Study On AF-hTERT-TK/GCV Targeted Gene Therapy And Bystander Effect On HCC

Telomerase is a ribonucleoprotein complex whose function is to add telomere repeats to chromosomal ends to make its stable and cell immortalization. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is the most important part of telomerase, which can catalyze the full-enzyme to be active, so the promoter is the core of its function. Because it only express in tumor cells which express telomerase activity, can be used as a tumor marker or can be targeted to cure cancers. Suicide gene may be used cure HCC by the features.Objective:To observe the target therapeutic effects of plasmid AF-pGL3-hTERT-TK on HepG2 cells which we have constructed before transfecting.Methods:1. Cell culture; 2. Constructing luciferase reporter plasmid group and therapeutic plasmid group by conjugating restrictive enzyme digestion product; 3. Transfecting HepG2 and normal hepatic cell L02 with AF conjugating liposome; 4. Observing the luciferase light and luciferase gene express ability under hTERT promoter after luciferase reporter plasmid transfection using fluorescent microscope and Liquid Sclintillation Analyzer; 5. Observing the cell growth, apoptosis and bystander effect under the influence of therapeutic plasmid; 6.Observing the expression of cell-circle protein by western blotting method.Results:Our results showed that when suicide gene TK can be effectively driven by hTERT promoter making the TK gene highly expressed in HCC cell. AF can make the therapeutic gene enter into HepG2 cell more easily by recognizing and combining the ASGPR receptor protein on HepG2 cell surface and induce its apoptosis and suicide, under the existence of bystander effect, the significant apoptosis rate 85%±3% were observed whereas the normal hepatic cell is only about 16%±2%; the tumor cells may be inhibited by the regulation mechanism of the cell-circle modulator cyclinD 1,Cdk2 and p21.Conclusion:AF-pGL3-hTERT-TK can target and attack HCC cell line and almost have no influence on normal L-02 hepatic cells. AF-pGL3-hTERT-TK has a potential in treating hepatocellular carcinomas.