双启动子调控的条件复制腺病毒制备及其体外抑瘤作用

Construction and development of a conditional replication-competent adenovirus driven by double tumor specific promoter and its oncolytic effect in Vitro

目的:构建并制备survivin及hTERT双启动子调控的条件复制腺病毒,探讨其特异性溶瘤作用。方法:PCR方法分别扩增肿瘤特异性survivin及hTERT启动子,分别克隆入腺病毒载体pXC1的两个复制必需基因E1A和E1B序列上游启动子区,构建出双肿瘤特异性启动子调控的条件复制腺病毒载体pXC1-SP-TP;脂质体法与pBHGE3骨架质粒共转染293E细胞进行重组腺病毒包装,稀释法测定腺病毒滴度;应用MTT、活细胞计数等方法观察其对肝癌细胞HepG2的特异性溶瘤作用并以正常人的血管内皮细胞ECV304作为对照。结果:测序及双酶切鉴定结果证实,成功构建了双肿瘤特异性启动子调控复制腺病毒载体;在293E细胞中获得了重组腺病毒Ad-SP-TP,滴度测定显示病毒滴度达到3.9×1010TCID50/ml;MTT结果显示,Ad-SP-TP可有效抑制肝癌细胞增殖而对正常细胞无增殖抑制作用;活细胞计数及细胞形态观察结果显示,重组腺病毒在肝癌细胞中选择性复制并发挥溶细胞作用。结论:双启动子调控的腺病毒具有显著的溶瘤作用但对正常人血管内皮细胞不发挥溶细胞作用,实验结果为肝癌靶向治疗提供了更为良好的条件复制型病毒载体及新的治疗策略。

Objective： To construct conditionally replicating adenovirus （CRAd） which is driven by two tumor spe- cific survivin and hTERT promoters, and then explore its specific oneolytic effect on human hepatoeellular carcinoma. Methods：The survivin and hTERT gene promoter regions were amplified by polymerase chain reaction and cloned into promoter regions of EIA and EIB gene of pXC1 vector respectively,to generate the plasmid pXC1 - SP - TP. Then pXC1 -SP -TP was cotransfected into 293E cells with pBHGE3 by LipofectamineTM 2000 to get packaged recombi- nant adenovirus of Ad - SP - TP. The titer of Ad - SP - TP was detected by TCID50. Specific oneolytie effect was in- vestigated in HepG2 hepatoma carcinoma cell by MTT and viable count methods, and normal vascular endothelial cells ECV304 were uesd as control. Results： Restriction endonuclease digestion analysis and DNA sequencing results showed that the conditionally replicating adenovirus vector was constructed in the sequence as designed. Recombinant adenovirus was obtained in 293E cells and virus titer was 3.9 ×10^10 TCID50/ml. MTT results showed that Ad - SP - TP could effectively inhibit proliferation of hepatoma carcinoma cell while no proliferation inhibition was observed in normal cells. Viable count and cell morphology results showed that recombinant adenovirus selectively replicating and inducing oncolytic effect in hepatoma carcinoma cells. Conclusion： Dual - promoter regulated adenovirus has signifi- cant oncolytic effect in hepatoma carcinoma cells compared with normal vascular endothelial cells. This study might provide a better CRAd vector and a new treatment strategy for liver cancer targeting therapy.