多重调控病毒.基因载体的构建及其体外抗肿瘤活性

目的 构建一种新型的病毒.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pSGS00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 CNHK500-hTRAIL的病毒滴度为2.39×1010pfu/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肺癌细胞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).MTr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 CNHKS00一hTRAIL是一种具备治疗肺癌潜力的新型病毒-基因治疗系统.

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

Objective To develop a triple-regnlated 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 eo-transfeeted with pSGS00 in 293 cells by site-specific recombination to generate recombinant conditionally replicating-competent adenovirus CNHK500-hTRAIL,in which EIA gene and E1B gene were driven by human telomerase reverse tran-seriptase promoter and hypoxia response promoter, respectively.Virus titer was measured by TCID50 meth-od.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 eytotoxicity in cultured cancer and normal cells was assayed by MTT.Results A novel gene-viral therapoutie system CNHK500-hTRAIL was constructed and its titer was 2.39 x 1010 pfu/ml.Proliferative test revealed that CNHK500-hTRAIL eould be selectively proliferated in the telomerase-positive NSCLC cells A549.Furthermore, in comparison with non-replieative adenovirus Ad-hTRAIL, the transgene expression of TRAIL in the suporna-tant of cultured cells A549 mediated with CNHK500-hTRAIL was significantly higher (183.12 μg/L vs 24.53 μg/L,P <0.O1).The MTT assay showed that CNHK5OO-hTRAIL killed the A549 cells more effec-tively than Ad-hTRAIL,and the inhibitory concentration 50% (IC50) MOI was 0.197 and 17.825, re-speetively.Conclusion The novel gene-viral therapeutic system CNHK500-hTRAIL holds potential for the treatment of NSCLC.