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溶瘤病毒(oncolytic viruses)可选择性地感染肿瘤细胞并在其中复制,使宿主细胞裂解并在体内产生强烈的免疫应答反应,从而抑制或者杀死肿瘤细胞。因其特有的溶瘤性和靶向性,溶瘤病毒成为肿瘤基因治疗的热门领域之一。基于Wnt信号的肿瘤特异性,本研究使用Wnt信号启动子TCF/LEF结合位点序列,调控腺病毒早期基因E1A,并删除E1A基因序列上能与Rb蛋白结合的24 bp片段。将抑癌基因TSLC1插入腺病毒的E3区,形成重组腺病毒Ad.wnt-E1A(Δ24)-TSLC1。通过双荧光素酶报告系统、Western blot实验、MTT法、结晶紫染色法、Hoechst染色实验分别检测Wnt信号活性、肿瘤细胞的存活率和凋亡的变化情况。结果发现,肿瘤细胞较正常肝细胞具有较高的Wnt通路活性和病毒敏感性,其中重组病毒处理Hep G2后杀伤效果显著,并可通过Caspase通路诱导细胞凋亡,为肝癌的临床治疗提供参考。
Oncolytic viruses selectively infect and replicate tumor cells, lyse host cells and produce a strong immune response in vivo, thereby inhibiting or killing tumor cells. Oncolytic virus has become one of the hot areas of tumor gene therapy due to its unique oncolytic and targeting properties. Based on the tumor specificity of Wnt signaling, this study used the Wnt signaling promoter TCF / LEF binding site sequence to regulate the adenovirus early gene E1A and delete the 24 bp fragment of the E1A gene that binds to the Rb protein. The tumor suppressor gene TSLC1 was inserted into the E3 region of adenovirus to form recombinant adenovirus Ad.wnt-E1A (Δ24) -TSLC1. The Wnt signaling activity, tumor cell viability and apoptosis were detected by dual luciferase reporter assay, Western blot, MTT assay, crystal violet staining and Hoechst staining. The results showed that the tumor cells had higher Wnt pathway activity and virus sensitivity than normal liver cells. The recombinant virus could kill Hep G2 cells significantly and induce cell apoptosis through Caspase pathway, which could provide a reference for the clinical treatment of liver cancer.