| 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。 |
| 关键词: Akt 腺病毒 SK-HEP-1细胞 肝细胞癌 |
| DOI：10.3724/SP.J.1008.2012.00332 |
| 投稿时间：2012-01-02修订日期：2012-03-12 |
| 基金项目:国家自然科学基金(30870955) . |
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| Construction of recombinant adenovirus vectors containing Akt gene and its effect on survival of hepatic cancer cells |
| CHEN Ke-he1△*,HAN Ze-long2△,LI Jian-jun3,PAN Jin-fei1,LIU An-ling2 |
(1. Clinical Oncology Center, Department of Hepatobiliary Pancreatic Surgery, the People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, Guangxi, China
2. Department of Cell Biology, College of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, Guangdong, China
3. Department of Health Care, Clinic of Outpatients, PLA General Political Department, Beijing 100120, China
*Corresponding author.) |
| 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. |
| Key words: Akt adenovirus SK-HEP-1 cells hepatocellular carcinoma |
