Effects Of Natural Killer Cells On Killing Breast Cancer Cell Lines In Vitro

Objective To investigate the most optimal conditions of culturing and proliferation of NK cells purified from PBMCs by negative depletion using immumomagnetic microbeads; and to investigate the relations between KIR on NK cells and HLA-Cw on the tumor cells, whether breast cancer cell lines would have enhanced susceptibility to allogenetic KIR-incompatible NK cells compared with their KIR-matched allo-genetic counterparts; to investigate the relations between NKG2 on NK cells and MIC on tumor cells with the killing activity of NK cells.To analyse the relationship between the expression level of MIC on tumor cell surfaces and the specific lysis by NK cells Methods 1. PBMCs were derived from 10ml healthy donors'perheral blood by Ficoll-Hypaque gradient and depletion of plastic-adherent cells in six-well plates. To obtain enriched NK cells, PBMCs were purified by negatice depletion using immumomagnetic beads system (CD56+ NK cell isolation kit. Melteny, Germany). 2. B95-8 cells were cultured in RPMI-1640(containing 10% FBS),and half the culture medium was replaced weekely. EB virus was obtained from the culture medium of B95-8 cells for 7 days and 14 days. PBMCs were cultured in 96 well U-bottom plate with RPMI-1640 which contained 10% FBS,20% EB virus,and 20μg/ml CyA. Half the culture medium was replaced in 3-4 days with new medium containing fresh CyA.After 4 weeks cuture, translated EBV-LCLs could be obtained. The analysis of the expression of CD 19 on the cells surface could judge whether PBMCs had been translated into EBV-LCLs successfully. Translated EBV-LCLs were irradiated(100Gy) before they were used as feeder cells in bulk NK cells expansion. NK cells were cultured in SCGM serum-free media (stem cell growth medium, CellGenix, Gaithersburg) containing IL-2 and EBV-LCLs.3.Genomic DNA was extracted from healthy donors' peripheral blood, as well as the tumor cell lines. The polymerase chain reaction-based sequence-specific primers(PCR-SSP) typing was used to detect the expression of KIR on NK cells surface and HLA-Cw on NK...