HSV-tk基因表达载体的构建及其对A549细胞的杀伤作用  

Construction of pcDNA3.0-tk eukaryotic expression vector and its induced suicidal effect on transfected human lung cancer cells

作　　者：高慧[1] 杨耀琴[1] 陶惠红[1] 赵培林[1] 

机构地区：[1]同济大学医学院肿瘤研究所,上海200092

出　　处：《同济大学学报（医学版）》2009年第5期24-28,31,共6页Journal of Tongji University(Medical Science)

基　　金：上海市教委科研创新重点项目(08ZZ19)

摘　　要：目的构建自杀基因HSV-tk表达质粒,研究自杀基因疗法对人肺癌细胞的杀伤作用,探讨其治疗肺癌的可行性。方法PCR扩增tk基因带EcoRⅠ、XhoⅠ酶切位点,然后构成建到pcDNA3.0载体中成pcDNA3.0-tk;将pcDNA3.0-tk转染A549肺癌细胞,应用细胞生长曲线和MTT法测定前药GCV对A549细胞的杀伤作用。结果成功获得pcDNA3.0-tk克隆,RT-PCR、Western印迹法证明A549细胞转染pcDNA3.0-tk后可表达自杀基因,GCV能特异性地杀伤A549/tk细胞。结论HSV-tk基因表达质粒构建成功;HSV-tK/GCV系统对肺癌A549细胞有一定的杀伤作用。Objective To construct eukaryotic expression vector containing suicide gene HSV-tk and to investigate the cytotoxic effects of tk gene on transfected human lung cancer cells A549 after prodrug treatment. Methods HSV-tk gene fragment with EcoR I , Xho I restriction sites was obtained by polymerase chain reaction （PCR） and followed by cloning the fragment into the corresponding sites of pcDNA3.0 to obtain pcDNA3.0-tk eukaryotic expression vector. Recombinant plasmid pcDNA3.0-tk was transfected into A549 ceils with Lipofectin-mediated method. The cytotoxic effect of a prodrug, Ganciclovir, on A549/tk cells was analyzed by cell growth curve and MTT assay. Results A549/tk cells expressed suicide tk gene on the levels of RNA and protein. Transfection with pcDNA3.0-tk conferred cytotoxicity to lung cancer cells in presence of Ganciclovir. Conclusion pcDNA3.0-tk eukaryotic expression vector has been successfully constructed, and HSV-TK/GCV system can effectively kill human lung cancer cells A549 in vitro.

关 键 词：自杀基因 胸苷激酶 基因治疗 肺肿瘤 

分 类 号：Q782[生物学—分子生物学]