双重调控增殖型腺病毒AdTPHre-hEndo的构建及鉴定  

Construction and identification of a double-regulated replicative adenovirus AdTPHre-hEndo

作　　者：方益锋[1] 单云峰[1] 高申孟[1] 张启瑜[1] 

机构地区：[1]温州医学院附属第一医院肝胆外科,325000

出　　处：《中华肝胆外科杂志》2011年第6期488-491,共4页Chinese Journal of Hepatobiliary Surgery

基　　金：卫生部科研基金资助项目（WKJ2007-2-019）

摘　　要：目的构建新型双重调控肿瘤特异性增殖型腺病毒载体治疗系统。方法通过基因重组技术构建受端粒酶逆转录酶启动子控制腺病毒E1A表达、缺氧反应元件启动子控制EIB表达并携带人内皮抑素基因的质粒pTPHre-hEndo。将质粒pTPHre-hEndo与含有腺病毒右臂的质粒pB-GHE3共转染至293细胞中同源重组得到带抗肿瘤基因的双重调控增殖型腺病毒AdTPHre-hEndo。用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。利用ELISA法检测人内皮抑素抗癌基因的表达。结果成功构建了由双重调控的选择增殖型腺病毒AdTPHre-hEndo，病毒滴度为3．25×10＾10pfu／ml。增殖实验结果证实AdTPHre-hEndo可以选择性地在端粒酶阳性的胰腺癌细胞中增殖。随着感染时间的延长，肿瘤细胞培养上清液中内皮抑素表达量不断增加，第7天达（310．25士1．41）ng／ml，明显高于携带该基因的非增殖型腺病毒载体（112．53±4．41）ng／ml。结论AdTPHre-hEndo具有高效表达人内皮抑素和在胰腺癌细胞内增殖的能力，为胰腺癌的生物治疗提供了一种新型的基因-病毒治疗系统。Objective To develop a double-regulated replicative adenovirus carrying the Human endostatin gene（hEndo）. Methods The plasmid pTPHre-hEndo was constructed by gene engineering technique, carrying human endostatin gene, in which E1A gene and E1B gene were driven by human telomerase reverse transcriptase （hTERT） promoter and hypoxia response element （HRE） promoter, respectively. The pTPHre-hEndo was co-transfected with pBHGE3 in 293 cells to generate recombinant adenovirus AdTPHre-hEndo. Virus titer was measured by the TCID50 method. Virus replication assay was performed to evaluate the selective replication ability of AdTPHre-hEndo. The transgene expression of endostatin was detected by ELISA assay. Results A novel gene-viral therapeutic system AdTPHre-hEndo was constructed by gene engineering technique and its titer was 3.25 × 10^10 pfu/ml. Proliferative test revealed that AdTPHre-hEndo could proliferate selectively in telomerase positive tumors. Furthermore, in comparison with non-replicative adenovirus Ad-hEndo, the transgene expression of endostatin mediated with AdTPHre-hEndo was significantly increased （P 〈0.01 ）. Conclusion The novel gene-viral therapeutic system AdTPHre-hEndo has the capacity to replicate in pancreatic cancer cells and expresses the endostatin efficiently, and may provide a new strategy for pancreatic cancer gene therapy.

关 键 词：腺病毒 端粒酶逆转录酶 缺氧反应元件 内皮抑素 

分 类 号：R73[医药卫生—肿瘤]