目的：构建编码融合基因 NT4p53(C22)Ant嵌合肽的重组腺病毒表达载体,并研究其对人肝癌 HepG2细胞的杀伤作用。方法使用分子克隆技术,通过同源重组获得重组腺病毒 rAVV-NT4p53(C22)Ant,收集上清、大量扩增并测定其滴度。感染人肝癌 HepG2细胞,采用免疫组化法检测 p53表达,MTT 实验、流式细胞仪观察 rAAV-NT4p53(C22)Ant对肿瘤细胞的杀伤作用。结果成功构建重组腺病毒表达载体,感染人肝癌 HepG2细胞后 p53表达率为(44.88±2.45)%；MTT检测显示,细胞存活率随作用时间延长明显降低,与空病毒组及对照组比较差异均有统计学意义(P<0.05),流式细胞仪检测显示 G0/G1期细胞比例及凋亡细胞较空病毒组及对照组增加。结论构建的 NT4p53(C22)Ant重组腺病毒在肝癌细胞中能有效表达,对肝癌 HepG2细胞有抑制增殖和促进凋亡的作用。%ABSTRACT:Objective To construct a recombinant adenovirus vector harboring fusion gene NT4p53(C22)Ant and study its killing effect on HepG2 tumor cells.Methods Using molecular cloning technology,the rAVV-NT4p53(C22)Ant was produced by homologous recombination.Then we collected virus supernatant and measured its titer after it was amplified by PCR.The effect of this fusion gene on HepG2 tumor cells was evaluated by IHC, MTT assay,PI staining and flow cytometry.Results The recombinant adenovirus was successfully constructed. The p53 expression rate in rAAV-NT4p53(C22)Ant group was (44.88±2.45)%.MTT assay showed that rAAV-NT4p53(C22)Ant could strongly suppress the growth of HepG2 tumor cells.Flow cytometry showed that rAAV-NT4p53(C22)Ant could induce obvious apoptosis of HepG2 tumor cells.Conclusion The recombinant adenovirus vector encoding gene NT4p53(C22)Ant has been successfully constructed and expressed in this experiment,and it can inhibit proliferation and induce apoptosis of HepG2 tumor cells.
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