携Apoptin和PEG3启动子双特异性溶瘤腺病毒对结直肠癌的抑制作用  

Anti-tumor effects of a dual cancer-specific oncolytic adenovirus carried Apoptin and PEG3 promoter on colorectal cancer in vitro and in vivo

作　　者：陈智飞[1,2] 李一权 胡宁宁[2] 兰添[2] 周晔[2,3] 李霄[2] 孙丽丽[2,4] 金宁一[2] 

机构地区：[1]吉林大学动物医学学院预防兽医学教研室,吉林长春130062 [2]军事医学科学院军事兽医研究所病毒研究室,吉林长春130122 [3]长春中医药大学附属医院检验科,吉林长春130103 [4]吉林省肿瘤医院头颈外一科,吉林长春130012

出　　处：《中国肿瘤生物治疗杂志》2015年第1期16-21,共6页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.81101140);国家科技重大专项子课题(No.2014ZX09304314-002);国家高技术研究发展计划(863计划)资助(No.2012AA02A407);吉林省重点科技攻关项目资助(No.20130206041NY)~~

摘　　要：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 anti-tumor effects of a dual cancer-specific oncolytic adenovirus Ad-ApoptinPEG3p-E1 a on colorectal cancer in vitro and in vivo. Methods: A dual cancer-specific oncolytic adenovirus vector carried Apoptin and PEG3 promoter( Ad-Apoptin-PEG3p-E1a) was constructed. In experiments in vitro,colon cancer SW1116 cells and gastric epithelial GES cells were infected with the constructed adenoviral vector. At various time points after infection,cell viability was assessed by MTT assay,apoptosis,levels of reactive oxygen species( ROS),mitochondrial membrane potential( MMP) and cytochrome C( Cyt C) by flow cytometry. In experiments in vivo,BALB / c mice bearing established subcutaneous transplantation tumors were treated with Ad-Apoptin-PEG3p-E1 a and a control vector respectively. Tumor growth kinetics and mean survival time were assessed. Results: In vitro,Ad-Apoptin-PEG3p-E1 a significantly suppressed proliferation,increased ROS production,reduced MMP levels and enhanced Cyt C release in a dose- and timedependent manner in SW1116 cells,as compared with the control vector. Ad-Apoptin-PEG3p-E1 a infection also resulted in increased apoptosis in SW1116 cells but had no significant effects on GES cells. In vivo,Ad-Apoptin-PEG3p-E1 a inhibited the growth of subcutaneous transplantation tumors significantly and extended the lifespan of the animals( 41. 0 ±0. 7 days),while Ad-PEG3p-E1a-,Ad-Apoptin- and Ad-mock-treated animals had shorter mean survival time( 34. 4 ±1. 6 days,33. 2 ± 1. 2 and 28. 4 ± 1. 4,respectively,P < 0. 05). Conclusion: The dual cancer-specific oncolytic adenovirus vector carrying Apoptin and PEG3 p may offer a promising gene therapy for colon cancer.

关 键 词：溶瘤腺病毒 凋亡素基因 进行性增量基因3 结直肠癌 SW1116细胞 GES细胞 

分 类 号：R735.3[医药卫生—肿瘤]