ABSTRACT
Objective: To construct the eukaryotic expression vector pSurp-EGFP regulated by the survivin gene promoter and to detect the specific expression of the promoter in human laryngeal cancer Hep-2 cells by green fluorescent protein assay.Methods: Thesurvivin gene promoter was generated by polymerase chain reaction(PCR) and the CMV promoter of the pShuttle vector replaced by the survivin gene promoter to generate the plasmid pSurp.The three plasmids pShuttle,pSurp and pEGFP-C1 were respectively double-enzyme digested so as to produce the plasmids pCMV-EGFP and pSurp-EGFP carrying the CMV or survivin promoter.The purified pCMV-EGFP and pSurp-EGFP were transfected into Hep-2 cell and vascular endothelial cell ECV304 using liposome transfection reagent and the expressions of EGFP detected by the fluorescent microscope.Results: Thesurvivin gene promoter was successfully cloned by PCR,and thesurvivin gene promoter-regulated pSurp-EGFP was constructed.Green fluorescence was observed in Hep-2 cells but not in ECV304. Conclusion: The high specific activity of the survivin gene promoter in Hep-2 cells that we successfully constructed attributes to the studies of tumor specific gene therapy.
ABSTRACT
Objective: To determine if fusion genes of HSV-tk gene and cytokine gene have synergy on the cell killing of the Hep-2 human laryngeal carcinoma cell line in vitro. Methods: Different fusion genes expressing vectors PL(TI) SN, PL(TT)SN and PL(TK)SN were generated by recombinant DNA technology. Hep-2 was infected by the recombinant retrovirus. The positive clones were obtained after G418 selection and were termed Hep/TI, Hep/TT and Hep/TK respectively. The integration and expression of fusion genes in Hep-2 cells were identified by RT-PCR and Southern blot. The growth state and GCV killing effect of fusion genes modified cells were used to investigate the expression of fusion genes and antitumour effect on Hep-2 cells. Results: RT-PCR and Southern blot analysis confirmed the integration and expression of fusion genes in Hep-2 cells. There was no significant difference in cell proliferation between the Hep/TI and Hep/ TK.but the growth of Hep/TT was restrained. After the treatment of GCV the Hep/TI, Hep/TT and Hep/TK all showed high sensitivity to GCV. The killing effect of GCV on Hep/TT was the most siginificant and bystander effects were observed siginificantly in vitro. Conclusion: The fusion genes of HSV-tk and cytokine gene have synergistic effects on killing Hep-2 cell after treatment of GCV in vitro,which might have therapeutic potentials for laryngocarcinoma.