hNIS/TK基因共转染介导^(131)Ⅰ/GCV对肝癌细胞株的毒性作用  

Toxicity of^(131)I/GCV to hepatocarcinoma following co-expression of human sodium iodide symporter gene and herpes simplex virus thymidine kinase gene

作　　者：郭国英[1] 陈立波[2] 刘天津[3] 郭礼和[3] 朱瑞森[2] 

机构地区：[1]浙江理工大学新元医学与生物技术研究所,浙江杭州310018 [2]上海交通大学附属第六人民医院,上海200233 [3]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200031

出　　处：《中国癌症杂志》2010年第11期826-831,共6页China Oncology

基　　金：国家自然科学基金项目(No:30700187);上海市科委青年科技启明星计划(No:08QA14040)

摘　　要：背景与目的:与传统的单纯疱疹病毒胸苷激酶(herpessimplex virus thymidine kinase,HSV-TK)/丙氧鸟苷(ganci clovir,GCV)肿瘤自杀系统相比,人钠/碘同向转运体(human sodium-iodidesymporter,hNIS)是近年来发现的新型治疗基因。然而,两者的疗效尚需进一步提高。本研究旨在探索hNIS介导的放射性核素体系与HSV TK/GCV自杀基因体系对肝癌细胞的联合毒性作用。方法:构建EF1-α启动子调控下的hNIS的表达载体与CMV启动予调控下的GFP和HSV-TK共表达载体,慢病毒包装后转染肝癌细胞HepG2,荧光显微镜下观察重组肿瘤细胞HepG2/NTG荧光蛋白的表达;采用RT-PCR技术进一步检测目的基因hNIS和HSV-TK的表达情况;MTT检测GCV对HepG2/NTG的杀伤作用;摄碘试验及流出试验评价HepG2/NTG对碘的摄取及滞留情况;最后通过克隆形成实验评价GCV和^(131)I对HepG2/NTG的单独及联合杀伤作用。结果:RT-PCR技术、荧光显像证实hNIS、GFP和HSV-TK基因在肝癌细胞中成功表达;与对照组相比,HepG2/NTG的摄碘高出76倍,20 mi n摄碘率最高,其后表现出碘外流,有效半衰期不到10 min,同时这种碘摄取能被NaC1O_4特异性抑制。GCV或^(131)I对HepG2/NTG的毒性作用呈现剂量依赖性:50μg/mL GCV作用72 h后,实验组细胞的存活率仅为(33.98±2.71)%;放射性浓度为7.4 MBq/mL的^(131)I处理7 h后,细胞的存活率仅为(41.17±0.72)%;GCV和^(131)I联合作用后,细胞的存活率仅为(8.55±1.22)%,较单一药物作用细胞存活率下降了6倍。结论:^(131)I/GCV对重组肝癌细胞产生显著的毒性作用,提示慢病毒介导hNIS/TK基因共转染介导肿瘤的放射化学治疗是可行的。Background and purpose： Herpes simplex virus thymidine kinase （HSV-TK） gene/ganciclovir （GCV） has been widely used as a form of traditional gene therapy whereas the sodium-iodide symporter gene （NIS） has been found to be a novel therapeutic gene. However, the therapeutic effects of both genes need to be enhanced. The purpose of this study was to investigate the feasibility of radiochemotherapy for hepatocarcinoma via the co-expression of the human sodium iodide symporter gene and the herpes simplex virus thymidine kinase gene. Methods： HepG2 cells were named HepG2/NTG after they were stably transfected with NIS, TK and GFP genes via a recombinant lentiviral vector. Gene expression was examined through fluorescence imaging, RT-PCR, MTT assay, and iodide uptake. The therapeutic effects were further assessed by clonogenic assay. Results： Gene expression was demonstrated through fluorescence imaging, RT-PCR, MTT assay, and radioiodide uptake. Within 20 minutes, stably transfected cells had a concentration of 125I up to 76-fold higher than in the wild-type cells while the efflux happened with a T1/2eff in under 10 minutes. The iodide uptake in HepG2/NTG cells was specifically inhibited by sodium perchlorate. Dosedependent toxicity to HepG2/NTG cells by either GCV or 131I was revealed through both clonogenic and MIT assay.

关 键 词：放射治疗 基因治疗 肝癌 钠/碘同向转运体 单纯疱疹病毒胸苷激酶 

分 类 号：R73-362[医药卫生—肿瘤]