The study on mechanism of enhancing bystander effect of HSV-TK/GCV system by Rb gene / 中华骨科杂志

ABSTRACT

Objective To explore an alternative strategy of ganciclovir-mediated cytotoxicity and study the mechanism of enhancing bystander effect of HSV-TK/GCV system by combination gene transfer of Rb gene and HSV-TK gene.Methods Recombinant eukaryotic expression plasmid pcDNA3.1-Rb harbour ing1.65kb of Rb gene was constructed.The pcDNA3.1-Rb and the plasmids tgCMV/HyTK car-ry ing HSV-TK gene were transfected respectively or co-transfected into the osteosarcoma cell line OS732by lipofection using DOTAP reagent.The mRNA expression of Rb gene and HSV-TK gene were detected by RT-PCR and Northern blot.Cell counting,cell cycle analysis and soft agar colony formation test were adopted to observe cell growth features.The expression of gap junction Connexin43gene was performed by RT -PCR and Western blot.The direct confirmation of gap junction intercellular commu ni cation was demonstrated by Lucifer yellow dye transfer.Cell sensitivity to GCV and″bystander effect″of HSV-TK/GCV system were measured by MTT assay.Results The eukaryotic expression plasmid pcD NA3.1-Rb was constructed successfully.The transfected cell lines OS732TK,OS732Rb and OS732RbTK were har-vested.mRNA expression of HSV-TK gene was demonstrated in OS732TK and OS732RbTK cells.Both exogenous and endogenous Rb gene expression could be detected in OS732Rb and OS732RbTK cells si-multa neously.Compared with parental cell OS732,OS732Rb and OS732RbTK cells changed their mor -phology and decreased the growth rate;the ability of soft agar colony formation reduced and the cell cycles were arrested at G 0 G 1 checkpoint.The Connexin43expression and gap junction in tercellular communica-tion en hanced in OS732Rb cell.GCV was of toxicity to only TK -positive cells,OS732TK and OS732RbTK,in a concentration dependent manner,the mixed coculture experiments of OS732and OS732Rb with the TK-negative cell,both showed sensitive to GCV,but the survival rate of OS732Rb cell was significantly lower than OS732cell under the same condition.Conclusion Coordinate ex pression of Rb andHSV-TK gene conferred a more po tent bystander effect by enhancing gap junction intercellular communica-tion and in hibiting prolifera tion.