携带mda7／IL24的三调控增殖腺病毒治疗肝癌的体外实验  

Therapeutic effect of triple regulated adenovirus vector expressing mda7/IL24 on liver cancer in vitro

作　　者：陈东锋[1] 那曼丽[1] 苏长青[2] 李林芳[2] 吴红平[2] 钱炎珍[2] 姜丽华[2] 吴孟超[2] 钱其军[1] 

机构地区：[1]浙江理工大学生命科学院新元医学生物研究所,杭州310018 [2]第二军医大学东方肝胆外科医院病毒与基因治疗实验室,上海200438

出　　处：《肿瘤》2008年第5期382-385,共4页Tumor

基　　金：国家自然科学基金资助项目(编号:30672402)

摘　　要：目的:构建缺失E1A区24 bp,由端粒酶反转录酶基因启动子和缺氧基因启动子调控的增殖腺病毒SG600-IL24,研究人白细胞介素24(interleukin 24,IL24)在肝癌细胞内的表达水平和SG600-IL24的增殖情况及其对肝癌细胞的杀伤作用。方法:利用DNA克隆和重组技术,构建SG600-IL24。ELISA检测IL24在肝癌细胞SMMC-7721、BEL-7404和正常成纤维细胞BJ内的表达。半数组织培养感染剂量(50%tissue culture infectious dose,TCID50)法检测SG600-IL24在3种细胞内48和96 h的增殖情况。MTT法和细胞病理效应(cytopathic effect,CPE)染色法检测SG600-IL24在不同MOI值对3种细胞的杀伤作用。结果:IL24在SMMC-7721和BEL-7404细胞内高表达而在BJ细胞内低表达。感染48和96 h后,SG600-IL24在SMMC-7721、BEL-7404和BJ细胞内分别增殖794和7940倍、622和7 810倍、20和200倍。MTT结果显示,杀伤50%和90%的SMMC-7721、BEL-7404和BJ细胞所需SG600-IL24的MOI值分别为0.3和5,3和20,50和150。CPE染色显示,SG600-IL24对肝癌细胞SMMC-7721和BEL-7404有明显杀伤作用,而对BJ细胞无明显影响,并且该病毒的杀伤能力比增殖腺病毒ZD55-IL24和非增殖腺病毒Ad-IL24强。结论:SG600-IL24高效感染肝癌细胞SMMC-7721和BEL-7404后,病毒增殖活跃,IL24的表达量显著升高。SG600-IL24对此2种肝癌细胞有良好的特异性杀伤作用,而对正常细胞没有明显影响,为应用该病毒治疗肝癌的体内研究建立了基础。Objective： To construct an E1A-deleted 24-bp triple regulated replicative adenovirus vector SG600/interleukin24 （IL24）, which was driven by both hTERT promoter and HRE promoter. The level of IL24 in liver cancer cells was determined and the replication capacity of SG600/IL24 and its killing effects on liver cancer cells were observed. Methods： SG600-IL-24 vector was constructed using DNA cloning and recombination techniques. The IL24 gene expression in liver cancer cell lines SMMC-7721 and BEL- 7404 and normal cell line BJ was detected by ELISA assay. The replications of SG600/IL-24 in different cell lines were determined by evaluating TCID50 （50% tissue culture infectious dose） at 48 and 96 h. In vitro cell-killing effects of SG600/IL24 on the three liver cancer cell lines were analyzed by MTT assay and CPE （cytopathic effect） staining method at different MOI values. Results： IL24 was over-expressed in both SMMC-7721 and BEL-7404 cells but was weakly expressed in BJ cells. At 48 and 96 h post infection the replication of SG600/IL-24 were 794 and 7940 folds in SMMC-7721 cells; 622 and 7 810 folds in BEL-7404 cells; 20 and 200 folds in BJ cells. MTT assay showed that the MOI values of SG600/IL24 for killing 50% and 90% cells were 0.3 and 5 for SMMC-7721 cells; 3 and 20 for BEL-7404 cells; 50 and 150 for BJ cells. CPE staining demonstrated that SG600/IL24 had significant killing effects on both liver cancer cells SMMC-7721 and BEL-7404 but had no significant influence on BJ cells. The cell-killing capability of SG600/IL24 was superior than that of replicative adenovirus ZD55/IL24 and non replicative adenovirus Ad-IL24. Conclusion： After SMMC-7721 and BEL- 7404 liver cancer cells are infected with SG600/IL24 at high efficiency, the virus replication is active and the expression of IL24 increases greatly. SG600/IL24 has specific cell-killing effects on the two liver cancer cell lines but has no significant influence on normal cells. This study provides a basis for further investigating the effect

关 键 词：肝肿瘤 实验性 白细胞介素24 增殖性腺病毒 缺氧反应元件 

分 类 号：R735.7[医药卫生—肿瘤] R73-362[医药卫生—临床医学]