上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

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

何峰，李帅，朱霞霞，杨吉成，盛伟华，缪竞诚   

  1. 苏州大学 医学部细胞教研室， 苏州 215123
  • 出版日期:2015-07-28 发布日期:2015-08-27
  • 通讯作者: 缪竞诚, 电子信箱: mjc@suda.edu.cn。
  • 作者简介:何峰(1990—)， 男， 硕士生; 电子信箱: hehua158002@163.com。
  • 基金资助:

    国家自然科学基金(81001016)

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

HE Feng, LI Shuai, ZHU Xia-xia, YANG Ji-cheng, SHENG Wei-hua, MIAO Jing-cheng   

  1. Department of Cell, Medical College of Soochow University, Suzhou 215123, China
  • Online:2015-07-28 Published:2015-08-27
  • Supported by:

    National Natural Science Foundation of China，81001016

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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1×109~1×1010 pfu/mL;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。

关键词: RGD腺病毒 RGD腺病毒, ING4基因, P53基因, 人肺腺癌细胞

Abstract:

Objective To construct recombinant adenovirus vectors carrying Ad.RGD-P53 and/or Ad.RGD-ING4-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-293A 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 reached 1×109-1×1010 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 (P<0.05). Results of real-time 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, BAX, and BCL-2 by double gene recombinant adenoviruses was more significant than that of single gene recombinant adenoviruses (P<0.05). Conclusion The Ad.RGD-P53 and Ad.RGD-ING4-P53 recombinant adenoviruses were constructed successfully. Expressions of target genes of recombinant adenoviruses can obviously inhibit the growth of human lung adenocarcinoma cells and induce its apoptosis. The effects of double gene recombinant adenoviruses are more significant than those of single gene recombinant adenoviruses. The molecular mechanism may be relevant to up-regulation of expressions of apoptosis related genes Caspase-3 and BAX and down-regulation of expression of BCL-2.

Key words: RGD adenovirus, ING4 gene, P53 gene, human lung adenocarcinoma cell