本文選題：自然殺傷細(xì)胞　切入點(diǎn)：造血干細(xì)胞移植　出處：《山西醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:就目前來(lái)說(shuō),異基因造血干細(xì)胞移植(allogeneic hematopoietic stem cell transplantaion,allo-HSCT)依然是治愈某些惡性血液病最有效的一種手段。但是,移植物抗宿主病(graft-versus-host disease,GVHD)是移植后最為嚴(yán)重的并發(fā)癥。殺傷細(xì)胞免疫球蛋白樣受體(killer cell immunoglobulin-like receptors,KIR)在alloHSCT的免疫中發(fā)揮重要作用。受者體內(nèi)缺乏供者抑制型KIR的相應(yīng)配體,可致供者自然殺傷(natural killer cell,NKs)細(xì)胞產(chǎn)生異源反應(yīng)性,殺傷受者樹(shù)突狀細(xì)胞(dendritic cell,DC)減低GVHD的發(fā)生率。目前,在體外通過(guò)封閉抑制型受體活化供者NK細(xì)胞,對(duì)受者DC細(xì)胞進(jìn)行殺傷,并將供者KIR基因型表達(dá)情況與臨床單倍體allo-HSCT預(yù)后相結(jié)合,探討供者KIR與受者HLA模式,為HSCT選擇最佳供者的研究并不多。本實(shí)驗(yàn)分為兩個(gè)部分:一部分在體外利用抗CD158a及CD158b的單克隆抗體對(duì)供者NK細(xì)胞表面抑制型受體KIR2DL1及KIR2DL2/L3進(jìn)行封閉,研究供者NK細(xì)胞對(duì)受者DC的殺傷作用;另一部分回顧分析了接受單倍體allo-HSCT的急性白血病患者一般資料,探討KIR/HLA受配體模式對(duì)單倍型HSCT預(yù)后的影響,為在臨床找尋最佳移植供者以及聯(lián)合NK細(xì)胞過(guò)繼輸注改善HSCT預(yù)后提供參考。第一部分體外修飾供者NK細(xì)胞殺傷受者DC減輕GVHD的作用研究方法:選取2010年5月至2014年12月行單倍體allo-HSCT的5例急性白血病患者及相應(yīng)供者分別作為研究對(duì)象。采用序列特異性引物聚合酶鏈反應(yīng)(即PCR-SSP)法,對(duì)5對(duì)供/受者移植前檢測(cè)KIR和HLA分型,分別采集供受者外周血單個(gè)核細(xì)胞,使用NK細(xì)胞分選試劑盒對(duì)供者NK細(xì)胞進(jìn)行富集,并以其作為效應(yīng)細(xì)胞,而取受者DC作為實(shí)驗(yàn)靶細(xì)胞,利用抗CD158單抗分別對(duì)供者表面KIR抑制型受體進(jìn)行封閉,并采用CCK-8比色法分別比較抑制性KIR封閉前后的NK細(xì)胞對(duì)DC的殺傷作用。結(jié)果:同一效靶比(10∶1)下,供者NK細(xì)胞表面抑制性受體封閉后,對(duì)受者DC的殺傷活性明顯升高,通過(guò)調(diào)整封閉抗體高濃度可提高其殺傷活性,組間差異有統(tǒng)計(jì)學(xué)意義(P0.05)。單一封閉KIR2DL2/L3受體表位,在同一效靶比(10∶1)下,供受者KIR2DL1/C1組對(duì)靶細(xì)胞殺傷效果高于受者為C2及C1/C2組,用不同濃度的CD158a單抗進(jìn)行封閉,隨封閉抗體濃度的增加,殺傷效果增強(qiáng)。第二部分單倍型異基因造血干細(xì)胞移植治療急性白血病療效觀察方法:回顧整理5例之前接受單倍體allo-HSCT的患者資料,移植前均使用改良白消安/環(huán)磷酰胺(Bu/Cy)+抗人胸腺細(xì)胞球蛋白(ATG)進(jìn)行預(yù)處理。結(jié)果:5例受者均成功完成供者干細(xì)胞的持久植入。中性粒細(xì)胞(需連續(xù)3d)計(jì)數(shù)≥0.5×109/L的中位時(shí)間是13d,血小板(不輸并且連續(xù)7d)計(jì)數(shù)≥20×l09/L的中位時(shí)間是18d。表達(dá)KIR2DL1、KIR2DL2/3且KIR2DS1陽(yáng)性供者對(duì)C2/C2組受者在移植后造血重建較快、急慢性GVHD發(fā)生率較低、移植后感染情況較輕。結(jié)論:利用抗CD158單抗封閉供者NK細(xì)胞表面抑制型受體KIR2DL1及KIR2DL2/L3可以提高供者來(lái)源NK細(xì)胞對(duì)受者DC的殺傷活性;供受者KIR2DL1/C1組對(duì)DC殺傷效果可能高于受者為C2及C1/C2組;在單倍型allo-HSCT中,供者同時(shí)表達(dá)KIR2DL1、KIR2DL2/3且KIR2DS1表達(dá)陽(yáng)性,可能對(duì)C2組受者的預(yù)后更好;探究供者KIR受者HLA模式能為之后移植供者的最優(yōu)選擇提供分子依據(jù)。
[Abstract]:Objective: at present, allogeneic hematopoietic stem cell transplantation (allogeneic hematopoietic stem cell transplantaion, allo-HSCT) is still one of the most effective means to cure some hematological malignancies. However, graft-versus-host disease (graft-versus-host disease GVHD) is the most serious complication after transplantation. The killer cell immunoglobulin like receptor (killer cell immunoglobulin-like receptors, KIR) play an important role in alloHSCT immune. The recipient is lack of corresponding ligand donor inhibitory KIR, can induce donor natural killer (natural killer, cell, NKs) cells to produce heterologous reactivity, killing recipient dendritic cells (dendritic, cell, DC) to reduce the incidence of GVHD at present, by blocking the inhibitory receptor activation of donor NK cells in vitro, on DC cells for destruction, and the donor KIR genotype and clinical expression of haploid allo-H The prognosis of SCT combination of donor and recipient KIR HLA mode, HSCT select the best donor research is not much. The experiment is divided into two parts: the first part of the anti CD158a and CD158b in vitro using monoclonal antibodies to donor NK cell surface inhibitory receptors KIR2DL1 and KIR2DL2/L3 are closed, donor NK cell killing effect on recipient DC; another part of the thesis analyses the general data of patients with acute leukemia underwent haploidentical allo-HSCT of KIR/HLA, receptor ligand model of haplotype HSCT prognosis, in search of the best clinical transplantation and combined NK cell adoptive infusion to provide reference to improve the prognosis of HSCT. The first part modified in vitro donor NK cell killing effect by method of DC reduction in GVHD: 5 cases of acute leukemia patients and their donors from May 2010 to December 2014 were selected as research on haploid allo-HSCT Like. Using polymerase chain reaction sequence specific primer (PCR-SSP) method, 5 pairs of donor / recipient before transplantation to detect KIR and HLA type, were collected from donor peripheral blood mononuclear cells were enriched for NK cells of donor NK cell sorting kit, which is a fine effect the cell, and DC as the target cell, respectively, to the donor KIR surface inhibitory receptors were closed using anti CD158 monoclonal antibody, and the cytotoxicity of CCK-8 colorimetric method were compared before and after the closure of the NK inhibitor KIR on DC cells. Results: the same effect target ratio (10: 1). Donor NK cell surface inhibitory receptors is closed, on the killing activity of DC was significantly increased by adjusting the high concentration of blocking antibodies can improve its cytotoxic activity, there was significant difference between the groups (P0.05). The single closed KIR2DL2/L3 receptor epitopes on the same target ratio (10: 1), for KIR2DL1/C1 group of recipients The target cell killing effect is higher than that for C2 and C1/C2 group, closed with different concentrations of CD158a monoclonal antibody, with the increase of blocking antibody concentration, enhance the killing effect. The second part haplotype allogeneic hematopoietic stem cell transplantation therapy of acute leukemia. Methods: 5 cases of patients undergoing review before data haploid allo-HSCT, before transplantation the use of modified busulfan / cyclophosphamide (Bu/Cy) + antithymocyte globulin (ATG) pretreatment. Results: 5 patients were successfully implanted donor stem cells. Persistent neutrophil (continuous 3D) median time count over 0.5 * 109/L 13D, platelet (not transmission and continuous 7D) median time count over 20 * l09/L 18d. and KIR2DL2/3 KIR2DL1 expression, KIR2DS1 positive donors to recipients in group C2/C2 in rapid hematopoietic reconstitution after transplantation, acute and chronic lower incidence of GVHD, than in the case of infection after transplantation Light. Conclusion: using anti CD158 monoclonal antibody blocking donor NK cell surface inhibitory receptors KIR2DL1 and KIR2DL2/L3 can improve the donor recipient NK cells on the killing activity of DC; KIR2DL1/C1 group of donor DC killing effect may be higher than the recipients were C2 and C1/C2 in allo-HSCT group; haplotype, donor and expression of KIR2DL1 KIR2DL2/3, and the positive expression of KIR2DS1 on the C2 group have a better prognosis; explore the donor KIR optimal HLA model can for donor selection and provide a molecular basis.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R457.7

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相關(guān)期刊論文 前1條

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本文編號(hào)：1565115