RESUMO
The aim of the present study was to investigate the therapeutic potential of a double suicide gene, thymidine kinase (TK) combined with cytosine deaminase (CD), mediated by generation of 5-polyamidoamine dendrimers (G5-PAMAM-D) on human Tenon's capsule fibroblasts (HTFs) as an anti-scarring agent. The pAcGFP1-Hyg-TK-CD plasmid was transfected into HTFs, and reverse-transcription polymerase chain reaction (RT-PCR) was used to detect TK-CD expression. MTT cell proliferation assay was used to evaluate the cytotoxic effects of ganciclovir (GCV) and 5-flurocytosine (5-FC) on HTFs. The optimal concentration of GCV and 5-FC in TK-CD transfected HTFs (HTF-TK-CD) was selected by accessing the lowest and highest cytotoxicity caused, respectively. The morphological changes of transfected HTFs following treatment with GCV and 5-FC were observed by light and transmission electron microscopy. Results demonstrated that the double suicide gene TK-CD mediated by the G5-PAMAM-D delivery system was successfully expressed in HTFs as determined by RT-PCR. A concentration of 3 µg/ml GCV and 200 µg/ml 5-FC was identified as optimal for these prodrugs. The growth rate and number of HTF-TK-CD cells decreased following treatment with GCV and 5-FC as revealed by light microscopy. Additionally, the prodrugs GCV and 5-FC not only demonstrated toxicity on transfected HTFs but also exerted a 'bystander effect'. The present study illustrated that the double suicide gene TK-CD delivery mediated by G5-PAMAM-D was effective in reducing HTF proliferation and inducing cell apoptosis. Furthermore, TK-CD delivery mediated by G5-PAMAM-D may be used as an anti-scarring agent and provide a therapeutic potential for patients requiring glaucoma filtration surgery.
