人端粒酶逆转录酶启动子介导的E1A和IL-24表达及其对肝癌细胞生长的抑制作用  

Expression of IL-24 and E1A Mediated by hTERT Promoter and Its Inhibiting Effect on SMMC-7721 Growth

作　　者：汪小华[1] 缪竞诚[2] 谢宇锋[2] 盛伟华[2] 韩亚丽[2] 杨吉成[2] 

机构地区：[1]苏州大学附属第一医院护理部,江苏苏州215006 [2]苏州大学医学部基础医学与生物科学学院细胞生物学系,江苏苏州215123

出　　处：《苏州大学学报（医学版）》2011年第4期554-558,568,共6页Suzhou University Journal of Medical Science

基　　金：江苏省高校自然科学基础研究面上项目(08KJB310011)

摘　　要：目的构建人端粒酶逆转录酶(hTERT)启动子介导的E1A和IL-24双基因靶向腺病毒载体,获得Ad-h-E1A-IL-24靶向重组病毒子,并探索其体外抑瘤作用。方法运用PCR扩增MCF-7乳腺癌细胞的DNA,XbaⅠ和HindⅢ酶切获得280 bp hTERT启动子,插入空载体构建成pTrack-hTERT;运用PCR及HindⅢ和XhoⅠ酶切获得E1A,与pTrack-hTERT构成pAdTrack-hTERT-E1A;运用PCR及NotⅠ和SalⅠ酶切获得IL-24,插入pTrack-PP-IRES;XbaⅠ和XhoⅠ酶切获得hTERT-E1A,与pTrack-PP-IL-24-IRES形成pTrack-hTERT-E1A-PP-IL-24-IRES,同源重组、包装和扩增获得Ad-h-E1A-IL-24重组靶向病毒子。用25 MOI重组靶向腺病毒感染SMMC-7721肝癌细胞,MTT法测定Ad-h-E1A-IL-24的细胞生长抑制作用。结果成功构建pTrack-hTERT-E1A-PP-IL-24-IRES,并获得Ad-h-E1A-IL-24重组病毒子。与非靶向双基因比较,Ad-h-E1A-IL-24组可明显抑制肿瘤细胞生长(P<0.05或0.01)。结论Ad-h-E1A-IL-24靶向腺病毒载体的体外抑瘤作用优于非靶向双基因载体。Objective To construct pAd-hTERT-E1A-IL-24 and explore the effect of Ad-h-E1A-IL-24 on anti-hepatocarcinoma（HCC） in vitro.Methods To amplify hTERT promoter by PCR using DNA of MCF-7 breast cancer cells as the template.hTERT promotor was cloned into pTrack at the XbaⅠand Hind Ⅲ site to form pTrack-hTERT,E1A into pTrack-hTERT at the Hind Ⅲ and XhoⅠsite to form pAdTrack-hTERT-E1A,IL-24 into pTrack-PP-IRES at the NotⅠand SalⅠsite.hTERT-E1A digested from pTrack-hTERT-E1A was inserted pTrack-PP-IL-24-IRES at the XbaⅠand XhoⅠsite to get pTrack-hTERT-E1A-PC-IL-24-IRES.pTrack-hTERT-E1A-PP-IL-24-IRES and pAdeasy-1 were co-transformed and packaged to obtain pAdeasy-1-pTrack-hTERT-E1A-PP-IL-24-IRES（Ad-h-E1A-IL-24）.SMMC-7721 cells were infected by Ad-h-E1A-IL-24 at 25MOI to explore the expression of E1A and IL-24 of SMMC-7721 cell infected with Ad-h-E1A-IL-24 by immunohistochemistry.The inhibition of Ad-h-E1A-IL-24 on proliferations of SMMC-7721 cell lines was observed in vitro by MTT assay.Results pAd-hTERT-E1A-IL-24 was successfully constructed.We favorably got the viruses contained with Ad-h-E1A-IL-24 recombinant vectors.IL-24 and E1A were a significantly amount expression in SMMC-7721 HCC infected with Ad-h-E1A-IL-24.Ad-h-E1A-IL-24 had significantly inhibitory effect on the growth of SMMC-7721 HCC.Conclusion hTERT promoter enhanced the synergetic effect of E1A and IL-24 in suppressing SMMC-7721 HCC growth in vitro.

关 键 词：构建 人端粒酶逆转录酶启动子 Ad-h-E1A-IL-24 SMMC-7721肝癌细胞 生长抑制 

分 类 号：R730.5[医药卫生—肿瘤] R730.3[医药卫生—临床医学]