Experimental Studies On Bystander Effect In Vitro And Tumor-killing Effect In Vivo Of HSV-tk/GCV Suicide Gene System On Laryngocarcinoma Cells

Laryngocarcinoma is the common malignancy tumor in head and neck. The managements remain to be surgical therapy, radiotherapy and chemotherapy. High frequent recurrence and bad efficiency in the late stage call for new therapies for it.In recent years, gene therapy of tumors has become the most noticeable research field. It's the fifth model followed surgical therapy, radiotherapy, chemotherapy and immunotherapy. The suicide gene therapy is one of the most promising gene therapies for the tumor. As the outstanding representative of suicide gene, the open reading frame of HSV-tk gene has 1128 nucleotide fromstart codon AUG to termination codon UGA. HSV-tk enzyme, which encoded by HSV-tk gene, can catalyze uni-phosphorylation, bi-phosphorylation and tri-phosphorylation of many chemicals similar to nucleotide, such as ACV, GCV and BvdU. The toxic tri-phosphoric acid molecules can repression DNA polyenzyme and competitive enter DNA chains of cells. As a result, DNA chains are broken and cells are killed.The aim of this study is to look for a new and effective therapy for the laryngocarcinoma. The experiment was done in the following ways:1. Recombinant retroviral vector pLXSN-tk was packaged with PA317 and selected in G418 to obtain the positive clones. Measure the viral titer by using NIH3T3 cells. High stable titer producing cells(1.6+106 /ml) were selected.2. Hep-2 was infected by the recombinant retrovirus. The positive clones were obtained and termed Hep-2/tk. PCR and RT-PCR demonstrated that tk gene was integrated into Hep-2/tk genome and expressed at the mRNA level. Observe the growth state of cells. The growth curves showed that there was no significant influence on the proliferous capacity of cells by transferring tk gene into cells.3. Hep-2/tk cells were mixed with Hep-2 cells in various percentages and exposed to GCV (5g/ml) after 24h. Cell survival rate was determined with MTT method after 6 days. The results show that HSV-tk/GCV suicide gene system has significant brstander effect on human laryngocarcinoma cells.4. Hep-2/tk, Hep-2/0 and Hep-2 cells (1+106cells /0.1ml) were inoculated separately into subcutaneous tissue of different nude mice. Begin to inject GCV (50mg/kg, 2 times/d) to abdominal cavity of nude mice and continuous measure the volume of tumor from the 8th day. At the 20th day, finish the experiment, kill nude mice and weigh tumor. The results showed that the average of volume andweight of tumors in Hep-2/tk group were significant smaller than that in Hep-2/0 and Hep-2 groups (p<0.05).The study showed that HSV-tk suicide gene system has significant tumor-repressive effect on human laryngocarcinoma cells. It will be the promising strategy for laryngocarcinoma therapy.