Objective: To observe anti-proliferative effect of herpes simplex virus thymidine kinase gene system (HSV-tk) on human Tenon capsule fibroblasts (HTFs) and it's mechanism.
Methods: Retroviral vector was constructed containing HSV-tk gene and transfer to packaging cell line PT67. The positive clones was selected with G418 and the supernatant was collected which contains virus particles. The titer of the virus was also calculated. The virus particles were verified by transmission electron microscope (TEM). PCR was carried out to detect the integrity of tk gene into viral DNA genome. HTFs cells were infected with the virus and the positive clones were selected for propagation culture. RT-PCR and Western blot were used to detect tk gene expression. The anti-proliferative effect of tk gene on HTFs cells was determined by MTT. Apoptosis of cells was detected by Hoechst 33258 DNA staining and flow cytometry. The morphologic changes of the cell were observed by TEM.
Results: The retroviral vector pL (tk) SN was constructed successfully which was verified by enzyme cutting. The shape of virus particles derived from the package cells appeared to be oval or spherical under TEM with the diameter around 100 nm. The tk gene was detected in the viral DNA genome by PCR. The virus titer was 4 x 10(7) cfu/ml. The expression of tk gene was detected by RT-PCR and Western blot both in mRNA and protein level. The anti-proliferative effect of tk gene on HTFs cells was dose-dependent, all cells were died 5 days after 5 x 10(-3) g/L GCV being added, IC50 = 6 x 10(-4) g/L. Both necrosis and apoptosis were observed in this study and the apoptosis rate was increased with increasing dose of GCV.
Conclusions: The proliferation of HTFs cells in vitro could be effectively inhibited by the killing effect of HSV-tk-GCV system through the pathway of necrosis and apoptosis.