多重调控病毒-基因载体的构建及其体外抗肿瘤活性

A novel triple-regulated oncolytic adenovirus carrying human tumor necrosis factor-related apoptosis-inducing ligand gene exerts potent antitumor efficacy on lung cancer in vitro

目的构建-种新型的病毒-基因治疗载体。方法通过克隆技术将人肿瘤坏死因子相关凋亡诱导配体（TRAIL）基因插入质粒载体pBHGE3的E3区，得到由主要晚期启动子（MLP）调控的腺病毒质粒pPE3-hTRAIL，再通过与质粒pSG500在293细胞中进行位点特异性重组得到E1A、E1B基因分别受hTERT启动子与HRE启动子双重调控的重组增殖型腺病毒，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。利用酶联免疫吸附试验（ELISA）检测人TRAIL基因的表达。并进行噻唑蓝（MTT）比色法实验检测其杀伤肿瘤细胞的能力。结果CNHKS00-hTRAIL的病毒滴度为2．39×10^10pfu／ml，增殖实验结果证实CNHK500-hTRAIL可以选择性地在端粒酶阳性的人8市癌细胞A549中增殖，其所携带的人TRAIL基因在A549中的表达量（183．12μg/L）明显高于携带该基因的非增殖型腺病毒载体Ad—hTRAIL（24．53μg/L，P〈0．01）。MTT显示CNHK500-hTRAIL对A549的杀伤能力明显高于携带该基因的非增殖型腺病毒载体Ad—hTRAIL，其半数抑制MOI值分别为17．825、0．197（P〈0．01）。结论CNHK500-hTRAIL是-种具备治疗肺癌潜力的新型病毒-基因治疗系统。

Objective To develop a triple-regulated replicative adenovirus carrying the human TRAIL gene, a novel gene-viral therapeutic system CNHK500-hTRAIL. Methods The human TRAIL gene was cloned into the upstream.of E3 of plasmid pBHGE3 to produce pPE3-hTRAIL that was regulated by major later promoter （MLP）. The plasmid pPE3-hTRAIL was co-transfected with pSG500 in 293 cells by site-specific recombination to generate recombinant conditionally replicating-competent adenovirus CNHK500-hTRAIL,in which E1A gene and EIB gene were driven by human telomerase reverse transcriptase promoter and hypoxia response promoter, respectively. Virus titer was measured by TCID50 method. Virus replication assay was performed to evaluate the selective replication ability of CNHK500- hTRAIL. ELISA assay was used to detect the transgene expression of TRAIL. The cytotoxicity in cultured cancer and normal cells was assayed by MTT. Results A novel gene-viral therapeutic system CNHK500- hTRAIL was constructed and its titer was 2.39 ×10^10 pfu/ml. Proliferative test revealed that CNHKS00- hTRAIL could be selectively proliferated in the telomerase-positive NSCLC cells A549. Furthermore, in comparison with non-replicative adenovirus Ad-hTRAIL, the transgene expression of TRAIL in the supernatant of cultured cells A549 mediated with CNHKS00-hTRAIL was significantly higher （ 183.12 μg/L vs 24.53 μg/L, P 〈 0.01 ）. The MTT assay showed that CNHKS00-hTRAIL killed the A549 ceils more effectively than Ad-hTRAIL,and the inhibitory concentration 50% （IC50） MOI was 0. 197 and 17. 825, respectively. Conclusion The novel gene-viral therapeutic system CNHK500-hTRAIL holds potential for the treatment of NSCLC.