论文部分内容阅读
目的:研究5/35嵌合型溶瘤腺病毒SG635在体外对肝癌HepG2和SMMC-7721细胞的特异性杀伤作用。方法:将SG600载体中5型腺病毒(Ad5)纤毛蛋白的knob和shaft结构域替换为35型腺病毒(Ad35)纤毛蛋白的相应结构域,构建成5/35嵌合型溶瘤腺病毒SG635。流式细胞术检测5/35嵌合型腺病毒Ad5/35-EGFP对HepG2和SMMC-7721细胞的感染效率,体外病毒增殖实验观察溶瘤腺病毒SG635的增殖能力,Western blotting检测SG635感染后肝癌细胞中E1A蛋白的表达,CCK-8实验检测SG635对肝癌HepG2和SMMC-7721细胞的杀伤作用。结果:在肝癌HepG2和SMMC-7721细胞中,Ad5/35-EGFP的感染效率明显强于5型腺病毒Ad5-EGFP;5/35嵌合型溶瘤腺病毒SG635在HepG2和SMMC-7721细胞中72 h的增殖倍数高于5型溶瘤腺病毒SG600(15 848.93vs6 309.57,6 309.57vs5 011.87,均P<0.01),而在人正常成纤维细胞BJ中几乎不增殖。SG635感染后,HepG2和SMMC-7721细胞中E1A蛋白表达高于SG600感染,在BJ中则无E1A表达。在一定MOI范围内,SG635对于HepG2细胞和SMMC-7721细胞的杀伤作用逐渐增强,且杀伤率明显强于SG600(MOI为1时,90%vs60%;MOI为10时,90%vs50%),对BJ无杀伤作用。结论:5/35嵌合型溶瘤腺病毒SG635能够高效感染并特异性杀伤肝癌细胞,具有较好的靶向性和安全性。
Objective: To study the specific killing effect of 5/35 chimeric oncolytic adenovirus SG635 on HepG2 and SMMC-7721 cells in vitro. METHODS: The knob and shaft domains of adenovirus type 5 (Ad5) of SG600 were replaced by the corresponding domains of adenovirus type 35 adenovirus (Ad35) and constructed into 5/35 chimeric oncolytic adenovirus SG635 . The infection efficiency of 5/35 chimeric adenovirus Ad5 / 35-EGFP on HepG2 and SMMC-7721 cells was detected by flow cytometry. The proliferative ability of oncolytic adenovirus SG635 was observed by in vitro virus proliferation assay. The hepatocellular carcinoma Cell E1A protein expression, CCK-8 test SG635 detection of HepG2 and SMMC-7721 cells killing effect. Results: Infection of Ad5 / 35-EGFP in HepG2 and SMMC-7721 cells was significantly stronger than that of adenovirus Ad5-EGFP. In the HepG2 and SMMC-7721 cells, 5/35 chimeric oncolytic adenovirus SG635 The multiplication rate of 72h was higher than that of type 5 oncolytic adenovirus SG600 (15 848.93 vs6 309.57,6 309.57vs5 011.87, all P <0.01), but hardly proliferated in human normal fibroblasts BJ. After SG635 infection, the expression of E1A protein in HepG2 and SMMC-7721 cells was higher than that in SG600, but not in BJ. In a certain range of MOI, the killing effect of SG635 on HepG2 cells and SMMC-7721 cells gradually increased and the killing rate was significantly higher than that of SG600 (90% vs 60% at MOI of 1, 90% vs 50% at MOI of 10) BJ no killing effect. Conclusion: The 5/35 chimeric oncolytic adenovirus SG635 can efficiently and specifically kill hepatoma cells with good targeting and safety.