寡核苷酸Dbait乏氧辐射双诱导重组质粒的构建及其对人宫颈癌HeLa细胞的乏氧放射增敏效应  

Construction of pcDNA 3.1(+)-HRE-Egr-1-Dbait and Its Radiosensitization Effect on Human Cervical Cancer HeLa Cells in Hypoxia Response

作　　者：温林春[1] 陆锡燕 尤传文[1] 辛勇[2] 章龙珍[2] 

机构地区：[1]南京鼓楼医院集团宿迁市人民医院肿瘤科,江苏宿迁223800 [2]徐州医学院附属医院放疗科

出　　处：《肿瘤防治研究》2013年第5期413-416,共4页Cancer Research on Prevention and Treatment

基　　金：国家自然科学基金资助项目(81071831)

摘　　要：目的构建携带新型放射增敏剂Dbait的乏氧辐射双诱导的重组质粒pcDNA 3.1(+)-HREEgr-1-Dbait,探讨乏氧条件下其对宫颈癌HeLa细胞的放射增敏作用,为Dbait的基因靶向放射增敏治疗提供实验依据。方法从C57BL/6裸鼠肝组织扩增Egr-1启动子,人工合成HRE增强子序列和新型寡核苷酸药物Dbait,通过基因重组分别将Egr-1、HRE和Dbait克隆入pcDNA 3.1(+)中,获得重组质粒pcDNA 3.1(+)-HRE-Egr-1-Dbait。转染宫颈癌HeLa细胞,采用集落形成试验观察在常氧和乏氧状态下宫颈癌HeLa细胞的放射敏感度。结果真核表达重组质粒pcDNA 3.1(+)-HRE-Egr-1-Dbait构建成功并通过PCR和测序鉴定。在常氧情况下宫颈癌HeLa细胞的D_0、D_q、SF2、α/β值分别为1.98、0.93、0.52、11.12。在乏氧情况下宫颈癌HeLa细胞的D_0、D_q、SF2、α/β值分别为1.74、1.46、0.43、15.82,氧增敏比OER为0.88。结论成功构建了携带新型放射增敏剂Dbait的乏氧辐射双诱导的真核表达质粒pcDNA 3.1(+)-HRE-Egr-1-Dbait,并验证了其在宫颈癌HeLa细胞中的乏氧辐射增敏效应。Objective To construct recombined eukaryotic expressional vector pcDNA 3.1（＋）-HRE-Egr-1-Dbait by hypoxia and radiation dual-induced promoter and to observe its radiosensitivity effect on HeLa cells in hypoxia response. Methods Egr-1 promoter elements were amplificated from C57BL/6 nude mice by PCR, Dbait oligonucleotide and HRE enhancer elements were gained by chemical synthesis.The recombined plasmid pcDNA 3.1（＋）-HRE-Egr-1-Dbait was constructed by gene reconstruction.pcDNA3.1（＋）-HRE-Egr-1-Dbait was transfected into HeLa cells. Colony Formation Assay was used to the detect the radiosensitivity effect transfectants. Results The recombinant plasmids pcDNA 3.1（＋）-HRE-Egr-1-Dbait was constructed and identified by enzyme digestion and sequencing. The D0, Dq, SF2,α/β value of HeLa cells in normal oxygen were 1.98, 0.93, 0.52, 11.12, respectively. The D0, Dq, SF2 value in hypoxia were 1.74,1.46, 0.43, 15.82. OER value was 0.88. Conclusion We successfully constructed the combined eukaryotic expressional plasmid vector pcDNA3.1（＋）-HRE-Egr-1-Dbait characterized by radiation and hypoxia dual-induced promoter. And this expression vector could significantly increase the radiosensitivity of human cervical cancer HeLa cells in hypoxic response.

关 键 词：乏氧 靶向 放射增敏 宫颈癌 

分 类 号：R737.33[医药卫生—肿瘤] R730.55[医药卫生—临床医学]