Killing Effect On HepG2 Cells Of HSV-tk/GCV System Controlled By HTERT Gene Promoter

Objectives: To establish the correlated eukaryotic expression vector of HSV-tk,and transfect the recombinant to tumor cell through lipidosome,then observe the killing effect of HSV-tk /GCV system on hepatoma carcinoma cell under the control of hTERT ( human telomerase reverse transcriptase ) core promoter.Methods: 1. The eukaryotic expression vectors pGL3-hTERT/luc,pGL3-basic/tk (Negative control) and pGL3-control/tk (Positive control) were constructed based on pGL3- hTERT/tk by recombinant DNA technology and Identificated by digestion after transformating into the E. coli JM109;2. Purified and amplified the correct sequence vector,then introduced it into hepatoma carcinoma cells (HepG2) and hepatocyte (L02) by lipidosome transfection.The transformants were contained different concentrations of G418 for selecting the positive clones. Get out RNA of the positive clones,then confirmed by RT- PCR. 3. Fluorescence microscope was used to detect the Activity of hTERT Promoter in HepG2 cells and L02 cells. 4.After GCV was added, terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling(TUNEL)and flow cytometrymethod were applied to investigate the apoptosis indexes and analyze cell cycles of HepG2 and L02. 5. We further detected the expression levels of cell cycle factors cyclinD1,CDK2,P21 by Western blotting.Results: 1. After digesting three groups of plasmid by HindⅢand XbaⅠ,we found that the gene fragments were correspond with what we expected. 2.After the recombinant vector pGL3- hTERT/tk was introduced into HepG2 cells by lipidosome transfection ,we have selected positice clones against G418(900μg/ml)and confirmed by RT-PCR. 3.From fluorescence microscopy,we found luc gene induced by hTERT promoter idio-expressed in tumor cells but which induced by SV40 promoter was not. 4. After treated with GCV,TUNEL showed that the cytoplasm was stained blue in negative control group but the tumor cells of pGL3- hTERT/tk group were decreased and its nucleolus were stained brown. FCM detected the apoptosis index of pGL3-basic/tk group is higher than pGL3-basic/tk group but lower than pGL3-control/tk group in hepG2,and the apoptosis index of pGL3-control/tk group is the highest in the three groups in L02,there were statistical significance among the three groups (P<0.05); The cell percentage of S in hepG2/tk group is lower than that of control group, Differences between the two groups exist statistical significance(P<0.05).It hinted that pGL3-hTERT-tk/GCV system influences the DNA replication and synthesis.5. the expression levels of cyclinD1 and CDK2 were increased and that of P21 was decreased by Western blot.Conclusions: To establish the associated eukaryotic expression vector of tk, transfect the recombinant to hepG2 tumor cell, can make tumor cell apoptosis and the cell cycle-related protein expression change.We can conclusion that hTERT gene core promoter is tumor-specific and may be useful in tk gene therapy of hepatoma carcinoma and in reducing the side effects of the therapy.