RESUMO
Objective: To observe the impact of transfection of human granulocyte macrophage colony stimulating factor (hGMCSF) mediated by hydroxyapatite(HA) nanoparticles on the growth of HepG2 cells in order to lay the foundation for further studying gene-modified tumor vaccines of hepatoma in clinic. Methods: The proliferation of HepG2 cells was measured by MTT assay. The plasmids containing hGM-CSF gene were transfected into HepG2 cells mediated by HA nanoparticles. Subsequently, resistant cells were selected by G418 screening. The monoclonal cells were selected by limiting dilution method. RT-PCR was used to identify the integration of hGM-CSF into HepG2 cells and transcription of hGM-CSF. ELISA was performed to detect the level of secreted hGM-CSF in cultured medium and the lasting time. The effect of hGM-CSF on cell cycle and apoptosis were assessed by flow cytometry (FCM) analysis. Results: MTT assay demonstrated that Nano-HA suspension liquid had no significant effects on the growth of HepG2 cells at the concentration below 80 μg/mL. RT-PCR demonstrated the hGM-CSF gene was successfully integrated and steadily expressed in the transfected HepG2 cells. ELISA indicated stable secretion of hGM-CSF from the stable transfected HepG2 cells [mean (216.22 ± 45.78) ng/106 cells per 24 h]. FCM analysis showed that hGM-CSF had no significant effects on the cell cycle and apoptosis of HepG2 cells. Conclusion: hGM-CSF gene could be safely transfected and stably expressed in HepG2 cells mediated by HA nanoparticles. Transfection of hGM-CSF gene mediated by HA nanoparticles has no effects on the growth of HepG2 cells. This study may lay a foundation for the further study of gene-modified hepatoma vaccines.