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目的:探讨携带凋亡素基因(Apoptin)和进行性增量基因3(progression-elevated gene 3,PEG3)启动子的双特异性溶瘤腺病毒Ad-Apoptin-PEG3p-E1a对结直肠癌的体内、外抑制效果。方法:以Ad-Apoptin-PEG3p-E1a、Ad-PEG3p-E1a、AdApoptin、Ad-mock(均为前期工作构建)分别感染人结直肠癌SW1116细胞和胃黏膜上皮GES细胞,MTT法检测感染后细胞的增殖水平;流式细胞术检测细胞的凋亡情况;采用DCFH-DA和Rho123染色流式细胞术分别检测细胞活性氧水平和线粒体膜电位,Western blotting检测细胞色素C的释放。建立BALB/c小鼠结直肠癌CT26细胞皮下移植瘤模型,观察Ad-ApoptinPEG3p-E1a对结直肠癌皮下移植瘤的抑制作用。结果:Ad-Apoptin-PEG3p-E1a抑制SW1116细胞增殖,并具有一定的剂效和时效关系,以MOI=100感染72 h后抑制率达到(56.23±6.64)%,其抑制能力明显强于Ad-p EG3p-E1a和Ad-Apoptin等对照组(均P<0.05)。Ad-Apoptin-PEG3p-E1a感染导致SW1116细胞活性氧水平上调、线粒体膜电位下降以及细胞色素C释放增加,最终诱导SW1116细胞凋亡,凋亡率为(37.97±3.78)%,但对正常GES细胞的上述各项指标均无明显影响。Ad-ApoptinPEG3p-E1a能显著抑制小鼠皮下移植瘤生长(P<0.05);有效延长模型动物平均生存期,Ad-Apoptin-PEG3p-E1a治疗组平均生存期为(41.0±0.7)d,显著高于Ad-PEG3p-E1a的(34.4±1.6)d、Ad-Apoptin的(33.2±1.2)d和Ad-mock的(28.4±1.4)d(均P<0.05)。结论:Ad-Apoptin-PEG3p-E1a可以特异性有效抑制结直肠癌细胞增殖及皮下移植瘤的生长。
Objective: To investigate the effect of Ad-Apoptin-PEG3p-E1a, a dual-oncolytic adenovirus carrying Apoptin and progression-elevated gene 3 (PEG3) promoters, , Outside inhibitory effect. Methods: Human colorectal cancer SW1116 cells and gastric epithelial GES cells were infected with Ad-Apoptin-PEG3p-E1a, Ad-PEG3p-E1a, AdApoptin and Ad-mock . The cell apoptosis was detected by flow cytometry. The levels of ROS and mitochondrial membrane potential were detected by DCFH-DA and Rho123 staining respectively. The release of cytochrome C was detected by Western blotting. The BALB / c mice model of colorectal cancer CT26 cells subcutaneous transplantation tumor was established to observe the inhibitory effect of Ad-ApoptinPEG3p-E1a on subcutaneous xenografts of colorectal cancer. Results: Ad-Apoptin-PEG3p-E1a inhibited the proliferation of SW1116 cells and had a dose-effect and time-dependent relationship. The inhibition rate of SW1116 cells was 56.23 ± 6.64% p EG3p-E1a and Ad-Apoptin and other control groups (all P <0.05). Infection with Ad-Apoptin-PEG3p-E1a resulted in the up-regulation of reactive oxygen species (ROS), the decrease of mitochondrial membrane potential and the increase of cytochrome C release in SW1116 cells, which eventually induced the apoptosis of SW1116 cells with a apoptotic rate of (37.97 ± 3.78)%, Of the above indicators have no significant impact. Ad-ApoptinPEG3p-E1a could significantly inhibit the growth of subcutaneous xenografts in mice (P <0.05), and prolong the average survival time of model animals. The average survival time of Ad-Apoptin-PEG3p-E1a treatment group was (41.0 ± 0.7) d, (34.4 ± 1.6) d for Ad-PEG3p-E1a, (33.2 ± 1.2) d for Ad-Apoptin and (28.4 ± 1.4) d for Ad-mock (all P <0.05). Conclusion: Ad-Apoptin-PEG3p-E1a can specifically inhibit the proliferation of colorectal cancer cells and the growth of subcutaneous xenografts.