重组复制缺陷型腺病毒Ad-p53AIP1的抗肿瘤效应及其作用机制  

Anti-tumor effect of recombinant replication-defective adenovirus Ad-p53AIP1 and its related mechanism

作　　者：姜云波[1] 陆应麟[1] 贾海泉[1] 徐元基[1] 陈惠华[1] 刘刚[1] 王妍[1] 杜芝燕[1] 

机构地区：[1]军事医学科学院基础医学研究所病理生物学研究室,北京100850

出　　处：《中国肿瘤生物治疗杂志》2008年第6期508-515,共8页Chinese Journal of Cancer Biotherapy

基　　金：国家重点基础研究发展规划(973)资助项目(No.2002CB513105)~~

摘　　要：目的:探讨外源性p53AIP1(p53-regulated apoptosis-inducing protein 1)基因抗肿瘤效应及其作用机制,以评估p53AIP1基因在肿瘤基因治疗中应用的可行性。方法:构建携带p53AIP1基因的重组复制缺陷型腺病毒Ad-p53AIP1,感染人肝癌细胞HepG2,应用MTT比色法、Western blotting、流式细胞术、罗丹明染色以及电镜等观察外源性p53AIP1基因表达对肿瘤细胞的作用及其相关机制。小鼠皮下接种感染Ad-p53AIP1的小鼠乳腺癌4T1细胞,观察Ad-p53AIP1对肿瘤细胞体内成瘤性的影响;建立4T1细胞皮下移植瘤小鼠模型,直接瘤内多点注射重组腺病毒,观察Ad-p53AIP1的抗肿瘤疗效。结果:感染Ad-p53AIP1的HepG2细胞能够高效表达P53AIP1蛋白。MTT检测显示Ad-p53AIP1对人肝癌细胞HepG2的生长抑制率达50%以上;流式细胞术分析证实p53AIP1使肿瘤细胞周期阻滞于G_2/M期;罗丹明染色、电镜观察以及凋亡相关蛋白PARP表达的检测均证实p53AIP1能诱导肿瘤细胞发生明显凋亡。感染Ad-p53AIP1的肿瘤细胞体内成瘤受到非常明显的抑制(P<0.01),Ad-p53AIP1瘤体注射对移植瘤生长也有明显的抑制作用(P<0.05)。Western blotting以及RT-PCR检测证实Ad-p53AIP1对p53 mRNA表达无影响,能下调mdm2基因的表达;p53AIP1能上调P53蛋白的表达、下调MDM2蛋白的表达水平,同时还影响P21等细胞周期相关蛋白和凋亡相关蛋白的表达。结论:p53AIP1有明显的体内外抗肿瘤作用,其作用机制与其反向调控P53蛋白、调控多种细胞周期和细胞凋亡相关蛋白、诱导细胞周期阻滞和细胞凋亡有关,该基因在肿瘤基因治疗中具有潜在的应用前景。Objective：To investigate the anti-tumor effect of exogenous p53-regulated apoptosis-inducing protein 1 （p53AIP1） and the related mechanism, so as to assess the feasibility of using p53AIP1 in tumor gene therapy. Methods： The recombinant replication-defective adenovirus Ad-p53AIP1 containing p53AIP1 gene was constructed and transfected into human hepatocellular carcinoma HepG2 cell line. The effects and the relevant mechanisms of exogenous p53AIP1 gene on cell growth were examined by MTT, Western blotting, flow eytometry, rhodamine staining and electron microscopy. Nude mice were subcutaneously inoculated with Ad-p53AIP1-infected mouse breast cancer cell line 4T1 to observe the effect of Ad-p53AIP1 on tumorigenesis of tumor cells. 4T1 breast cancer xenograft models were established in mice and intratumoral injections of Ad-p53AIP1 were given to observe the anti-tumor effect of Ad-p53AIP1. Results： Over-expression of p53AIP1 protein was confirmed in HepG2 cells infected with Ad-p53AIP1. Cell growth of HepG2 cells which contain wild-type p53 gene was inhibited by over 50% after infection. Flow cytometry showed transfection with p53AIP1 gene resulted in cell cycle arrest at G2/M. Results of PARP protein examination, rhodamine staining and electron microscopic observation demonstrated that p53AIP1 induced obvious apoptosis in tumor cells. Moreover, the infection with Ad-p53AIP1 significantly inhibited the tumorigenesis of 4T1 cells in vivo （P 〈 0.01 ）and the growth of tumors in vivo after intratumorial injection（P 〈 0.05）. Furthermore, Western blotting and RT-PCR confirmed that Ad-p53AIP1 had no influence on p53 mRNA expression and downregulated mdm2 gene and protein ; it also up-regulated P53 protein expression. In addition, Ad-p53AIP1 could regulate cell cycle-related proteins and apoptosis-related proteins such as P21. Conclusions： Ad-p53AIP1 possess obvious tumor inhibitory effect in vitro and in vivo; the mechanism is related to its regulation of P53 protein, cell cycle and apoptosis-relat

关 键 词：p53AIP1 肝癌细胞 细胞周期 细胞凋亡 抗瘤效应 基因治疗 

分 类 号：R737.5[医药卫生—肿瘤] R730.54[医药卫生—临床医学]