同种异体NK细胞对不同肿瘤细胞的体外杀伤活性及其机制的初步探讨

为了观察同种异体NK细胞对不同肿瘤细胞的体外杀伤活性,并初步探讨其分子机制。以K562细胞为对照,应用LDH释放法检测不同效靶比时同种异体NK细胞杀伤CNE2、KG1a和U251细胞的活性。应用RT-PCR和流式细胞仪分别检测4种细胞MHCI类链相关分子(MICA/B)和人巨细胞病毒糖蛋白UL16结合蛋白(ULBP1~3)基因和分子的表达情况。效靶比20∶1时用AMO-1、BMO-1、M295、M310和M551单抗分别阻断肿瘤细胞表面MICA、MICB、ULBP1、ULBP2和ULBP3分子,观察NK细胞对其杀伤活性的变化。结果:NK细胞对K562、CNE2和KG1a细胞均有杀伤活性,对U251细胞无杀伤活性。在mRNA水平4种细胞均表达MICA/B和ULBP1~3基因。K562细胞表达MICA/B和ULBP1~3全部分子;KG1a和U251细胞均不表达5种分子;CNE2细胞表达MICA/B和ULBP2,不表达ULBP1和ULBP3。CNE2、KG1a和U251细胞均高表达HLAI分子,而K562细胞不表达。用单抗分别阻断靶细胞表面相应的NKG2D配体分子,NK细胞对KG1a和U251细胞的杀伤活性无变化。NK细胞对K562和CNE2细胞的杀伤活性可部分被封闭。同种异体NK细胞在体外对不同肿瘤细胞的杀伤活性不同,其杀伤机制也不完全相同。

Cytotoxic activities of allogenic NK cells on different tumor cells in vitro and their possible mechanism

It is decided to observe the cytotoxicity of allogenic NK cells on different tumor cells in vitro and preliminarily explore its molecular mechanism,in which the K562 cells sensitive to cytotoxicity were used as the positive control,and the cytotoxic activities of NK cells isolated from 5 healthy volunteers on the tumor cell lines CNE2,KG1a and U251 cells were analyzed by LDH releasing assay at different effector-target cell ratio(E∶T). Meanwhile,the expressions of genes encoding MHC I class chain-associated molecule(MICA/B) and HCMV-glycoprotein UL16-binding protein(ULBP1-3) of these cell lines were assayed by RT-PCR and flow cytometry. In blocking experiments,monoclonal antibodies against different NKG2D ligands,such as AMO-1,BMO-1,M295,M310 and M551,were added to the target cells at E∶T ratio of 20∶1 in order to observe the differences on cytotoxicity. Comparing with K562 cells,the CNE2 and KGla cells were relatively sensitive to allogenic NK cells,however,the U251 cells was resistant to the cytotoxic effect of allogenic NK cells. The MICA/B and ULBPI-3 genes were expressed on these cell lines at mRNA level. On the surface of these cell lines different levels of expressions of protein MICA/B,ULBP1-3 and HLA I molecules could be detected. As demonstrated from the results of blocking experiments,it was showed that the cytotoxic activities of NK cells on K562 and CNE2 cells were partially inhibited in comparison with those on KGla and U251 cells,in which the latter was not influenced. From the data mentioned,it is evident that the cytotoxic activities of allogenic NK cells on different kinds of tumor cell lines vary a great deal and their molecular mechanisms may be also different.