| Increasing publications focused on the killing of tumor cells by nature killer (NK) cells. However, the reverse effect of tumor cells against NK cells is ignored. This study is to uncover the possible molecular mechanism of primary NK cell apoptosis induced by erythroleukemia cell line K562.Primary NK cells are isolated from peripheral blood by the MACS columns and cultured in stem cell groth medium including recombinant human interleukin-2(RhIL-2). Then NK cells and K562cells are mixed and cocultured at different E:T ratios and different times. The apoptosis of NK cells and K562cells are detected by flow cytometry using the PE-An-nexinV/7-AAD reagents. The action of FasL/Fas signaling pathway in NK cells apoptosis induced by K562cells is explored by coculturing. Flow cytometry is used to detect the level (MFI, mean fluore scence intensity) of membrane bound Fas on NK cells and membrane bound FasL on K562cells during NK cells apoptosis induced by K562cells. NK cells and K562cells at1:5adding anti-Fas neutralizing antibody and anti-Fas activating antibody respectively, and observing the apoptosis of NK cells by DAPI staining and flow cytometry.The purity of NK cells isolated from MACS reaches (93.99±4.22)%. At the same E:T ratio, the apoptotic rate of NK cells induced by K562cells is significantly increased in a time-dependent manner. When the E:T ratio is reduced, the apoptotic rate of NK cells is gradually increased, and the cytotoxic activity of NK cells to K562is found to decrease. The mean fluorescence intensity (MFI) level of Fas on NK cells is significantly increased, but the change of FasL MFI on K562cells has no statistical significance. FasL/Fas pathway is blocked by anti-Fas neutralizing antibody ZB4and the experimental group of NK cells apoptosis induced by K562cells is significantly decreased. When the concentration of CH11gradually increases, it is found that the nucleus edge of the NK cells becomes more irregular and the chromosomes gradually concentrates, resulting in a strong blue fluorescence, followed by nuclear pyknosis.Taken together, these data indicate that K562cells can induce activated NK cells apoptosis via the FasL/Fas pathway, which could be involved in immuno escape. |
