可溶性HLA-G1与NK-92细胞表面ILT2及KIR2DL4受体相互作用的研究  

Interactions between sHLA-G1 and its receptors ILT2, and KIR2DL4 on NK-92 cells

作　　者：韦娇[1,2] 张海泉 鱼艳荣[3] Muti-Ur-Rehman Khan 汪蕴[3] 邓力[2] 丰美福[3] 

机构地区：[1]四川大学华西药学院药物化学教研室,成都610041 [2]四川大学华西医院生物治疗国家重点实验室干细胞与组织工程研究室 [3]中国科学院动物研究所生物膜与膜生物工程国家重点实验室

出　　处：《中国医药生物技术》2007年第6期428-434,共7页Chinese Medicinal Biotechnology

摘　　要：目的探讨可溶性HLA-G1(sHLA-G1)对人NK-92细胞杀伤活性的抑制与细胞表面免疫球蛋白样转录分子2(ILT2)和杀伤细胞免疫球蛋白样受体2DL4(KIR2DL4)受体的关系。方法①通过原核表达技术获得sHLA-G1重组蛋白(重组蛋白),并采用蛋白质印迹法进行鉴定。②取NK-92细胞,加入终浓度20μg/ml的重组蛋白分别培养10、30min,再分别加入抗HLA-G1/G5、抗ILT2和抗KIR2DL4抗体,采用流式细胞术检测各组NK-92细胞表面sHLA-G1和ILT2、KIR2DL4受体表达阳性率;以NK-92细胞单独培养作为对照组。③以人白血病K562细胞为靶细胞,以经不同方式处理的NK-92细胞为效应细胞,效靶比为5:1,共同培养2h,采用流式细胞术检测NK-92细胞对K562细胞的杀伤率。NK-92细胞处理方式为单纯重组蛋白处理(分别加入终浓度为0、10、20μg/ml的重组蛋白培养30min)和表面受体封闭+重组蛋白处理(分别加入抗ILT2、抗KIR2DL4、抗LT2+抗KIR2DL4抗体培养30min,再分别加入终浓度为0、10、20μg/ml的重组蛋白培养30min)。结果①蛋白质印迹分析表明所获重组蛋白为带有组氨酸标签的特异蛋白。②NK-92细胞与20μg/ml重组蛋白共培养30min后,sHLA-G1表达阳性率明显高于而ILT2、KIR2DL4受体表达阳性率均明显低于对照组(均P<0.05)。③以终浓度0、10、20μg/ml的重组蛋白处理的NK-92细胞对K562细胞的杀伤率分别为39.79%±2.00%、27.79%±0.75%、21.36%±0.67%(两两比较,均P<0.01);单独封闭ILT2受体,杀伤率分别为23.09%±1.63%、21.13%±0.38%、18.42%±0.47%(两两比较,均P<0.01);单独封闭KIR2DL4受体,杀伤率分别为30.74%±0.44%、26.03%±0.38%、21.15%±0.35%(两两比较,均P<0.01)。结论sHLA-G1通过与NK-92细胞表面ILT2和KIR2DL4受体直接结合而抑制NK-92细胞的杀伤活性。Objective To investigate the inhibitory effect of soluble HLA-G1 (sHLA-G1) on the cytolytic function of NK-92, and the relation of sHLA-G1 receptors, ILT2 and KIR2DL4, to the inhibition. Methods ①Recombinant sHLA-G1 was obtained by prokaryotic expression and verified by Western blot. ②NK-92 cells were treated with the recombinant sHLA-G1 at 20 μg/ml for 10 or 30 min, and then added with, anti-HLA-G1/G5 mAb, anti-ILT2 Ab, and anti-KIR2DL4 mAb. NK-92 without treatment served as a positive control. The expression of sHLA-G1, ILT2, and KIR2DL4 on the surface of NK-92 cells was analyzed with flow cytometry. ③Human leukemia cells K562 was used as target cells and the NK-92cells treated with recombinants sHLA-G1 as the effecters. The effecters and target cells was co-cultured for 2 h at a ratio of 5:1. Then, flow cytometry was used to detect the death rate of K562 cells. Before the co-culture, the effecter, NK-92 cells were divided into two groups. In one group, the NK-92 was treated with 0, 10, or 20μg/ml recombinant sHLA-G1 for 30 min; and in the other group, it was first incubated with anti-ILT2 Ab, or anti-KIR2DL4 mAb, or both, for 30 min, and then co-cultured with 0, 10, or 20μg/ml recombinant sHLA-G1 for 30 min. Results ①The recombinant protein was verified as having a histidine tag by Western blot. ②Compared with the control, the positive rate of sHLA-G1 expression was significantly increased and that of the ILT2 and KIR2DL4 was significantly decreased in the NK-92 cells treated with 20 μg/ml recombinant sHLA-G1 for 30 min (P < 0.05). ③After being co-cultured with 0, 10, or 20 μg/ml recombinant sHLA-G1-treated NK-92, the death rate of the K562 reached 39.79% ± 2.00%, 27.79% ± 0.75%, or 21.36% ± 0.67% (one-to-one compared, P < 0.05). When the ILT2 was blocked, the death rate was 23.09% ± 1.63%, 21.13% ± 0.38%, and 18.42% ± 0.47% (one-to-one compared, P < 0.05); and when the KIR2DL4 was blocked, it was 30.74% ± 0.44%, 26.03% ± 0.38%, 21.15% ± 0.35% (one-to-one compared, P < 0.05). Conclusi

关 键 词：HLA抗原 杀伤细胞 天然 受体 细胞表面 重组融合蛋白质类 

分 类 号：R392[医药卫生—免疫学]