Experiment of the effect and safety of mdr1 gene transferring into k562 cells / 重庆医科大学学报

ABSTRACT

Objective:To transfer full-length human mdrlcDNA into K562 cells and assess the feasibility and safety in vitro.Methods:PA317 cells were transducted via a virus vector,pHaMDR1/A,containing full-length human mdr1cDNA by calcium phosphate precipitation.The K562 cells were infected with the virus supernatant.The transfection rate was determined by using CFU selection,FACS and SP immunohistochemical methods respectively.The foreign Pgp function was tested by MTT assay and DNR exclusion with FACS analysis .The cellular cycle and the expression of bcl-2 and c-myc gene were detected by using CFU sclcction,FACS and SP immunohistochemical mcthods respcctively.The forcign Pgp function was tested by MTT assay and DNR exclusion with FACS analysis.The cellular cycle and the expression of bcl-2 and c-myc gene were detected by using PI via FCM and SP stain respectively.These data were analyzed with statistic graph and T-test for match pairs.Results:①The highest transfection rate of K562/MDR6-18 cells were 34%,even up to 84% after colchicine selection.②The exogenous Pgp expression of K562/MDR6-18 cells lasted for more than 4 months with a slow decrease after 10 generations.③The exogenous Pgp got an exclusive pump function.④K562/MDR6-18 cells appeared 1.46-2.22 times cross multidrug resistance phenotype.⑤K562/MDR6-18 cellular proliferation showed a mild,short,restored change.There was no abnormal apoptosis or proliferation.Conclusions:It implies a feasible,attractive and safe way that mdr1 gene transfers into target cells to get exogenous effective MDR.They also provide the basic protocol for using mdr1 gene modifying bone marrow transplantation against the myelosuppressive effects of anticancer drugs.