腺病毒介导IL-24-E1A双基因载体的构建及其体外抑制SMMC-7721肝癌细胞生长作用初探

Construction of adenovirus vector expressing IL-24 and E1A and its inhibition of SMMC-7721

摘要:

构建IL-24和E1A双基因腺病毒载体, 获得Ad-IL-24-E1A重组腺病毒子, 分析其体外抑瘤作用。PCR及BglⅡ和SalⅠ酶切获得IL-24, 与空载体构建成pAdTrack-IL-24-IRES。Xho I和EcoR V酶切获得E1A片段, 与pAdTrack-IL-24-IRES连接后成功构建pAdTrack-IL-24-IRES-E1A, 同源重组、包装和扩增获得Ad-IL-24-E1A重组病毒子。用50 MOI重组腺病毒感染 SMMC-7721肝癌细胞, MTT法测定Ad-IL-24-E1A的细胞生长抑制作用; 流式细胞仪分析细胞凋亡情况。本研究成功获得Ad-IL-24-E1A重组病毒子。与其他组相比, Ad-IL-24-E1A明显抑制肿瘤细胞生长, 感染48 h后凋亡率达52%, 而抑制正常细胞作用不明显。研究提示Ad-IL-24-E1A双基因重组载体明显抑制SMMC-7721肝癌细胞生长。

Abstract:

We constructed the recombinant adenovirus vector expressing IL-24 and E1A (Ad-IL-24-E1A) and investigated the inhibition of Ad-IL-24-E1A on SMMC-7721 hepatocellular carcinoma in vitro. We amplified IL-24 gene by PCR using pAdTrack-IL-24 as template. The IL-24 gene was cloned into pAdTrack-IRES at the BglⅡand SalⅠsite to form pAdTrack-IL-24-IRES. E1A digested from pAdTrack-E1A was cloned into the pAdTrack-IL-24-IRES at the Xho I and EcoR V site to form the pAdTrack-IL-24-IRES-E1A. We co-transformed both pAdTrack-IL-24-IRES-E1A and pAdeasy-1 digested by Pme I and packaged to obtain Ad-IL-24-E1A. Ad-IL-24-E1A at 50 MOI infected SMMC-7721 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay determined cell proliferation. Flow cytometry detected Cell apoptosis. The apoptotic rate of SMMC-7721 cells was 52% 48 h after infection with Ad-IL-24-E1A. The result showed that the growth of SMMC-7721 cells was significantly inhibited by Ad-IL-24-E1A at the MOI of 50.