KIR2DS1受体对人类NK细胞杀伤白血病细胞的作用研究

Effect of KIR2DS1-mediated human natural killer cells cytotoxicity against leukemia cells

目的 探讨活化性杀伤细胞免疫球蛋白样受体（KIR）家族成员KIR2DS1在自然杀伤（NK）细胞杀伤白血病细胞中的作用.方法 采集健康人群外周血单个核细胞,采用NK细胞分选试剂盒富集NK细胞,将分选的NK细胞进行体外扩增培养,以作为效应细胞.取初治确诊的急性髓细胞白血病（AML）患者新鲜骨髓液,分离后的白血病细胞作为靶细胞.采用CCK8比色法检测NK细胞对白血病细胞的杀伤率.采用聚合酶链反应和顺序特异性引物（PCR-SSP）基因分型法分别检测NK细胞及靶细胞的KIR基因和HLA-Cw;根据KIR2DS1表达与否将NK细胞分为KIR2DS1阳性NK细胞与KIR2DS1阴性NK细胞;用抗-KIR2DS1抗体封闭NK细胞的KIR2DS1受体,用CCK8比色法检测封闭前后KIR2DS1阳性组NK细胞对靶细胞的杀伤率.根据HLA-Cw将NK细胞和靶细胞分别分为C1纯合子组（表达HLA-Cw 01、03、07、08、12、14、16）、C2纯合子组（表达HLA-Cw02、04、05、06、15、17、18）和C1/C2杂合子组（既表达C1组的等位基因又表达C2组的等位基因）.结果 经过富集后NK细胞的纯度为（91.2±5.94）％;不同效靶比下,KIR2DS1阳性NK细胞的杀伤率明显高于阴性组;当效靶比为10：1时,KIR2DS1阳性NK细胞组对白血病细胞的杀伤率（42.82±6.81）％高于KIR2DS1阴性组（28.61±5.14）％;抗-KIR2DS1封闭后NK细胞对白血病细胞杀伤作用明显减弱（t=-3.00,P＜0.05）;KIR2DS1阳性NK细胞C1纯合子组对靶细胞C2纯合子组的杀伤率（54.39±3.46）％明显高于靶细胞C1组（41.22±3.68）％（t=8.33,P＜0.05）和C1/C2组（41.32±5.09）％（t=6.37,P＜ 0.05）.结论 KIR2DS1阳性NK细胞对白血病细胞的杀伤效力高于KIR2DS1阴性NK细胞,即KIR2DS1可以有效介导NK细胞杀伤白血病细胞;HLA-C1纯合子的KIR2DS1阳性NK细胞对HLA-C2纯合子的靶细胞杀伤作用强.

Objective To study the killing effects of the killer cell immunoglobulin-like receptors （KIR） KIR2DS1-positive natural killer （NK） cells against leukemia cells.Methods High-purified NK cells separated by RosetteSep NK cell enrichment kit from healthy donor peripheral blood were taken as effector cells,and the freshly isolated bone marrow mononuclear cells from newly diagnosed acute myelogeneous leukemia （AML） patients were taken as target cells.The cytotoxic activity of NK cells were detected by CCK8 kit assay.HLA-Cw,KIR gene of the healthy donors and patients were detected by polymerase chain reaction and sequence specific primer （PCR-SSP） genotyping techniques,respectively.NK cells were divided into KIR2DS1-positive group and KIR2DS1-negevitive group,and then anti-KIR2DS1 mononuclear antibody was used to block KIR2DS1 of NK cells.Meanwhile based on HLA-Cw,KIR2DS1-positive NK cells and target cells were divided into C1 homozygote group（expressing HLA-Cw 01,03,07,08,12,14,16 alleles）,C2 homozygote group （expressing HLA-Cw 02,04,05,06,15,17,18 alleles） and the C1/C2 heterozygote group （co-expressing the alleles in C1 group and C2 group）.Results The purity of NK cells was （91.2±5.94） % through flow cytometry analysis.At the same effector-target ratio,cytotoxicity of KIR2DS1-positive NK cells against target cells was higher than that of KIR2DS1-negetive NK cells.While the E：T =10：1,cytotoxicity of KIR2DS1-positive NK cells against target cells [（2.82±6.81） %] was significant higher than that of KIR2DS1-negetive NK cells [（28.61±5.14） %] against target cells.The killing effects of KIR2DS1-positive NK cells was significantly weakened after KIR2DS1 blockaded with specific antibody （t =-3.00,P =0.05）.When focusing on the C1 group NK cells,KIR2DS1-positive NK cells against C2 group [（4.39±3.46） %] target cells was significantly higher than than against C1 group [（41.22±3.68） %] （t =8.33,P 〈 0.05） and C1/C2 group [（41.32±5.09） %] （t =6.37,P 〈 0.05） target cells.Conclusions The killing effects of KIR2DS1-positive NK cells are significantly higher than that of KIR2DS1-negetive NK cells.The HLA-C1 group KIR2DS1-positive NK cells could recognize HLA-C2 loci and then kill the target cells.