The Study Of Transfecting Fe₃O₄ MNP-mediated CD Gene Into U251 Glioma Cells In Vitro

Objective To investigate the killing effects of cytosine deaminase/5-fluorocytosine(CD/5-FC) suicide gene therapy system on human malignant glioma cells in vitro through transfecting cytosine deaminase gene containing pCMVCD plasmid into U251 glioma cells by magnetic nanoparticle of Fe304.Methods Amplification,restriction enzyme digestion and pCMVCD plasmid DNA sequencing using plasmid pCMVCD the CD gene in vitro;The pCMVCD plasmid was transfected into the U251 human malignant glioma cells mediated by Fe₃O₄MNP.Resistant clones (named U251/CD cells) were isolated by screening with G418 presence; Using immunofluorescence test to detect the CD protein levels expressed by U251/CD cells.U251/CD cells were incubated with 5-FC in different concentrations to determine the number of viability cells(or cytotoxicity assay),and measured by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay.Results Fe₃O₄MNP-mediated plasmid containing CD gene pCMVCD successfully transfected into the U251 cells,obtained with CD gene U251/CD cells,immunocytochemical staining showed that the success of U251/CD cells expressed the protein CD;The untreated U251 cells were insensitive to 5-FC,with the IC₅₀ about 6500μmol/L. After the transfection,the IC50 was dramatically reduced to about 10μmol/L.Therefore,gene transfection made G418-resistant clones (U251/CD cells) highly sensitive to 5-FC.Containing different concentrations of 5-FC in the culture medium,U251/CD cells in the transmission electron microscope under the typical apoptotic morphology, the apoptotic rate was dose-dependent manner.Conclusion The killing effects of U251 cells modified by CDgene were induced by 5-FC,and the CD/5-FC system was feasible to treat glioma.