Research On Amplification Of Natural Killer Cells In Vitro In A Large Scale And Cytotoxic Mechanism

Objective:To research on amplification of natural killer cells in a large scale and cytotoxic mechanism. Methods:There are four programs in amplificating peripheral blood. The first program is traditional program of amplification for NK cells. IL-18was added in first program which was called the second program and IL-7was added in first program which was called the third program.They all compared with the forth program, which was called traditional program of amplification for CIK cells. The proportion of different lymphocyte subsets in the amplified products, especially NK cells and NK-T cells by flow cytometry on the day0, day5, day10and day15. We also compared the absolute counts and change folds of total cells, NK cells and NK-T cells after15days. We compared the antitumor cytotoxicity among the four programs by LDH release. Cytokine secretion(IFN-γ and TNF-a) was detected by ELISA in the day5and day15and compared among the four difference programs. MHC missing cell line (K562), MHC mismatched cell line (MCF-7) and MHC matched cell line (MDA-MB435) were killed by purified NK cells and NK-T cells, compared with the NK cells and NK-T cells blocked KIR or NKG2D. Result:The NK cells increased significantly (P=0.043) in the second program, while the NK-T cells increased significantly (P=0.010) in the forth program. The proportion of NK cells in second program(72.2%±21.93%) is significantly higher than other programs (P=0.012,0.034,0.002). The change folds of total cells were33.6±20.51、38.07±23.61、39.57±23.76、63.67±26.13respectively after15days in vitro. The change folds of NK cells were40.48±31.93、203.46±77.04、55.89±28.66、14.63±14.21respectively. The change folds of NK cells in second program is apparently higher than the first, third and forth program (P=0.002,0.004,0.001). The absolute counts of total cells were from2.5×106to (8.4±6.2)×107(9.5±7.2) X107、(9.9±7.2)×107(1.6±0.8) X108respectively. While the absolute counts of NK cells were from (5.0±1.4)×105to (2.3±2.1)×107(7.9±6.7)×107、(3.0±2.0)×107(7.2±7.9) X106respectively. The cytotoxicty of amplified productions in second program was higher than other programs after15days in vitro. In E:T=40:1, the cytotoxicty of amplified productions in second program significantly higher than in the third and forth program (P=0.016,0.001). The cytotoxicty of purificated NK cells against K562was significantly higher than MDA-MB435cells and MCF-7cells (P=0.019,0.022), while NK-T cells against MDA-MB435cells was significantly higher than K562cells and MCF-7cells (P=0.001,0.005). The cytotoxicty of purificated CD3-CD56+NK cells against K562cells was significantly higher than NK-T cells (P=0.012). KIR and NKG2D receptor all play an important regulation effect for NK cells (P=0.014,0.037). But for NK-T cells, NKG2D receptor plays an important regulation effect, while KIR did not. Conclusion:The second program for amplifying NK cells (IL-2+IL-15+IL-18) could efficiently amplify NK cells which posse higher killing activity. NK cells were based on amplified product, which contains NK-T cells. NK cells and NK-T cells have difference cytotoxic mechanism. NK cells posse higher cytotoxity against K562than other target cells which express MHC. KIR and NKG2D play an important role in activating and killing of NK cells. The cytotoxity of NK-T cells was MHC-restricted similar as T cells. And NKG2D receptor, rather than KIR receptor effect the MHC-restricted for NK-T cells.