中文摘要：

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○RCMV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24h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。

英文摘要：

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.