免疫球蛋白样抑制性受体KIR基因与HLA-Cw的匹配对NK细胞活性影响的实验研究

发布时间：2018-05-01 13:31

本文选题：免疫球蛋白样抑制性受体 + CD158　；参考：《苏州大学》2009年硕士论文

【摘要】： 目的:探索自然杀伤(natural killer,NK)细胞免疫球蛋白样受体(killerimmunoglobulin-like receptor,KIR)基因和HLA-Cw配体匹配数对NK细胞活性的影响;研究NK细胞KIR2DS1基因表达对AML(急性髓系白血病)靶细胞杀伤活性的影响作用及探讨NK细胞KIR/HLA-Cw(human leucocyte antigen-Cw,人类白细胞抗原Cw位点)错配的杀伤机制。方法:采用聚合酶链式反应和顺序特异性引物(PCR-SSP)基因分型技术分别检测27例健康供者及30例患者HLA-Cw、KIR基因;NK细胞磁珠分选试剂盒负向分选高纯度供者来源的NK细胞作为效应细胞;AML患者新鲜骨髓分离的单个核细胞作为靶细胞;抗CD158a,CD158b单克隆抗体封闭NK细胞KIR2DL1,KIR2DL2/3受体;MTT比色法检测KIR受体封闭前后,KIR基因与HLA-Cw不同组合的NK细胞对AML细胞的杀伤率。结果:磁珠负选NK细胞,经流式细胞术检测,测的分选后CD3-/CD56+16+细胞含量为(90.8±6.08)%;NK细胞KIR基因与靶细胞HLA-Cw的匹配数少NK细胞的杀伤功能强,0个匹配的杀伤率为(50.66±8.40)%,1个匹配与2个匹配的杀伤率分别为(38.28%±6.71)%,(19.84±4.32)%,F=20.226,P＜0.001;1个相合的KIR/HLA-Cw配对组中＞50%表达抑制性KIRs的NK细胞杀伤率为10%,25%-50%的杀伤率20%,＜25%杀伤率55%,F=16.276,p＜0.001;NK细胞抑制性KIR受体封闭后,NK细胞对靶细胞的杀伤作用增强t=-3.00,P=0.005;C1组KIR2DS1+的NK细胞对C2组靶细胞的杀伤作用高于C1组及C1/C2组的靶细胞,杀伤率分别为(57.370±1.400)%、(44.190±4.666)%、(36.770±6.56)%,F=11.87,P=0.021,KIR受体封闭后差异更为明显,F=18.72,P=0.009。结论:NK细胞对AML白血病细胞的杀伤机制遵循KIR/HLA-Cw配体不相合学说;KIR/HLA-Cw的匹配数,KIRs表达水平与NK的活性相关,匹配数少,NK细胞的活性强,杀伤AML白血病靶细胞的能力强:活化性基因KIR2DS1+的NK细胞对靶细胞的杀伤率高于KIR2DS1-的NK细胞;C1组KIR2DS1+的NK细胞对C2组靶细胞的杀伤率高于对C1、C1/C2组靶细胞的杀伤作用。
[Abstract]:Objective: To explore the effect of natural killer (NK) cell immunoglobulin like receptor (killerimmunoglobulin-like receptor, KIR) gene and HLA-Cw ligand matching number on NK cell activity, and to investigate the effect of NK cell KIR2DS1 gene expression on the killing activity of AML (acute myeloid leukemia) target cells. Man leucocyte antigen-Cw, human leukocyte antigen Cw locus) mismatch killing mechanism.
Methods: polymerase chain reaction and sequence specific primer (PCR-SSP) genotyping were used to detect the HLA-Cw, KIR gene of 27 healthy donors and 30 patients, and NK cells with high purity donor source were negatively selected as effector cells by NK cell magnetic beads sorting kit, and the mononuclear cells isolated from fresh bone marrow of AML patients were used as target cells. Anti CD158a, CD158b monoclonal antibody closed NK cells KIR2DL1, KIR2DL2/3 receptor, and MTT colorimetric assay to detect the killing rate of NK cells with KIR gene and HLA-Cw different combinations of NK cells to AML cells before and after KIR receptor closure.
Results: the magnetic bead negative selected NK cells were detected by flow cytometry. The content of CD3-/CD56+16+ cells was (90.8 + 6.08)% after the flow cytometry, and the KIR gene of NK cells and the target cell HLA-Cw were less lethal, 0 matched killing rates were (50.66 + 8.40)%, and 1 matching and 2 matched (38.28% + 6.71)%, respectively, (19.84 + 4.32. )%, F=20.226, P < 0.001; the killing rate of NK cells expressed in the 1 matched KIR/HLA-Cw pairing group was 10%, the killing rate of 25%-50% was 20%, the killing rate of < 25% was 55%, F=16.276, P < 0.001, NK cell inhibitory KIR receptor closed, NK cells enhanced the target cells. The killing effect of the cells was higher than the target cells in the C1 group and the C1/C2 group. The killing rate was (57.370 + 1.400)%, (44.190 + 4.666)%, (36.770 + 6.56)%, F=11.87, P=0.021, and the difference was more obvious after the KIR receptor was closed, F=18.72, P=0.009.
Conclusion: the killing mechanism of NK cells to AML leukemia cells follows the theory of KIR/HLA-Cw ligand incompatibility; the matching number of KIR/HLA-Cw, the expression level of KIRs is related to the activity of NK, the matching number is less, the activity of NK cells is strong and the ability to kill the target cells of AML leukemia is strong: the killing rate of NK cells with active gene KIR2DS1+ is higher than that of KIR2DS1- N. K cells; C1 group KIR2DS1+ NK cells on C2 group target cell killing rate is higher than C1, C1/C2 group target cell killing effect.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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