本文選題：免疫球蛋白樣抑制性受體 + CD158�。� 參考：《蘇州大學》2009年碩士論文

【摘要】： 目的:探索自然殺傷(natural killer,NK)細胞免疫球蛋白樣受體(killerimmunoglobulin-like receptor,KIR)基因和HLA-Cw配體匹配數(shù)對NK細胞活性的影響;研究NK細胞KIR2DS1基因表達對AML(急性髓系白血病)靶細胞殺傷活性的影響作用及探討NK細胞KIR/HLA-Cw(human leucocyte antigen-Cw,人類白細胞抗原Cw位點)錯配的殺傷機制。 方法:采用聚合酶鏈式反應和順序特異性引物(PCR-SSP)基因分型技術(shù)分別檢測27例健康供者及30例患者HLA-Cw、KIR基因;NK細胞磁珠分選試劑盒負向分選高純度供者來源的NK細胞作為效應細胞;AML患者新鮮骨髓分離的單個核細胞作為靶細胞;抗CD158a,CD158b單克隆抗體封閉NK細胞KIR2DL1,KIR2DL2/3受體;MTT比色法檢測KIR受體封閉前后,KIR基因與HLA-Cw不同組合的NK細胞對AML細胞的殺傷率。 結(jié)果:磁珠負選NK細胞,經(jīng)流式細胞術(shù)檢測,測的分選后CD3-/CD56+16+細胞含量為(90.8±6.08)%;NK細胞KIR基因與靶細胞HLA-Cw的匹配數(shù)少NK細胞的殺傷功能強,0個匹配的殺傷率為(50.66±8.40)%,1個匹配與2個匹配的殺傷率分別為(38.28%±6.71)%,(19.84±4.32)%,F=20.226,P＜0.001;1個相合的KIR/HLA-Cw配對組中＞50%表達抑制性KIRs的NK細胞殺傷率為10%,25%-50%的殺傷率20%,＜25%殺傷率55%,F=16.276,p＜0.001;NK細胞抑制性KIR受體封閉后,NK細胞對靶細胞的殺傷作用增強t=-3.00,P=0.005;C1組KIR2DS1+的NK細胞對C2組靶細胞的殺傷作用高于C1組及C1/C2組的靶細胞,殺傷率分別為(57.370±1.400)%、(44.190±4.666)%、(36.770±6.56)%,F=11.87,P=0.021,KIR受體封閉后差異更為明顯,F=18.72,P=0.009。 結(jié)論:NK細胞對AML白血病細胞的殺傷機制遵循KIR/HLA-Cw配體不相合學說;KIR/HLA-Cw的匹配數(shù),KIRs表達水平與NK的活性相關(guān),匹配數(shù)少,NK細胞的活性強,殺傷AML白血病靶細胞的能力強:活化性基因KIR2DS1+的NK細胞對靶細胞的殺傷率高于KIR2DS1-的NK細胞;C1組KIR2DS1+的NK細胞對C2組靶細胞的殺傷率高于對C1、C1/C2組靶細胞的殺傷作用。
[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.

【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

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