摘要
目的探讨供者KIR分子表达差异对NK细胞杀伤人脐血内皮细胞系ECV304活性的影响,并观察参与杀伤的活化信号通路。方法RT-PCR及流式细胞仪检测ECV304细胞NKG2D配体MICA/B、ULBP1-3表达,PCR-SSP法行HLA-A、B、Cw分型。自8例健康供者分离外周血NK细胞,流式细胞仪检测KIR2DL1的表达率,LDH释放法测定NK细胞在效靶比20∶1时对ECV304细胞的杀伤活性及anti-KIR2DL1mAb对NK细胞杀伤活性的影响。结果ECV30细胞在mRNA水平表达MICA/B、ULBP1-3,但在膜蛋白水平均不表达。HLA分型表明,ECV304表达KIR2DL1的配体,而不表达KIR2DL2/3、KIR3DL1的配体。8例健康供者NK细胞KIR2DL1表达率有较大差异,对ECV304细胞的杀伤活性也有不同,双变量相关分析示个体KIR2DL1表达率与NK细胞对ECV304的杀伤率存在负相关(rS=-0.994,P=0.000)。anti-KIR2DL1mAb明显增强NK细胞对ECV304的杀伤活性(t=-4.860,P=0.002)。结论NK细胞对ECV304细胞的杀伤分子机制主要为HLA-KIR信号系统错配,目前已知的NKG2D配体MICA/B、ULBP1-3并不参与,这有助于临床活体器官移植时在遗传指导下选择供体。
Objective To analyze the influence of KIR2DL1 expression on the cytotoxicity of allo-NK ceils against ECV304 ceils and to investigate potential activation signals involved in the cytotoxicity. Methods RT-PCR and flow cytometry were used to detect the expressions of MICA/MICB and ULBP1-3 in ECV304 cells, while PCR-SSP was employed to type HLA-A, B, and Cw. Expression of KIR2DL1 on the surface of NK from 8 healthy donors was measured by flow cytometry. Cytotoxicity of allo-NK cells against ECV304 cells was detected by LDH releasing method at effect-to-target cell ratios （E：T） of 20： 1. In blocking experiments, anti-KIR2DL1 monoclonal antibody （mAb） was added to the NK cells before performance of lysis experiment. Results Expression of KIR2DL1 was different among donors; MICA/B and ULBP1-3 were expressed at mRNA level but not on the surface of ECV304; the ligand of KIR2DL1 but not KIR2DL2/3 or KIR3DL1 expressed on ECV304. Spearman correlation analysis showed that KIR2DL1 expression was negatively correlated with cytotoxicity of allo-NK cell against ECV304 （ rS= - 0.994, P = 0.000）. When blocked with KIR2DL1 mAb, NK cells displayed significantly higher cytotoxicity against ECV304 （ t = - 4.860, P = 0. 002）. Conclusion Cytotoxicity of allo-NK cells against ECV304 is mainly determind by KIR-ligand mismatch, Ligands of NKG2D as MICA/B and ULBP1-3 do not participate in the cytotoxicity, which benefits the donor selection guided by inberitance intiving organ transplantation.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第6期649-651,654,共4页
Immunological Journal
关键词
NK细胞
脐静脉内皮细胞
HLA
Ⅰ类分子
杀伤细胞免疫球蛋白样受体
Natural killer cells
Umbilical vein endothelial cells
HLA class Ⅰ molecule
Killer cell immunoglobulin-like receptor
