携带TRAIL基因的新型溶瘤病毒体外诱导肝癌细胞凋亡  

A novel oncolytic adenovirus carrying human tumor necrosis factor-related apoptosis-inducing ligand gene induces hepatocellular carcinoma cell line apoptosis in vitro

作　　者：刘永靖[1] 陈飞虎[2] 苏长青[3] 王星华[3] 钱炎珍[3] 钱其军[3] 

机构地区：[1]中国人民解放军第105医院胸心外科,安徽省合肥市230031 [2]安徽医科大学药学院,安徽省合肥市230032 [3]中国人民解放军第二军医大学东方肝胆外科医院病毒-基因实验室,上海市200438

出　　处：《世界华人消化杂志》2009年第3期241-246,共6页World Chinese Journal of Digestology

基　　金：南京军区重大专项课题资助项目;No.07Z006;国家自然科学基金国际合作重大资助项目;No.30120160823~~

摘　　要：目的:评价携带TRAIL基因的肿瘤特异性增殖型腺病毒CNHK500-hTRAIL在人肝癌细胞株中介导TRAIL基因表达及对肝癌细胞的凋亡诱导作用.方法:CNHK500-hTRAIL感染人肝癌细胞株HepG2、Hep3B以及人正常肝细胞株WRL-68,通过增殖实验观察病毒的选择性增殖能力,并进行酶联免疫吸附试验(ELISA)检测TRAIL蛋白的表达量,四甲基偶氮唑蓝(MTT)比色法检测其杀伤肝癌细胞的功效,流式细胞术(FCM)检测其对细胞早期凋亡的影响.结果:CNHK500-hTRAIL能选择性地在HepG2、Hep3B细胞内高效增殖;感染72h后HepG2、Hep3B细胞培养上清中TRAIL的表达量分别为167.4、173.22ng/L;在MOI=0.1时,CNHK500-hTRAIL即可明显杀伤HepG2、Hep3B细胞,并选择性地诱导细胞早期凋亡,均显著高于空病毒CNHK500及非增殖型腺病毒Ad-hTRAIL;而对人正常肝细胞株WRL-68则无明显杀伤作用.结论:携带TRAIL基因的肿瘤特异性增殖型腺病毒载体对肿瘤细胞的杀伤能力和目的基因的表达,明显优于单纯的病毒载体及传统的非增殖型腺病毒载体,应用前景广阔。AIM： To evaluate the efficiency of transgene expression and inducing apoptosis on tumorspecific replication-competent adenovirus carrying human tumor necrosis factor-related apoptosis-inducing ligand gene, a novel geneviral therapeutic system CNHK500-hTRAIL, in hepatocellular carcinoma （HCC） cell lines in vitro. METHODS： HCC cell lines HepG2, Hep3B and normal hepatocyte lines WRL-68 were transfected with CNHK500-hTRAIL. Virus replication assay was performed to evaluate the selective replication ability of CNHK500-hTRAIL. ELISA assay was used to detect the transgene expression of TRAIL. The cytotoxicity in cultured HCC and normal ceils was evaluated by Methyl thia- zolyl tetrazolium （MTT）. Flow cytometry （FCM） was used to detect the early apoptotic induced by CNHK500-hTRAIL. RESULTS： CNHK500-hTRAIL was selectively proliferated in the telomerase-positive HCC cell lines HepG2 and Hep3B. 72 hours after infection with CNHK500-hTRAIL, the expression of TRAIL in the supernatant of cultured cell lines HepG2 and Hep3B were 167.4 and 173.22 ng/L, respectively; CNHK500-hTRAIL killed more HepG2 and Hep3B cells significantly with MOI = 0.1 and induced obvious apoptosis but not in WRL-68 even with MOI = 50. The ability of transgene expression and inducing apoptosis were significantly stronger than that of replication-competent adenovirus CNHK500 or replication-defective Ad-hTRAIL. CONCLUSION： Replication-competent adenovirus carrying human TRAIL gene is more effective than simplex oncolytic adenovirus or replication-defective adenovirus carrying human TRAIL gene in both cytotoxicity and efficiency of gene transfer in HCC, and holds great promise in the area of HCC therapy.

关 键 词：基因治疗 腺病毒 肿瘤坏死因子相关凋亡诱导配体 凋亡 肝细胞癌 四甲基偶氮唑蓝 流式细胞术 

分 类 号：R735.7[医药卫生—肿瘤] R373[医药卫生—临床医学]