶单击模型确定的Do、Dq值判定肝癌细胞的放射敏感性。结果放射线照射前,Adv组与HepG2组细胞浆内IκBα呈低值,照射后更减低;而细胞核内NF-κB活性照射前为(+),照射后为(++),呈持续激活状态;细胞凋亡指数在照射前分别为1.4、1.6,照射后升高至8.9、11.7。AdmIκBα组在照射前、后细胞浆内IκBα均呈高值,均为Adv组的3倍;而细胞核内NF-κB活性在照射前、后均呈阴性;照射前细胞凋亡指数为18.2,照射后升高至88.3。3组中AdmIκBα组细胞存活分数最低,为0.301;而SER(放射增敏比)最大,为2.099;Do值最小,为1.468,Dq值最小,为0.709。结论用mIκBα基因转染肝癌细胞HepG2,可抑制肝癌细胞内NF-κB的抗凋亡活性,增强肝癌细胞的放射敏感性。

Objective To investigate the efficacy of transfection with a mutant IκBα gene （mIκBα） in enhancing the radiosensitivity of hepatocellular carcinoma cells. Methods The hepatocellular carcinoma Hep G2 cells were divided into 3 group and transfected with the adenovirus containing mIκBα vector （Ad-mIκBα group）, Ad vector （Adv group）, or without treatment （parental control cells）. Before and after irradiation of the cells with 6 Gy high-energy X ray, Western blotting was performed to measure the expression level of Iid3a in the cytoplasm, and electrophoresis mobility shift assay （EMSA） carried out to evaluate the nuclear factor-κB （NF-κB） activity in the cell nuclei, with the cell apoptosis detected using TUNEL assay. The radiosensitivity of the HepG2 cells were determined by comparison between the 3 groups in term of the surviving cell fractions （SFz） after 2-Gy X-ray exposure, and of the Do and Dq values obtained using the single-hit multi target model. Results Before the X-ray exposure, the Hep G2 cells in Adv group and the control group showed low levels of IκBαabsorbance in the cytoplasm, which were further decreased after the exposure. The NF-κB activity in the nuclei of the cells in the two groups was positive （＋） before irradiation, and substantially enhanced （＋＋） after the exposure and maintained the stably activated state. The apoptotic index of the cells in the two groups was 1.4 and 1.6 before irradiation, and increased to 8.9 and 11.7 after the irradiation, respectively. The cells in the Ad-mIκBα group, however, exhibited high levels of IκBα absorbance either before or after the irradiation, which were approximately 3 times that of the Adv group, but the NF-id3 activity remained negative irrespective of the irradiation. The apoptotic index of the cells in the Ad-mIκBα group was 18.2 before irradiation, was increased to 88.3 after the irradiation. Among the 3 groups, Ad-mIκ Ba group had the smallest SF2 value of 0.301 but the highest sensitivity enhancement ratio （SER） of 2.99, with the lowest Do and Dq values （1.468 and 0.709, respectively）. Conclusion mhd3a gene transfection into HepG2 cells inhibits the anti-apoptotic activity of NF-κB to enhance the radiosensitivity of the cells.