封闭CD158受体表位对供者NK细胞杀伤受者成熟DC细胞的研究

发布时间：2018-03-04 09:29

本文选题：自然杀伤细胞　切入点：造血干细胞移植　出处：《山西医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:就目前来说,异基因造血干细胞移植(allogeneic hematopoietic stem cell transplantaion,allo-HSCT)依然是治愈某些恶性血液病最有效的一种手段。但是,移植物抗宿主病(graft-versus-host disease,GVHD)是移植后最为严重的并发症。杀伤细胞免疫球蛋白样受体(killer cell immunoglobulin-like receptors,KIR)在alloHSCT的免疫中发挥重要作用。受者体内缺乏供者抑制型KIR的相应配体,可致供者自然杀伤(natural killer cell,NKs)细胞产生异源反应性,杀伤受者树突状细胞(dendritic cell,DC)减低GVHD的发生率。目前,在体外通过封闭抑制型受体活化供者NK细胞,对受者DC细胞进行杀伤,并将供者KIR基因型表达情况与临床单倍体allo-HSCT预后相结合,探讨供者KIR与受者HLA模式,为HSCT选择最佳供者的研究并不多。本实验分为两个部分:一部分在体外利用抗CD158a及CD158b的单克隆抗体对供者NK细胞表面抑制型受体KIR2DL1及KIR2DL2/L3进行封闭,研究供者NK细胞对受者DC的杀伤作用;另一部分回顾分析了接受单倍体allo-HSCT的急性白血病患者一般资料,探讨KIR/HLA受配体模式对单倍型HSCT预后的影响,为在临床找寻最佳移植供者以及联合NK细胞过继输注改善HSCT预后提供参考。第一部分体外修饰供者NK细胞杀伤受者DC减轻GVHD的作用研究方法:选取2010年5月至2014年12月行单倍体allo-HSCT的5例急性白血病患者及相应供者分别作为研究对象。采用序列特异性引物聚合酶链反应(即PCR-SSP)法,对5对供/受者移植前检测KIR和HLA分型,分别采集供受者外周血单个核细胞,使用NK细胞分选试剂盒对供者NK细胞进行富集,并以其作为效应细胞,而取受者DC作为实验靶细胞,利用抗CD158单抗分别对供者表面KIR抑制型受体进行封闭,并采用CCK-8比色法分别比较抑制性KIR封闭前后的NK细胞对DC的杀伤作用。结果:同一效靶比(10∶1)下,供者NK细胞表面抑制性受体封闭后,对受者DC的杀伤活性明显升高,通过调整封闭抗体高浓度可提高其杀伤活性,组间差异有统计学意义(P0.05)。单一封闭KIR2DL2/L3受体表位,在同一效靶比(10∶1)下,供受者KIR2DL1/C1组对靶细胞杀伤效果高于受者为C2及C1/C2组,用不同浓度的CD158a单抗进行封闭,随封闭抗体浓度的增加,杀伤效果增强。第二部分单倍型异基因造血干细胞移植治疗急性白血病疗效观察方法:回顾整理5例之前接受单倍体allo-HSCT的患者资料,移植前均使用改良白消安/环磷酰胺(Bu/Cy)+抗人胸腺细胞球蛋白(ATG)进行预处理。结果:5例受者均成功完成供者干细胞的持久植入。中性粒细胞(需连续3d)计数≥0.5×109/L的中位时间是13d,血小板(不输并且连续7d)计数≥20×l09/L的中位时间是18d。表达KIR2DL1、KIR2DL2/3且KIR2DS1阳性供者对C2/C2组受者在移植后造血重建较快、急慢性GVHD发生率较低、移植后感染情况较轻。结论:利用抗CD158单抗封闭供者NK细胞表面抑制型受体KIR2DL1及KIR2DL2/L3可以提高供者来源NK细胞对受者DC的杀伤活性;供受者KIR2DL1/C1组对DC杀伤效果可能高于受者为C2及C1/C2组;在单倍型allo-HSCT中,供者同时表达KIR2DL1、KIR2DL2/3且KIR2DS1表达阳性,可能对C2组受者的预后更好;探究供者KIR受者HLA模式能为之后移植供者的最优选择提供分子依据。
[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.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R457.7

【参考文献】