减毒沙门氏菌介导的NK4基因对HepG2细胞致癌作用的影响(英文)

摘要

Objective:The aim of the study was to construct a stable strain of recombined attenuated Salmonella typhimurium expressing NK4 gene,and observe the effect of the strain on the metastatic potentiality of HepG2 cells.Methods:The NK4 cDNA was isolated from PCAGGS/hNK4 plasmid by PCR,and subcloned into eukaryotic expression vector pcDNA4.The recombinant plasmid was electro-transferred into attenuated Salmonella typhimurium Ty21a to obtain the recombinant strain encoding NK4 gene(TPN).Simultaneously,the recombinant attenuated Salmonella typhimurium carrying GFP gene(TPG) was also constructed.After the TPG and TPN were transferred into HepG2 cells,the transfection rate and the expression level of NK4 protein were detected by flow cytometry and ELISA,and the effects of expression product on the proliferation and migration of HepG2 and angiogenesis were observed.Results:The TPN and TPG were successfully constructed.Fortyeight hours after transfection with TPG,the infection rate was 82.58% ± 1.74%,and the expression level of NK4 protein in supernatant was(181.5 ± 11.7) ng/6 × 105 cells.The supernatant had obviously depressant effect on the proliferative activity of HepG2 cells(P < 0.05),and could obviously restrain the hepatocyte growth factor-mediated migration of tumor cells(P < 0.01).The inhibitory effect of the expression product on the tumor angiopoiesis was obviously observed(P < 0.05),without a dosage-effect relation.Conclusion:The TPN could effectively transfer tumor cells in vitro and express interest NK4 protein.The expression product could effectively inhibit the proliferation and migration of hepatocellular carcinoma cells and the tumor angiopoiesis.