The Study Of Targetable Killing And Wounding On HCC By HTERT-TK/GCV Combined With HTERT-CD/5-Fc

Gene therapy opens a broad prospect in tumor therapy. The research in suicide gene of liver cancer has made great progress, which is an relatively effective and potential gene therapy in clinical application. However, the medicinal effect is not so satisfied. Multiplied treatment of gene is expected to be the new target in treatment of liver cancer.Currently multiplied treatment of gene adopts herpes simplex virus.thymidine kinase,HSV-TK and E coli cytosine deaminase,CD. Meanwhile, manifestation of suicide gene needed in gene therapy of tumor is strictly bounded to target cell, doesn't influence the growth of normal cells. So searching target of tumor cell is a practical problem demanding prompt solution.Telomerase is a kind of reverse transcriptase keeping the length of telomere, which plays a vital role in proliferation, aging, immortality and canceration of cells. It has been proved that telomerase is positive in 85% of tumor cells, is negative in nearly all the adults'cells. In three subunits of telomerase, htert is the key procedure in slowing its activity, so htert promoter will be the ideal switch for target manifestation of gene.It has been proved that suicide gene CD,TK has gained preliminary results. However, there is little research on combination of these two, especially under such a circumstances that htert promoter target cancel cell . This paper is to observe and evaluate the high-efficiency target manifestation of liver cancer cells by combination of these two suicide gene driven by htert promoter.Objective: The aim of this study was to observe the targetable killing and wounding effects of HCC cell line HepG2's growing and apoptosis which was caused by hTERT-TK/GCV combined with hTERT-CD/5-Fc.Methods: 1. Cell culture; 2. conjugated the plasmid of pGL3-hTERT-CD 3. hTERT-TK/GCV combined with hTERT-CD/5-Fc and then transfected HepG2 and normal hepatic cell L-02 at the same time; 4. We watched the effects of HCC cell 's survival rate which was caused by drug level through the method of MTT. 5. we watched the influence of HCC cell 's growing and apoptosis which were lead by suicide gene through the way of flowing cytometry .6. we watched the bystander effect and influence to hepatoma carcinoma cell 's growth and aoptosis which caused by suicide-gene through the way of TUNEL.Results:1.The way of MTT showed that the promoter of hTERT could drive two suicide gene and expressed hepatoma carcinoma cell HepG2.When GCV was 20μg/ml, 5-FC was 200μg/ml, it had the optima lethal effect to the cell of HepG.2.2. The way of flowing cytometry showed that two suicide gene-system and single suicide gene of TK respectively drived by the promotor of hTERT and expressed HCC cell, significant apoptosis rate 82.43% and 63.37% were respectively observed whereas the normal hepatic cell were only 17.42% and 14.62%.3. The way of TUNEL showed that in hepatic cell, suicide gene could be drived and expressed high efficiently by the promoter of hTERT.The result indicated that apoptotic bodies could produced and the rate of apoptosis was obvious while the normal transfected hepatic celldid not had this phenomenon.Conclusion: hTERT-TK/GCV combined with hTERT- CD/5-Fc could targetable attack HCC cell and plays no obvious effect on normal hepatic cell. The bystander effect was obvious.This method retrieves the low transfected efficient of gene therapy and play role on future's experiment and clinical.