KIR受体基因在中国汉族人群中的分布及其在异基因无关造血干细胞移植中的作用

发布时间：2018-01-30 15:39

  本文关键词： NK细胞 KIR基因 2DS4等位基因 异基因造血干细胞移植 移植物抗宿主病　出处：《苏州大学》2011年硕士论文　论文类型：学位论文

【摘要】：【目的和意义】造血干细胞移植(Hematopoietic Stem Cell Transplantation, HSCT)是目前治疗恶性血液病的最有效方法之一,供者和患者人类白细胞抗原系统(Human Leukocyte Antigen, HLA)相合与否被认为是影响造血干细胞移植成败的决定性因素。但是,即使是在HLA高分辨基因分型全相合的造血干细胞移植中,移植预后在受者间也存在着很大的差异。因此,近年来,除了HLA基因相合外,自然杀伤细胞(Natural Killer, NK)受体系统日益受到关注。杀伤细胞免疫球蛋白样受体(killer Ig-like receptor, KIR)是表达在NK细胞及部分T细胞表面的一类受体,在异基因造血干细胞移植中起着重要的作用。KIR基因的分布在不同人种不同个体间存在很大的差异,目前尚缺乏对中国汉族人群KIR基因分布规律的了解,因而,研究KIR基因遗传学背景的差异对进一步探讨其对疾病的影响具有重要意义。KIR基因有两种单体型:单体型A和单体型B。单体型A存在唯一的活化型受体基因2DS4,其中只有2DS4*001 (包括2DS4*0010101、*0010102、*0010103、*00102、*00103)编码NK细胞表面有功能的受体,其他等位基因(如2DS4*003, *004, *006, *007, *008,*009)在第5外显子有22个碱基的缺失,导致移框突变使其所编码的蛋白缺失了跨膜区和胞内区,从而无法锚定在细胞膜上,以功能尚不明确的游离蛋白的形式存在。但是,到目前为止,国内外尚无针对KIR2DS4基因与造血干细胞移植相关的研究。随着对KIR受体识别和杀伤肿瘤细胞重要作用的不断了解,人们逐渐认识到供者KIR与受者HLA遗传背景影响了HSCT预后,由抑制型KIR (inhibitory KIR receptors, iKIR) -HLA“错配模式”引起的NK细胞异源反应性在HSCT中促进移植物植入以及增强移植物抗白血病效应(graft-versus-leukemia, GVL)的作用,已经成为国内外学者研究的热点。同时,供者来源的活化型KIR受体(activating KIR receptors, aKIR)传导的活化信号对NK细胞活性的调节可能起到在临床治疗中如何控制移植物抗宿主病(graft-versus-host disease, GVHD)和调节移植物抗白血病效应的作用。 【方法】研究对象为150例中国汉族人,75对接受异基因造血干细胞移植的供者及受者,患者白血病分型为ALL29例、CML24例、AML19例、其他恶性血液病3例。 此项研究共分为三部分:⑴采用基因测序(Sequence-based typing, SBT)对HLA-A, -B, -C, -DRB1和-DQB1位点进行高分辨分型;采用序列特异性引物聚合酶链反应(PCR-sequence-specific primers PCR-SSP)进行KIR基因分型,研究了KIR基因以及KIR单体型在中国汉族人群中的分布情况以及与其他人种进行了比较,并且在此基础上分析了供者KIR和受者HLA的配受体关系。⑵建立了SBT结合TOPO TA克隆的方法对2DS4等位基因进行全长测序,证实了2DS4完整型与缺失型等位基因的存在,研究KIR2DS4等位基因在中国人群中的分布,分析了中国人群KIR2DS4等位基因的分布特点并发现了三个新等位基因。⑶建立了供者KIR-受者HLA的配受体模式,分析了“缺失配体”模式对异基因HSCT预后的影响;采用实时定量PCR (Real-time PCR),对30例存在“缺失配体”的移植动态随访了受者体内KIR2DL1、2DS1、3DL1和3DS1基因在移植后的mRNA表达;并结合临床资料,分析KIR单体型对移植预后的影响,同时进一步分析了其中35例供者为KIR单体型A纯合的移植中供者来源2DS4等位基因与移植后急性GVHD发生的关系,并且从转录水平研究了KIR2DL1/3DL1基因与异基因HSCT预后的相关性。 【结果】⑴17种KIR基因均能够被检测出,比例从21.3%至100%不等,所有样本都存在框架基因3DL3、3DP1、2DL4和3DL2,除框架基因以外,2DL1和2DP1是在中国汉族人群中分布最广的基因,而比例最低的是2DL2和2DS3;44.0%的个体KIR分型为A/A,56.0%的个体KIR分型为B/x。KIR基因与KIR单体型在中国汉族人群中的分布比例与日本人群相似,而与高加索人群和非洲裔美国人群存在较大差异。⑵分析供者抑制型KIR受体与受者HLA配体分子,75例移植中有53例供者存在KIR2DL1基因而受者没有HLA-C2组位点;2例供者存在KIR2DL12/L3基因而受者没有HLA-C1组位点;24例供者存在KIR3DL1基因而受者没有HLA-Bw4位点。⑶中国汉族人群在KIR基因及2DS4等位基因分布上具有其独特性。150例样本中139例(92.7%)存在KIR2DS4基因,对2DS4基因全长测序后发现4个2DS4等位基因:KIR2DS4*00101、*003、*004和*007;78.4%(109/139)的个体存在2DS4*00101;2DS4缺失型(个体存在1个或缺失2DS4*00101基因)与完整型(个体存在两个2DS4*00101基因)的存在比率为1:2。在139例2DS4阳性的个体中,66例KIR分型为A/A,73例KIR分型为B/x型。在A/A组中,42.4% (28/66)的个体为完整型/缺失型2DS4杂合,明显高于B/x组(11.0%, 8/73);12.1% (8/66)的个体为缺失型2DS4纯合,这个比例在B/x组中为30.1% (22/73)。发现了3个KIR2DS4新基因。⑷研究KIR基因对75例进行HSCT预后的影响,60例移植供受者均为HLA-A, -B, -C, -DRB1, -DQB1位点高分辨全相合,15例有单个HLA-C等位基因分型不相合。存在“缺失配体”模式的移植在A/A组和B/x组中所占的比例分别为88.6%和87.5%。采用多因素分析研究“缺失配体”模式移植的比例对移植预后的影响没有发现统计学差异。75例移植中,当供者KIR分型为B/x (n=40)时,移植后总体生存率(Overall survival, OS)要明显高于供者为A/A组(n=35) (RR 3.1 [95% CI 1.1-8.6], P= 0.007)。在其中35例供者为KIR单体型A/A纯合的移植中,当供者2DS4等位基因为完整型(n=14)时,移植后急性GVHD的发生率要明显高于供者2DS4等位基因为缺失型(n=21)的情况(RR 9.0 [95% CI 1.2-66.9], P= 0.010),其中以Ⅲ-Ⅳ度aGVHD尤为明显(P=0.006)。Real-time PCR动态随访患者移植后2DL1、2DS1、3DL1和3DS1基因mRNA表达水平发现:发生aGVHD的患者组在移植后30-90天,2DL1和3DL1和的mRNA拷贝数相比无aGVHD组明显升高(P值分别为0.021和0.005);当供者与受者均为KIR单体型A纯合时,这一差异更为显著:在发生aGVHD的患者组与不发生aGVHD的患者组之间,比较移植后90天时KIR2DL1和3DL1基因的mRNA表达水平,具有统计学上的差异(z=2.558, P=0.011)。结果提示,移植早期(90天) KIR2DL1和3DL1基因的mRNA高表达与aGVHD的发生呈正相关。 【结论】中国汉族人群在KIR基因及2DS4等位基因分布上具有独特性;供者为KIR单体型(B/x)的移植预后较好;供者来源完整型2DS4等位基因以及患者移植早期2DL1和3DL1基因mRNA高表达增加了移植后aGVHD的发生风险。因此,移植前在HLA分型的基础上结合KIR基因分型和KIR2DS4等位基因分型,可能对临床上选择合适的供者提供有利依据;移植后动态随访患者KIR2DL1/3DL1基因mRNA表达有利于预测aGVHD的发生;探索供者KIR和受者HLA遗传背景与异基因造血干细胞移植预后的关系,为遗传学指标指导下供者的优化选择提供分子依据,也为临床制定治疗方案提供参考。
[Abstract]:[Objective] and the significance of hematopoietic stem cell transplantation (Hematopoietic Stem Cell Transplantation, HSCT) is one of the most effective methods for the treatment of malignant hematological diseases, donors and patients with human leukocyte antigen system (Human Leukocyte Antigen, HLA) the decisive factor is not consistent with hematopoietic stem cell transplantation success. However, even if in the HLA high resolution genotyping of HLA mismatched hematopoietic stem cell transplantation, transplantation and prognosis are also of great difference in between. Therefore, in recent years, in addition to the HLA gene matched natural killer cells (Natural, Killer, NK) receptor system has attracted more and more attention. The killer cell immunoglobulin like receptor (killer Ig-like receptor, KIR) is a kind of expression in NK cells and T receptor on the surface of cells in allogeneic hematopoietic stem plays a role for the distribution of important.KIR based on different cell transplantation For there are great differences between different individuals, there is still a lack of China Han population KIR gene distribution, therefore, the difference of KIR gene genetic background the significance of the.KIR gene has two haplotypes to further explore its effect on disease has activated receptor gene 2DS4 only exist monomer type and A haplotype B. and haplotype A, only 2DS4*001 (including 2DS4*0010101, *0010102, *0010103, *00102, *00103) encoding functional NK cell surface receptor, other alleles (such as 2DS4*003, *004, *006, *007, *008, *009) in the fifth exon of the 22 bp deletion, resulting in frameshift the mutation of encoding protein lacking transmembrane and intracellular domains, which can not be anchored in the cell membrane, the presence of free protein in the form of function is not clear. However, so far, there is no domestic and made for KIR2DS4 gene Blood stem cell transplantation related research. With the continuous understanding of KIR receptor recognition and killing tumor cells plays an important role, people gradually realize that the donor KIR and the genetic background of HLA affect the prognosis of HSCT by inhibiting KIR (inhibitory, KIR receptors, iKIR) alloreactive NK cells induced by -HLA mismatch model to promote engraftment and enhanced GVL effect in HSCT (graft-versus-leukemia, GVL) role, has become a hot research at home and abroad. At the same time, activated donor type KIR receptor (activating KIR receptors, aKIR) activation of signal transduction regulation on the activity of NK cells may play how to control graft-versus-host disease in clinical treatment (graft-versus-host disease, GVHD) and the regulation of graft versus leukemia effect.
[Methods] the subjects were 150 Han Chinese and 75 donors and recipients who received allogeneic hematopoietic stem cell transplantation. The patients were classified into ALL29 cases, CML24 cases, AML19 cases, and other malignant hematologic diseases in 3 cases.
This study is divided into three parts: firstly by gene sequencing (Sequence-based typing, SBT) of HLA-A, -B, -C, -DRB1 and -DQB1 loci typing; using polymerase chain reaction sequence specific primers (PCR-sequence-specific primers PCR-SSP) KIR gene typing of KIR gene and KIR distribution haplotype in China Han population and other populations were compared and analyzed on the basis of donor KIR and recipient HLA ligand receptor relationship. It has built SBT combined with TOPO TA clone of 2DS4 alleles were sequenced, confirmed the 2DS4 intact and deletion allele the presence of the gene distribution of KIR2DS4 alleles in the Chinese population, analyzes the distribution of population Chinese KIR2DS4 alleles and three new alleles. Established by donor KIR- ligand receptor HLA Model to analyze the impact of "missing ligand" model for allogeneic HSCT prognosis; using quantitative real-time PCR (Real-time PCR), 30 cases of the existence of "dynamic transplantation follow-up missing ligand" by the expression in the mRNA after transplantation in vivo KIR2DL1,2DS1,3DL1 and 3DS1 gene; combined with clinical data, analysis of the influence of KIR haplotype of transplantation prognosis, further analysis of 35 cases of donor KIR haplotype A homozygous transplant donor 2DS4 allele of GVHD gene and the occurrence of acute graft, and at the transcription level of KIR2DL1/3DL1 gene associated with allogeneic HSCT patients.
[results] the 17 kinds of KIR gene could be detected, the proportion from 21.3% to 100% range, all samples are framework genes 3DL3,3DP1,2DL4 and 3DL2, in addition to gene 2DL1 and 2DP1 framework, is the most widely distributed in the Han population in China gene, and is the lowest proportion of 2DL2 and 2DS3; 44% individuals KIR type A/A, 56% individuals of the KIR type was the distribution ratio of B/x.KIR gene and KIR haplotype in Chinese Han population and similar to those of Japanese, but there is a big difference between Caucasian and African American population. The analysis of donor and recipient inhibitory KIR receptor HLA ligands, 75 cases of transplantation 53 cases of donors and recipients without KIR2DL1 gene group HLA-C2 loci; 2 cases of donors and recipients without KIR2DL12/L3 gene group HLA-C1 loci; 24 cases of donor and recipient without the presence of the KIR3DL1 gene HLA-Bw4 locus. The China Han population in KIR group 鍥犲強2DS4绛変綅鍩哄洜鍒嗗竷涓婂叿鏈夊叾鐙�鐗规�，

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