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

【摘要】： 目的:探索自然殺傷(natural killer,NK)細(xì)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(xì)胞活性的影響;研究NK細(xì)胞KIR2DS1基因表達(dá)對AML(急性髓系白血病)靶細(xì)胞殺傷活性的影響作用及探討NK細(xì)胞KIR/HLA-Cw(human leucocyte antigen-Cw,人類白細(xì)胞抗原Cw位點(diǎn))錯(cuò)配的殺傷機(jī)制。 方法:采用聚合酶鏈?zhǔn)椒磻?yīng)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(xì)胞磁珠分選試劑盒負(fù)向分選高純度供者來源的NK細(xì)胞作為效應(yīng)細(xì)胞;AML患者新鮮骨髓分離的單個(gè)核細(xì)胞作為靶細(xì)胞;抗CD158a,CD158b單克隆抗體封閉NK細(xì)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(xì)胞對AML細(xì)胞的殺傷率。 結(jié)果:磁珠負(fù)選NK細(xì)胞,經(jīng)流式細(xì)胞術(shù)檢測,測的分選后CD3-/CD56+16+細(xì)胞含量為(90.8±6.08)%;NK細(xì)胞KIR基因與靶細(xì)胞HLA-Cw的匹配數(shù)少NK細(xì)胞的殺傷功能強(qiáng),0個(gè)匹配的殺傷率為(50.66±8.40)%,1個(gè)匹配與2個(gè)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(gè)相合的KIR/HLA-Cw配對組中＞50%表達(dá)抑制性KIRs的NK細(xì)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(xì)胞抑制性KIR受體封閉后,NK細(xì)胞對靶細(xì)胞的殺傷作用增強(qiáng)t=-3.00,P=0.005;C1組KIR2DS1+的NK細(xì)胞對C2組靶細(xì)胞的殺傷作用高于C1組及C1/C2組的靶細(xì)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(xì)胞對AML白血病細(xì)胞的殺傷機(jī)制遵循KIR/HLA-Cw配體不相合學(xué)說;KIR/HLA-Cw的匹配數(shù),KIRs表達(dá)水平與NK的活性相關(guān),匹配數(shù)少,NK細(xì)胞的活性強(qiáng),殺傷AML白血病靶細(xì)胞的能力強(qiáng):活化性基因KIR2DS1+的NK細(xì)胞對靶細(xì)胞的殺傷率高于KIR2DS1-的NK細(xì)胞;C1組KIR2DS1+的NK細(xì)胞對C2組靶細(xì)胞的殺傷率高于對C1、C1/C2組靶細(xì)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(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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本文編號：1829584