沉默乏氧诱导因子-1α和生存素基因联合X射线照射对乏氧细胞的体外抑瘤效应

为探讨RNA干扰（RNA interference，RNAi）沉默乏氧诱导因子-1α（Hypoxia induced factor -1α，HIF-1α）和生存素（Survivin）基因联合X射线照射对乏氧人肝癌细胞SMMC-7721的体外抑瘤效应，利用双靶向HIF-1α和Survivin基因载体，采用阳离子脂质体介导法转染SMMC-7721细胞，经乏氧培养36h后，分别以RT-PCR和Western blot法检测HIF-1α和Survivin基因的mRNA及其蛋白表达，分别以四甲基偶氮唑盐（MTT）法和流式细胞术检测细胞存活分数及细胞凋亡。结果表明，转染质粒pGenesil-Survivin-HIF的SMMC-7721细胞中，HIF-1α和Survivin基因mRNA表达水平与对照和阴性干扰组相比明显降低（p〈0．05），也未检测到HIF-1α和Survivin蛋白的表达。单、双干扰联合放疗组存活分数较照射组明显降低（p〈0．01），双干扰联合放疗组存活分数较其它组也明显降低（p〈0．01）。单、双干扰联合放疗组凋亡细胞百分数较对照组明显升高（p〈0．01），双干扰联合放疗组凋亡细胞百分数较其它组也明显升高（p〈0．01）。结果提示，质粒pGenesil—Survivin—HIF可有效地干扰乏氧SMMC-7721细胞HIF-1α和Survivin基因的mRNA及其蛋白表达。双干扰联合放疗组对乏氧SMMC-7721细胞的体外抑瘤效应明显优于单干扰联合放疗组。

Anti-tumor effect of silencing Hif-lα and Survivin genes combined with X-ray irradiation on hypoxic tumor cells in vitro

In order to investigate the anti-tumor effect of RNA interference silencing HIF- 1α and Survivin genes combined with X-ray irradiation on hypoxic SMMC-7721 cells in vitro, we detected mRNA and protein expression of HIF-1α and Survivin genes in hypoxic SMMC-7721 ceils after transfection with pGenesil-Survivin-HIF carried by liposome by RT-PCR and Western blot respectively. Survival fraction and apoptosis of hypoxic SMMC-7721 cells in vitro were detected by MTT and FCM assay respectively. The results show that mRNA expression of HIF-1α and Survivin genes in hypoxic SMMC-7721 cells transfected with pGenesil-Survivin-HIF is significantly lower than that of control and negative interference group （p〈0.05）. Protein expression of HIF-1α and Survivin genes in hypoxic SMMC-7721 cells after transfectlon with pGenesil-Survivin-HIF can not be detected. Survival fraction of pGenesil-Survivin＋5 Gy group, pGenesil-HIF＋5 Gy group and pGenesil-Survivin-HIF＋5 Gy group is significantly lower than that of 5 Gy group （p〈0.01）. Survival fraction of pGenesil-Survivin-HIF＋5 Gy group is also significantly lower than that of other groups （p〈0.01）. Percentage of apoptosis of pGenesil-Survivin＋5 Gy group, pGenesil-HIF＋5 Gy group and pGenesil-Survivin-HIF＋5 Gy group is significantly higher than that of control group （p〈0.01）. Percentage of apoptosis fraction of pGenesil-Survivin-HIF＋5 Gy group is significantly higher than that of other groups （p〈0.01）. These results can be suggested that the pGenesil-Survivin-HIF could silence mRNA and protein expression of HIF-1α and Survivin genes in hypoxic SMMC-7721 cells. Anti-tumor effect of pGenesil-Survivin-HIF＋5 Gy group is more powerful than that of pGenesil-Survivin＋5 Gy group or pGenesil-HIF＋5 Gy group with hypoxic SMMC-7721 cells in vitro.