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E1B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3.25×1010pfu/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±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.

abstractsObjective 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 El A 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 X 1010 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.