Targeted Killing Effect On Human Breast Cancer Cells Of Suicide Gene HSV-TK Controlled By HMAM Promoter And Enhancer

Objective:1.To construct recombinant adenovirus carrying reporter gene EGFP at the downstream of hMAM promoter and enhancer .2.To construct recombinant adenovirus carrying suicide gene HSV-Tk at the downstream of hMAM promoter and enhancer .3 . To confirm the breast-cancer-cell-specific regulation effect of hMAM promoter and enhancer.4.To explore new methods of targeted therapy for breast cancer by suicide gene under the control of hMAM promoter and enhancer.Methods:1.hMAM promoter and enhancer DNA is synthesized according to Genebank andinserted into plasmid vector pMD19-T simple.2.Recombinant plasmid vectors pMD19-T simple-EP-EGFP and pMD19 simple -T-EP-TK are constructed, which respectively carries reporter gene EGFP or suicide gene HSV-TK at the downstream of hMAM promoter and enhancer.3.Recombinant adenoviru skeleton cosmids pAxcwit2-EP-EGFP and pAxcwit2 -EP-TK are obtained through transfering target gene from recombinant plasmids to adenovirus skeleton cosmid pAxcwit2.4.Recombinant adenovirus Ad-EP-EGFP and Ad-EP-TK are obtained through transfecting linearized recombinant adenovirus skeleton cosmids pAxcwit2-EP-EGFP and pAxcwit2-EP-TK into HEK293 cells.5.Recombinant adenovirus Ad-EP-EGFP is infected into breast cancer cells T-47D which express hMAM,breast cancer cells ZR-75-30 and nasopharyngeal cancer cells 5-8F which don't express hMAM separately in vitro.The espression of GFP is observed under fluorescence microscope to confirm the breast-cancer- cell-specific expression regulation function of hMAM promoter and enhancer.6.Recombinant adenovirus Ad-EP-EGFP and Ad-HSV-TK adenovirus are infected into breast cancer cells T-47D,ZR-75-30 and nasopharyngeal cancer cells 5-8F respectively in vitro, then prodrug GCV is given. T-47D,ZR-75-30 and 5-8F cells uninfected with adenovirus are also given GCV as control group.Killing effect on cells of suicide gene HSV-TK driven by hMAM promoter and enhancer is then observed through Trypan Blue Staining and cell counting.Results:1.GFP can be observed under fluorescence microscope in breast cancer cells T-47D but can't be observed in other cells.2.Human breast cancer cells T-47D can be targeted killed by suicide gene HSV-TK under the regulation of hMAM promoter and enhancer, but no cytotoxicity is shown to control group T-47D cells without recombinant adenovirus infection and with recombinant EGFP adenovirus infection after being given GCV.There is no killing effect to other cells in any case.Conclusion:1.hMAM promoter and enhancer has breast-cancer-cells-special expression regulation function.2.Human breast cancer T-47D cells can be targeted killed by suicide gene HSV-TK driven by hMAM promoter and enhancer.