Akt重组腺病毒载体的构建及其对肝癌细胞生存的影响
CSTR:
作者:
作者单位:

作者简介:

通讯作者:

中图分类号:

基金项目:

国家自然科学基金(30870955) .


Construction of recombinant adenovirus vectors containing Akt gene and its effect on survival of hepatic cancer cells
Author:
Affiliation:

Fund Project:

Supported by National Natural Science Foundation of China(30870955).

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    目的 构建携带Akt基因的重组腺病毒载体,初步研究其对肝癌细胞生存的影响。方法 分别将Akt-wt及Akt-dn基因亚克隆至穿梭质粒pacAd5 CMVK-NpA,采用RAPAd R○ CMV腺病毒表达系统构建重组腺病毒并用PCR鉴定,以蛋白质印迹检测Akt及其下游GSK3β、p-GSK3β的表达,后将Akt重组腺病毒感染肝癌SK-HEP-1细胞,检测血清饥饿24 h后细胞存活情况。结果 酶切pacAd5 CMV-Akt-wt和pacAd5 CMV-Akt-dn均获得大小为6 kb和1.44 kb(Akt)的两个片段。PCR 扩增得到1.44 kb左右的条带。蛋白质印迹结果 显示两组中p-GSK3β表达有明显差别,肝癌SK-HEP-1细胞存活率在感染pacAd5 CMV-Akt-dn组较感染Akt-wt腺病毒组明显降低。结论 成功构建了Akt-wt及Akt-dn基因重组腺病毒载体,并初步发现其有促进肝癌细胞死亡作用,为体内外进一步研究Akt基因及其相关信号通路在肝癌发生发展中的作用奠定了基础。

    Abstract:

    Objective To construct a recombinant adenovirus encoding the Akt gene and to investigate its influence on the survival of hepatic cancer patients. Methods The Akt-wt and Akt-dn genes were subcloned into pacAd5 CMVK-NpA plasmid separately, and then the adenoviral recombinant adenovirus encoding the Akt gene was constructed with the RAPAdR○ CMV Adenoviral Expression System and was identified by PCR. The expression of Akt, GSK3β and p-GSK3β protein was detected by Western blotting analysis. SK-HEP-1 cells were infected with pacAd5 CMV-Akt-wt, pacAd5 CMV-Akt-dn and pacAd5 CMV-GFP, respectively. The cell survival was observed under fluorescence microscope after 24 h-starvation. Results pacAd5 CMV-Akt-wt and pacAd5 CMV-Akt-dn were enzymatically digested into two fragments(6 kb and 1.44 kb). PCR result of the viral supernatant yielded a band of about 1.44 kb. Western blotting analysis showed p-GSK3β expression in the Akt-wt group was stronger than that in the Akt-dn group. The survival rate of the SK-HEP-1 cells significantly decreased in the Akt-dn group than in the Akt-wt group. Conclusion We have successfully constructed the recombinant adenoviruses pacAd5-Akt-wt and pacAd5-Akt-dn, and they have pro-death effect in hepatic cancer cells, which paves a way for further studying Akt gene and the related signal pathway in hepatocellular carcinoma.

    参考文献
    相似文献
    引证文献
相关视频

分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2012-01-02
  • 最后修改日期:2012-03-12
  • 录用日期:2012-03-20
  • 在线发布日期: 2012-03-21
  • 出版日期:
文章二维码
重要通知
友情提醒: 近日发现论文正式见刊或网络首发后,有人冒充我刊编辑部名义给作者发邮件,要求添加微信,此系诈骗行为!可致电编辑部核实:021-81870792。
            《海军军医大学学报》编辑部
关闭