Construction of recombinant adenovirus vectors containing Akt gene and its effect on survival of hepatic cancer cells
10.3724/SP.J.1008.2012.00332
- Author:
Ke-He CHEN
1
Author Information
1. Clinical Oncology Center, Department of Hepatobiliary Pancreatic Surgery, The People's Hospital of Guangxi Zhuang Autonomous Region
- Publication Type:Journal Article
- Keywords:
Adenovirus;
Akt;
Hepatocellular carcinoma;
SK-HEP-1 cells
- From:
Academic Journal of Second Military Medical University
2012;33(3):332-335
- CountryChina
- Language:Chinese
-
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 RAPAd® 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.
