Investigation Of Purging Effect Of CD3AK/iNOS On Leukemic Cells In Vitro

Objective: TO construct immunocytal nitric oxide donor CD3AK/iNOS through transferring the inducible nitric oxide synthase(iNOS) gene into human CD3AK by retroviral vector,and investigate purging effect of CD3AK/iNOS on 1eukemic cells in autologous peripheral blood stem cell transplantation in vitro. Methods:The amphotropic packaing cell line PA317 transfected with the iN0S gene is cultivated,amplified and screened by G418.The Viral titer is determined with the NIH3T3 cell.PA317/INOS cells are observed by scanning electron microscope.Human peripheral blood mononuclear cells are isolated and activated by anti-CD3 monoclonal antibody in vitro .CD3AK cells are incubated with viral supernatant,then they are selected by G418.The amount of nitric oxide and the activity of INOS in the cultured supematant of CD3AK/iNOS are evaluated by the methods of Griess.The killing activities of CD3AK/iNOS,CD3AK/NEO and CD3AK for cell line K562 and K562/ADM in vitro were evaluated by MTT.Results:Anti-G418 positive packaging cell 1ine PA317 harboring the iNOS gene clones can stably synthesize and excrete recombinant retroviral vectors.The titer of recombinant retrovira1 vectors is 1.0x105CFU/ml .Recombinant retroviral vectors on the surface of PA317/INOS cells are observed by scanning electron microscope.The content of N0 and activity of iNOS that synthesized and excreted by CD3AK/iNOS are 1argely increased compared to those of CD3AK.And CD3AK/iNOS possessed higher effect of cytotoxicity for K562 and K562/ADM as determined by MTT method than that of CD3AK. Conclusion:The construction CD3AK/iNOS has more significant cytotoxic activity on K562 cell line and its multi-drug resistant cell line(K562/ADM )than CD3AK,which may become a more efficient method of purging minimal residual leukemic cells in autologous hematopoietic stem cell transplantation in vitro.