KIR2DS1介导同种异体自然杀伤细胞对树突状细胞杀伤作用的研究

目的 研究体外活化性杀伤细胞免疫球蛋白样受体KIR2DS1介导的自然杀伤（NK）细胞对树突状细胞（DC）的杀伤作用,分析体外KIR2DS1阳性的NK细胞与C2表位DC相互作用后C-C趋化因子受体类型7（CCR7）的表达,探究异源反应性NK细胞预防移植物抗宿主病（GVHD）的机制.方法 采集健康人外周血单个核细胞,通过NK细胞分选试剂盒富集NK细胞作为效应细胞;取初诊急性髓系白血病（AML）患者外周血,提取单个核细胞,诱导DC作为靶细胞;通过PCR-序列特异性引物（SSP）基因分型技术和直接测序（SBT）法检测NK细胞和靶细胞KIR基因和人类白细胞抗原（HLA）-Cw基因位点.CCK-8比色法检测KIR2DS1阳性和阴性的NK细胞对DC的杀伤作用;流式细胞术检测与DC作用后NK细胞表面CCR7的表达.结果 流式细胞术检测富集后的NK细胞纯度达（94.20±1.23）％.同一效靶比（10：1）下,KIR2DS1阳性的NK细胞对DC杀伤作用高于KIR2DS1阴性的NK细胞（t=3.70,P＜ 0.05）;同一效靶比（10：1）下,HLA C1/C1组KIR2DS1阳性的NK细胞对C2/C2组DC的杀伤作用高于其对C1/C1组和C1/C2组DC的杀伤作用,杀伤率分别为（15.06±1.81）％、（10.07±1.03）％和（8.65±0.93）％（F=56.368,P=0.001）.与HLA-C2＋ DC共培养后,KIR2DS1阳性NK细胞表面CCR7阳性表达率增加.结论 活化性的KIR2DS1基因能够在体外有效介导供者NK细胞杀伤受者DC,可能用于预防GVHD;同时能够使NK细胞捕获CCR7,体外获得定向迁移能力.

Effect of natural killer cells expressing KIR2DS1 receptor on efficiently killing dendritic cells in vitro

Objective To study the killing effects of the killer cell immunoglobulin-like receptors （KIR）2DS1-mediated natural killer （NK） cells against dendritic cells （DCs） and analysis the expression of chemokine receptor CCR7 after KIR2DS1-positive NK cells interact with the C2 epitope DCs in vitro,and explore the mechanism of alloreactive NK cell to limit graft-versus-host disease （GVHD）.Methods NK cells were separated by Rosettesep NK sorting kit from healthy donor peripheral blood and taken as effector cells.The mononuclear cells from peripheral blood of newly diagnosed acute myeloid leukemia （AML） patients,and DCs were induced as target cells.Polymerase chain reaction and sequence specific primers （PCR-SSP） genotyping techniques and direct sequencing SBT were used to detect KIR gene,HLA-Cw of the healthy donors and patients.The cytotoxic activity of KIR2DS1-positive NK cells or KIR2DS1-negative NK cells against DCs were detected by CCK-8 assay.The expression of CCR7 after NK cells interacted with DCs was detected by flow cytometry.Results The purity of NK cells analyzed by flow cytometry was （94.20±1.23） %.Under same target ratio （10∶1）,the killing effect of KIR2DS1＋ NK cell against DCs was higher than that in KIR2DS1-NK cells （t =3.70,P 〈 0.05）.Under same target ratio （10∶1）,the cell lysis in C1/C1 group,KIR2DS1＋ NK cells against C2/C2 group DCs （15.06±1.81） % was significantly higher than that against C1/C1 group （10.07±1.03） % and C1/C2 group （8.65±0.93） % DCs （F =56.368,P =0.001）.KIR2DS1 ＋ NK cells efficiently acquired CCR7 upon coculture with C2 epitope DCs.Conclusions NK cell expressing the KIR2DS1-activating receptor efficiently kill DCs,prevent GVHD,while enable NK cell acquire CCR7,obtaining migratory properties in vitro.