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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。
Objective To construct a recombinant adenovirus carrying Akt gene and study its effect on the survival of hepatocellular carcinoma cells. Methods Akt-wt and Akt-dn genes were subcloned into the shuttle plasmid pacAd5 CMVK-NpA respectively. The recombinant adenovirus was constructed by RAPAd ○ RCMV adenovirus expression system and identified by PCR. Western blotting was used to detect Akt and its downstream GSK3β, p-GSK3β After the recombinant adenovirus of Akt was infected into SK-HEP-1 cells, the survival of the cells was detected 24h after serum starvation. Results Two fragments of size 6 kb and 1.44 kb (Akt) were obtained by digestion with pacAd5 CMV-Akt-wt and pacAd5 CMV-Akt-dn. PCR amplification of about 1.44 kb band. The results of western blotting showed that the expression of p-GSK3β in the two groups was significantly different. The survival rate of SK-HEP-1 cells in hepatocellular carcinoma was significantly lower than that in the adenovirus group infected with pacAd5 CMV-Akt-dn. Conclusions The recombinant adenovirus vectors of Akt-wt and Akt-dn were successfully constructed and found to promote the death of hepatocarcinoma cells, which laid the foundation for the further study on the role of Akt gene and its related signaling pathways in the development of hepatocellular carcinoma .