survivin启动子有效片段的克隆及其在胃癌细胞AGS中的转录活性研究  

Cloning of survivin promoter and its specific transcriptional activities in gastric cancer cell line AGS

作　　者：徐迪晖[1] 王继德[1] 姜泊[1] 张以洋[1] 智发朝[1] 陈利[1] 

机构地区：[1]南方医科大学附属南方医院消化病研究所,广东广州510515

出　　处：《中国现代医学杂志》2006年第18期2734-2737,共4页China Journal of Modern Medicine

摘　　要：目的克隆survivin启动子的有效片段,并检测它在人胃癌细胞株AGS和人正常胃上皮细胞GES-1中的转录活性,为该启动子在胃癌的靶向性基因治疗中奠定基础。方法用PCR扩增survivin基因的启动子片段,克隆入荧光素酶报告质粒pGL3-Basic,构建pGL3-sur-pro重组质粒,用脂质体法瞬时转染AGS及GES-1中,检测survivin启动子在细胞中的转录活性。同时构建含CMV启动子的pGL3-CMV-pro重组质粒作为阳性对照。结果琼脂糖凝胶电泳显示PCR扩增的survivin启动子片段长约440bp,测序结果与GenBank中survivin启动子序列一致。成功构建了pGL3-sur-pro重组质粒。荧光素酶活性检测显示,survivin启动子片段在细胞株AGS中有较高转录活性,而在GES-1中几无转录活性。结论本实验克隆的survivin启动子有效片段在胃癌细胞株AGS中有转录活性,在正常胃上皮细胞中无活性。其有可能作为调控元件用于胃癌的靶向性基因治疗。[Objective] To clone DNA sequence of survivin promoter, and study its transcriptional activities in human gastric cancer cell and normal gastric cell. [Methods] The fragment of survivin promoter was acquired by PCR amplification and inserted into luciferase reporter vectors （pGL3-Basic） to reconstruct a recombinant plasmid named pGL3-sur-pro. Then the reconstructed plasmid was transiently transfected into human gastric cancer cell lines AGS and normal gastric cell lines GES-1. The transcriptional activities of survivin promoter in various cells were determined by measuring the luciferase activities after 48 hours of transfection. A pGL3-CMV-pro, containing the CMV promoter controlled luciferase gene, was used as positive control. [Results] Electrophoresis demonstrated that cloned survivin promoter was about 440 bp and DNA sequencing showed a same sequence as registered in GenBank, and the length was 440 bp. The recombinant plasmid pGL3-sur-pro was comfirmed by double digestion and PCR method with correct results. Luciferase assay showed there were transcriptional activities in the AGS, but not in the GES-1. [Conclusion] The survivin promoter cloned in this study is more highly activated in gastric cancer cell than in normal cell. This makes it a potential candidate promoter in the gene therapy of gastric cancer.

关 键 词：胃癌 SURVIVIN 启动子 基因治疗 

分 类 号：R735.2[医药卫生—肿瘤]