【摘要】 目的研制出一种新的基因-病毒治疗系统。方法通过基因操作技术将主要晚期启动子(MLP)调控的人干扰素-γ基因插入腺病毒E1A基因受hTERT启动子调控、E1B基因受HRE启动子调控的增殖病毒载体质粒PXC70-HRE-TP的E1A上游,得到腺病毒质粒pSG500-hγ。通过pSG500-hγ与质粒pBHGE3在293细胞中同源重组得到重组病毒CNHK500-hγ。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法检测人干扰素-γ抗癌基因的表达。结果CNHK500-hγ的病毒滴度为4×109pfu/ml,增殖实验结果证实CNHK500-hγ可以选择性地在端粒酶阳性的肝癌细胞中增殖,CNHK500-hγ所携带的人干扰素-γ基因在肝癌细胞株中的表达量(442μg/L)明显高于携带该基因的非增殖型腺病毒载体(120μg/L)Ad-hγ(P<0.005)。结论CNHK500-hγ是一种具备治疗肝癌潜力的新型基因-病毒治疗系统。更多还原

【Abstract】 Objective To develop an double-regulated repplicative adenovirus carrying the human interferon gamma (hγ).Methods The hγ gene regulated by major late promoter (MLP) was cloned into the upstream of E1A of plasmid PXC70-HRE-TP,which E1A gene and E1B gene were driven by telomerase reverse transcriptase promoter and hypoxia response promoter respectively to produce plasmid pSG500-hγ.The plasmid pSG500-hγ was co-transfected with pBHGE3 in 293 cells to generate recombinant adenovirus CNHK500-hγ.Virus titer was measured by TCID50 method.Virus replication assay was performed to evaluate the selective repplication ability of CNHK500-hγ.By ELISA assay the transgene expression of hγ was detected.Results A novel gene-viral therapeutic system CNHK500-hγ was constructed and its titer was 4×109 pfu/ml.Proliferative test revealed that CNHK500-hγ could selectively be proliferated in tumors positive for telomerase.Furthermore,in comparison with non-replicative adenovirus Ad-hγ,the transgene expression of hγ mediated with CNHK500-hγ was significantly higher (P< 0.005).Conclusion The novel gene-viral therapeutic system CNHK500-hγ holds potential for the treatment of HCC.更多还原