Human endostatin gene transfected adult skin melanoma cells

Objective To study the biological characteristics of human endostatin(hEndo) gene transfected adult skin melanoma cells in vitro and in vivo.Methods The plasmid pcDNA3.1(-)-hEndo was transfected into adult skin melanoma cells by electroporation,and then the stable clones were selected with G418.The transcription and expression of hEndo gene in the transfected melanoma cells were verified by RT-PCR and agarose gel electrophoresis analysis and Western blot.The biological activities of hEndo protein were investigated by MTT in vitro.Stable clones expressing endostatin were subcutaneously injected into the right flank of BALB/c-nu/nu mice of 4~6 weeks old.Then the growth of transduced tumors in vivo was investigated.Results The bands of 624bp and 5.4kb were identified from digested plasmid pcDNA3.1(-)-hEndo.The stable clones were selected with G418 after the eletroporation,the expression of hEndo mRNA was verified by RT-PCR,and Western blot displayed the expression product of hEndo was about 20kD in the transfected melanoma cells.MTT showed that the conditioned medium of melanoma cells transduced with recombination human endostatin expression vector could inhibit the proli-(feration) of human umbilical vein endothelial cells in vitro.The growth of transduced cells in vivo showed that transfected melanoma cells grew in vivo at a slower rate than the control cells(P0.05).RT-PCR showed that endostatin expressed in the transduced tumors.Conclusion Adult skin melanoma cells in vitro transfected with exogenetic hEndo gene can express and secrete active hEndo,and inhibit the growth of transduced tumors in vivo.