端粒酶逆转录酶启动子调控的肿瘤增殖腺病毒CNHK300对肝癌细胞的杀伤作用  

hTERT promoter regulated replication-selective adenovirus CNHK300 in treatment of hepatocellular carcinoma

作　　者：李月敏[1] 宋三泰[1] 江泽飞[1] 张琪[2] 苏长青[2] 徐建明[1] 钱炎珍 钱其军[2] 

机构地区：[1]军事医学科学院附属三七医院肿瘤中心,北京100039 [2]第二军医大学东方肝胆外科医院病毒和基因治疗中心 [3]上海新霁生物科技有限公司

出　　处：《中华医学杂志》2005年第7期468-472,共5页National Medical Journal of China

基　　金：国家863高技术发展计划重点基金资助项目(2001AA217031);国家自然科学基金国际合作资助项目(30120160824)

摘　　要：目的观察端粒酶逆转录酶(hTERT)启动子调控的肿瘤增殖腺病毒CNHK300对肝癌细胞的杀伤作用。方法采用Western印迹检测了腺病毒E1A在细胞中的表达;通过病毒增殖实验和细胞生长抑制实验,验证CNHK300选择性复制和杀伤能力,并与ONYX 015(E1B5 5000蛋白缺失的2型和5型嵌和型腺病毒)和wtAd5(野生型腺病毒)进行比较;用携带绿色荧光蛋白的CNHK300 EGFP感染BJ和Hep3B,直观的观察其增殖过程。结果CNHK300病毒48h在Hep3B和HepGII中的增殖倍数分别为40 625和65 326倍,与wtAd5增殖能力相近,较ONYX 015强几倍至十几倍。在BJ正常成纤维细胞中CNHK300病毒增殖能力减弱,CNHK300病毒48h增殖倍数为180倍,而wtAd5增殖能力仍可高达4000倍。CNHK300在MOI0 5集落形成单位/细胞以下就可以有效地杀伤肝癌细胞, MOI0 002集落形成单位/细胞时就可以杀伤半数Hep3B细胞,较ONYX 015具有更强的肿瘤杀伤能力。CNHK300对正常成纤维细胞的杀伤力明显弱于wtAd5, CNHK300在MOI100集落形成单位/细胞时BJ细胞存活率近50%。结论肿瘤选择性增殖腺病毒CNHK300可选择性地在端粒酶阳性的肝癌细胞中复制,并产生溶瘤作用,在正常细胞中复制能力和杀伤能力明显减弱,且无论选择性和杀伤能力均优于ONYX 015。Objective To evaluate the therapeutic efficiency of replicative adenovirus CNHK300 targeted at telomerase-positive hepatocellular carcinoma. Methods Human liver cancer cell line HepGII and Hep3B, human embryonic kidney cell line 293, and normal human fibroblasts of the line BJ were cultured and added with adenoviruses CNHK300, ONYX-015 (55 000 protein deleted adenovirus), or wtAd5 (wild type 5) with different multiplicity of infection (MOI) for 7 days. 293 cells were used to measure the titer of the filial generation virus from different cells. The cell survival rate was calculated by MTT method 2, 4, 6, and 8 days after. Different cells were added with CNHK300 virus and then the E1A protein in the cytoplasm was measured by western blotting. Fluorescence microscopy was used to observe the CNHK300-EGFP proliferation after the cells were cultured and added with the virus for 1.5 hours.Results^The replicative viruses CNHK300 and wtAd5 proliferated rapidly in HepGII and Hep3B cells since 24 hours after inoculation and proliferated 40625 and 65326 times respectively with a proliferation potential similar to that of the wild-type adenovirus and much higher than that of the ONYX-015 virus. CNHK300 of the MOI of 0.0002 killed half of the cancer cells, especially those of the line Hep3B, within 5~6 days, and CNHK300 virus of the MOI of 0.5 pfu/cell killed almost all the HepGII cells in the 8th day, with a killing power lower than that of the wild-type virus and higher than that of the ONYX-015 cells. The IC_~50 was as low as MOI of 0.002 pfu/cell for the Hep3B cell and was as high as MOI of 100 pfu/cell for the BJ cell. CNHK300 was a less powerful killer of fibroblasts than wild-type virus. E1A expression was shown by western blotting in ~293 cells and CNHK300-infected liver cancer cells, but not in the CNHK300-infected normal human fibroblasts. Fluorescence microscopy showed only isolated fluorescence-positive fibroblasts till the 10th day of infection, but obvious proliferation of CNHK300-EGFP virus since the 3rd day an

关 键 词：肝癌细胞 腺病毒 ONYX-015 端粒酶逆转录酶 集落形成 肿瘤增殖 杀伤 增殖倍数 病毒增殖 调控 

分 类 号：R735.7[医药卫生—肿瘤] R730[医药卫生—临床医学]