腺病毒介导ING4或/和P53基因表达对人肺腺癌细胞的生长抑制作用  

Inhibition effect of expressions of ING4 and/or P53 mediated by adenovirus on growth of human lung adenocarcinoma cell

作　　者：何峰[1] 李帅[1] 朱霞霞[1] 杨吉成[1] 盛伟华[1] 缪竞诚[1] 

机构地区：[1]苏州大学医学部细胞教研室,苏州215123

出　　处：《上海交通大学学报（医学版）》2015年第7期953-960,共8页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(81001016)~~

摘　　要：目的在本室已构建Ad.RGD空载体和Ad.RGD-ING4腺病毒基础上,再构建Ad.RGD-P53和Ad.RGD-ING4-P53单双基因重组腺病毒,研究其对人肺腺癌细胞的生长抑制作用。方法 PCR克隆P53基因,构建pAd.RGD-P53和pAd.RGD-ING4-P53同源重组腺病毒质粒,用QBI-293A细胞进行包装扩增和检测效价。将Ad.RGD、Ad.RGD-ING4、Ad.RGD-P53、Ad.RGD-ING4-P53分别感染A549细胞后,流式细胞仪检测各组重组腺病毒对A549细胞的感染效率。Western blotting检测A549和PC-9细胞中ING4或/和P53蛋白的表达。MTT法检测A549细胞生长,流式细胞仪检测各组A549和PC-9细胞的凋亡率。real-time PCR检测A549细胞内凋亡相关基因Caspase-3、BAX及BCL-2 mRNA的表达水平。结果 PCR和酶切鉴定结果表明：成功构建了pAd.RGD-P53和pAd.RGD-ING4-P53同源重组腺病毒质粒,包装扩增后经效价检测可达1×109~1×1010pfu/m L;各组重组腺病毒感染A549细胞后,感染效率可达90%~95%。Western blotting检测结果表明ING4或/和P53蛋白可在A549和PC-9细胞中成功表达。MTT法检测发现ING4或/和P53单双基因重组腺病毒均能明显抑制A549细胞生长,流式细胞仪检测表明ING4或/和P53基因表达均可诱导A549和PC-9细胞凋亡,且Ad.RGD-ING4-P53双基因重组腺病毒组较Ad.RGD-ING4和Ad.RGD-P53单基因组更为明显（P〈0.05）。real-time PCR检测表明腺病毒介导的ING4或/和P53基因表达能够上调A549细胞内Caspase-3、BAX mRNA表达,下调BCL-2 mRNA的表达,且双基因组较单基因组更为明显（P〈0.05）。结论成功构建了Ad.RGD-P53和Ad.RGD-ING4-P53重组腺病毒。各重组腺病毒目的基因表达均能明显抑制人肺腺癌细胞的生长,诱导细胞凋亡,且双基因组较单基因组效果更优,其分子机制可能与细胞内凋亡相关基因Caspase-3、BAX表达上调及BCL-2表达下调有关。Objective To construct recombinant adenovirus vectors carrying Ad.RGD-P53 and/or Ad.RGDING4-P53 based on constructed empty vector Ad.RGD and adenovirus Ad.RGD-ING4 and explore the inhibition effect on human lung adenocarcinoma cells.Methods The P53 gene was obtained by PCR and pAd.RGD-P53 and pAd.RGD-ING4-P53 homologous recombinant adenovirus plasmids were constructed.Plasmids were packaged and amplified by using QBI-293 A cell and the titer was detected.A549 cells were infected by Ad.RGD,Ad.RGD-ING4,Ad.RGD-P53,and Ad.RGD-ING4-P53,respectively.The efficiency of infection of recombinant adenoviruses towards A549 cells was detected by flow cytometry.Protein expressions of ING4 and/or P53 in A549 and PC-9 cells were detected by Western blotting.The growth of A549 cells was detected by MTT and the apoptosis of A549 and PC-9 cells were detected by flow cytometry.The mRNA expressions of apoptosis related genes Caspase-3,BAX,and BCL-2 in A549 cells were detected by real-time PCR.Results The results of PCR and enzyme digestion showed that pAd.RGD-P53 and pAd.RGD-ING4-P53 homologous recombinant adenovirus plasmids were successfully constructed.The titer detection reached1×109-1 ×10（10） pfu/mL after being packaged and amplified.The efficiency of infection of recombinant adenoviruses towards A549 cells was 90%-95%.Results of Western blotting showed that ING4 and/or P53 protein successfully expressed in A549 and PC-9 cells.MTT found that ING4 and/or P53 recombinant adenoviruses significantly inhibited the growth of A549 cells.Flow cytometry confirmed that expressions of ING4 and/or P53 could induce the apoptosis of A549 and PC-9 cells.The induction of apoptosis by double gene recombinant adenoviruses was more significant than that of single gene recombinant adenoviruses（P0.05）.Results of realtime PCR showed that expressions of ING4 and/or P53 induced by adenoviruses up-regulated mRNA expressions of Caspase-3 and BAX in A549 cells and down-regulated mRNA expression of BCL-2.The regulation of expressions of Caspase-3

关 键 词：RGD腺病毒 ING4基因 P53基因 人肺腺癌细胞 

分 类 号：R734.2[医药卫生—肿瘤]