沉默乏氧诱导因子-1α基因对乏氧人肝癌细胞SMMC-7721放射敏感性的影响  

Effect of Silencing Hypoxia Induced Factor-1α on the Radiosensitivity of Hypoxic Human Hepatoma SMMC-7721

作　　者：杨巍[1] 朱巍[1] 曹建平[1] 刘芬菊[1] 陈秋[1] 

机构地区：[1]苏州大学医学部放射医学与公共卫生学院放射生物学教研室,江苏苏州215123

出　　处：《苏州大学学报（医学版）》2011年第2期173-178,238,共7页Suzhou University Journal of Medical Science

基　　金：国家自然科学基金青年项目(30600160);国家自然科学基金面上项目(81071958);教育部"长江学者和创新团队发展计划项目"(IRT0849);苏州大学引进人员启动资助项目(Q4126803)

摘　　要：目的探讨RNA干扰乏氧诱导因子-1α(HIF-1α)基因对CoCl2诱导的乏氧人肝癌细胞SMMC-7721放射敏感性的影响及其机制。方法利用分子生物学技术构建靶向HIF-1α基因的RNA干扰质粒pGenesil-HIF,脂质体介导转染SMMC-7721细胞后进行乏氧培养,分别采用RT-PCR和Western blot法检测HIF-1α基因的mRNA和蛋白表达,四甲基偶氮唑盐(MTT)法检测细胞增殖活性,流式细胞术检测细胞周期和细胞凋亡,克隆存活实验检测细胞放射敏感性的变化。结果酶切鉴定和测序证实重组质粒pGenesil-HIF构建正确,转染质粒后乏氧培养24 h,SMMC-7721细胞HIF-1α基因的mRNA和蛋白表达水平明显低于对照组和阴性干扰组(P<0.05或P<0.01);乏氧培养24 h、48 h和72 h后,HIF-1α干扰组细胞增殖活性明显低于对照组(P<0.05或P<0.01),细胞阻滞于G0/G1期(P<0.01);乏氧培养48 h和72 h后HIF-1α干扰组细胞凋亡百分率明显高于对照组(P<0.01);乏氧培养72 h后HIF-1α干扰组SMMC-7721细胞放射增敏比为1.45。结论 RNA干扰HIF-1α基因可增强乏氧人肝癌细胞SMMC-7721的放射敏感性,其机制可能与RNA干扰HIF-1α基因抑制乏氧细胞增殖和诱导凋亡有关。Objective To investigate the effect of RNA interference of hypoxia induced factor-1α（HIF-1α） gene on radiosensitivity of hypoxic human hepatoma SMMC-7721 induced by CoCl2 and its mechanism.Methods RNA interference plasmid pGenesil-HIF targeting HIF-1α gene was constructed with molecular biology technique.mRNA and protein expression of HIF-1α gene were detected in hypoxic SMMC-7721 cells after transfection with pGenesil-HIF mediated by liposome by RT-PCR and Western blot respectively.Cell viability was detected by MTT assay.Cell cycle distribution and apoptosis of SMMC-7721 cells were detected by FCM assay.Radiosensitivity of SMMC-7721 cells was determined by clonogenic assay.Results Recombinant plasmid pGenesil-HIF was identified by enzyme digestion and sequencing.mRNA and protein expression of HIF-1α gene in hypoxic SMMC-7721 cells was significantly lower than that in the control and negative interference group 24 h after transfection with pGenesil-HIF（P0.05 or P0.01）.Cell viability in the pGenesil-HIF group was significantly lower than that in the control group after cultured in hypoxic condition for 24,48 and 72 h（P0.05 or P0.01） and cells arrested in the G0/G1 phase of cell cycle（P0.01）.The percentage of apoptosis of RNA interference in the HIF-1α group was significantly higher than that in the control group after cultured in hypoxic condition for 48 and 72 h（P0.01）.The radiosensitization ratio of SMMC-7721 cells in RNA interference in the HIF-1α group was 1.45 after cultured in hypoxic condition for 72 h.Conclusion RNA interference of HIF-1α gene could enhance radiosensitivity of hypoxic human hepatoma SMMC-7721 cells,which is associated with inhibitting hypoxic cells proliferation and inducing appoptosis.

关 键 词：RNA干扰 乏氧诱导因子-1Α 肝癌 放射敏感性 

分 类 号：R730.55[医药卫生—肿瘤] R735.7[医药卫生—临床医学]