AIM: To investigate killing effect of herpes simplex virus thymidine kinase/ganciclovior system(HSV-TK/GCV) on osteosarcoma cell and its mechanisms.METHODS: Recombinant retroviral vector (DORHyTK) containing hygromycin phosphotransferase-thymidine kinase fusion gene(HyTK) was constructed and introduced into human osteosarcoma cell line OS732 with DOTAP. DNA and total RNA extracted from HyTK expressing cells (OS732TK) were tested by PCR and RNA dot blot analysis. A chemosensitivity of OS732TK cells to GCV and "bystander effect" were measured by means of MTT colorimetric assay. Hoeschst 332258 staining and flow cytometric analysis were performed for mechanism of HSV-TK/GCV system gene therapy. RESULTS: DORHyTK was constructed successfully; the HyTK gene existed and expressed in OS732TK cells; All OS732TK cells were killed after 5 days of exposure to 5 mg/L GCV. The "bystander effect" was observed in both a high population and a low population, but the former was stronger than the latter. Hoeschst 332258 staining revealed the characteristic hallmarks of apoptosis, however necrosis also existed. Flow cytometric analysis of DNA content showed a G 0-G 1 phase blockade. CONCLUSION: HSV-TK/GCV gene therapy system showed its strong killing effects on osteosarcoma cells OS732; The phenomenon of "bystander effect" was also very apparent. GCV exposure induced both necrotic and apoptotic death in HSV-TK expressing cells, and perturbed the cell cycle. HSV-TK/GCV gene therapy system may provide a new therapeutic approach for treatment of osteosarcoma.

AIM: To establish a method of in vitro donor heart perfusion in murine cardiac transplantation during preservation and apply it in adenovirus mediated gene transfection for donor heart. METHODS: Donor heart was transfected with recombinant adenovirus and stored for 2 hours after harvest, then it was transplanted heterotopically in abdomen. The grafts were appraisal by palpitation. Marked gene products were determined by X-Gal staining, aod T cell infiltration was determined by immunohistochemistry. The activation markers of recipients' lymphocytes were examed by cytometry. RESULTS: The grafts survival rate is 100% after perfusion and cold storage. The LacZ staining became strong 1 week after transplantation. The grafts remained an intact structure and no apparent T cell infiltration. The activation status of recipients' lymphocytes were not enhanced by transfected cardiac graft. CONCLUSION: In vitro perfusion during graft cold preservation is feasible for adenovirus mediated gene transfection. [