NK细胞杀伤ARH-77细胞的活性及机制的初步探讨

Cytotoxicity of allogenic NK cells against myeloma cells in vitro and its mechanism

目的探讨同种异体NK细胞杀伤人骨髓瘤ARH-77细胞的活性及机制。方法 LDH释放法检测NK细胞杀伤ARH-77细胞的活性;RT-PCR方法和流式细胞仪分别检测K562和ARH-77细胞NKG2D配体和HLA-I基因和分子。阻断K562和ARH-77细胞NKG2D配体,观察NK细胞杀伤活性的变化。结果相同效靶比时NK细胞杀伤ARH-77细胞的活性明显低于杀伤K562细胞。两种细胞均表达MICA/B和ULBP1～3基因。K562细胞高表达MICA/B和ULBP1～3分子,不表达HLA-Ⅰ类分子;ARH-77细胞低表达ULBP1～3分子,高表达HLA-Ⅰ类分子。ARH-77细胞HLA基因型为A2、3,B15、35,Cw3、4。阻断NKG2D配体,NK细胞对K562细胞的杀伤活性明显降低,对ARH-77细胞的杀伤活性无明显改变;阻断HLA-Ⅰ类分子后,NK细胞对K562细胞的杀伤活性无变化,对ARH-77细胞的杀伤活性明显提高。结论 ARH-77细胞对NK细胞的杀伤敏感性低,其机制与其高表达HLA-Ⅰ类分子、低表达NKG2D配体有关。

Objective To observe the cytotoxicity of allogenic natural killer（NK） cells against myeloma cells in vitro and preliminarily explore its molecular mechanism.Methods Cytotoxicity of NK cells against ARH-77 cells was analyzed by LDH releasing assay at different effector-to-target cell ratios（E：T）.The genes and proteins of NKG2D ligands on K562 and ARH-77 cell surface were measured by RT-PCR and flow cytometry,respectively.The genes of HLA-I molecules in ARH-77 cell and KIR of NK cells were assayed by PCR-SSCP.In blocking experiments,monoclonal antibodies of HLA-I molecule and NKG2D ligands were added to the target cell at E∶T of 20∶1.Results Compared with K562 cells,ARH-77 cells resisted NK cell cytolysis.The mRNA genes of MICA/B and ULBP1-3 were positive in two cell lines.K562 cell lines highly expressed MICA/B and ULBP1-3 protein but weakly expressed HLA-I molecule,while ARH-77 cell lines did the opposite.The HLA-I genotypes of ARH-77 cells were A2,3;B15,35;Cw3,4.NKG2D ligands blocking experiments revealed that the cytotoxicity of NK cells against K562 cell was partially inhibited,but that against ARH-77 cell was not affected.Conversely,the cytotoxicity of NK cells against ARH-77 cell was obviously improved,and that against K562 cell was not affected when HLA-I molecules were shaded.Conclusion Weak expression of NKG2D ligands and high expression HLA-I molecules by NK cells may contribute to the reduced cytotoxicity of NK cells against myeloma cell ARH-77.