基因-病毒治疗系统CNHK500-p53的构建和体外初步研究  

CONSTRUCTION OF A NOVEL GENE-VIRAL THERAPEUTIC SYSTEM CNHK500-p53 AGAINST LIVER CANCER

作　　者：赵红川[1] 张琪[1] 陈颖华[1] 陆敏强[1] 杨扬[1] 蔡常洁[1] 陈规划[1] 

机构地区：[1]中山大学附属第三医院,广州510630

出　　处：《肝胆外科杂志》2006年第6期454-457,共4页Journal of Hepatobiliary Surgery

基　　金：国家973项目资助;项目编号:2003CB515507

摘　　要：目的构建一种新的基因-病毒治疗系统。方法通过基因操作技术将目的基因表达盒mCMV启动子+p53基因+SV40polyA插入腺病毒E1A基因受端粒酶逆转录酶(hTERT)启动子调控、E1B基因受缺氧反应元件(HRE)启动子调控的增殖病毒载体质粒pSG500的E1A下游,得到腺病毒质粒pSG500-p53;通过pSG500-p53与5型腺病毒右臂的质粒pBHGE3在293细胞中同源重组得到重组病毒CNHK500-p53;利用Westernblot和ELISA法检测病毒E1A、E1B基因和p53抑癌基因的表达;TCID50方法测定病毒滴度;通过增殖实验观察重组病毒的选择性增殖能力;应用细胞病理效应(CPE)实验检测病毒抗肿瘤作用。结果CNHK500-p53的病毒滴度为2×1010pfu/ml,增殖实验证实CNHK500-p53可以选择性地在端粒酶阳性和缺氧微环境的肝癌细胞中增殖,CNHK500-p53所携带的p53基因在肝癌细胞株中的表达量388±34.6μg/L明显高于非增殖型腺病毒载体Ad-p53的76.3±13.17μg/L(P<0.005),并显示了较强的抗肿瘤效应。结论CNHK500-p53是一种具备治疗肝癌潜力的新型基因-病毒治疗系统。Objective To develop an double-regulated replicative adenovirus carrying the p53 gene against liver cancer. Methods The p53 gene regulated by mCMV promoter was cloned into the downstream of E1A of pSG500, which E1A gene and E1B gene were driven by human telomerase reverse transcriptase （hTERT） promoter and hypoxia response element （HRE） promoter respectively to produce pSG500-p53. The pSG500-p53 was co-transfected with pBHGF_3 in 293 cells to generate recombinant adenovirus CNHK500- p53, and was verified by PCR. The E1A, E1B protein of virus was measured by Western blot analysis, The transgene expression of 1353 was detected by ELISA assay. Virus titer was measured by TCID50 method. Virus replication assay was performed to evaluate the selective replication ability of CNHK500-p53. Cytopathic effect （CPE） was used to detect viral antitumor activity. Results A novel gene-viral therapeutic system CNHK500-p53 was constructed by gene engineering technique and its titer was 2 ×10^10 pfu/ml. Western blot analysis showed CNHK500 E1A protein could be detected in tumor cells but not in normal cells under normoxia. E1B protein could only be detected under hypoxia condition at a MOI of 1. Furthermore, in comparison with non-replicative adenovirus Ad-p53, the transgene expression of p53 mediated with CNHK500-p53 was significantly higher （388 ±34. 6 μg/L versus 76. 3 ± 13.17 μg/L, P 〈 0. 005）. Proliferative test revealed that CNHK500-p53 could selectively be proliferated in tumors positive for telomerase. The replicafive multiples of CNHK500-p53 in HepG Ⅱ , Hep3B and SMMC7721 after 48h of virus proliferation were 12700, 311380 and 213649 fold respectively,similar to those of WtAdS. However, CNHK500-p53 demonstrated more significant attenuated replicative ability in normal cell lines than WtAdS. CNHK500-p53 replicated only 5.6 fold at 48h, while the WtAd5 still reached 5451.5 fold. CNHK500- p53 could kill liver cancer cells but spare normal cells at a low MOI. Conclusion The novel gene-viral therapeuti

关 键 词：腺病毒 端粒酶逆转录酶 缺氧反应元件 P53基因 肝癌 

分 类 号：R657.3[医药卫生—外科学]