本文關鍵詞： NK細胞 KIR基因 2DS4等位基因 異基因造血干細胞移植 移植物抗宿主病　出處：《蘇州大學》2011年碩士論文　論文類型：學位論文

【摘要】：【目的和意義】造血干細胞移植(Hematopoietic Stem Cell Transplantation, HSCT)是目前治療惡性血液病的最有效方法之一,供者和患者人類白細胞抗原系統(tǒng)(Human Leukocyte Antigen, HLA)相合與否被認為是影響造血干細胞移植成敗的決定性因素。但是,即使是在HLA高分辨基因分型全相合的造血干細胞移植中,移植預后在受者間也存在著很大的差異。因此,近年來,除了HLA基因相合外,自然殺傷細胞(Natural Killer, NK)受體系統(tǒng)日益受到關注。殺傷細胞免疫球蛋白樣受體(killer Ig-like receptor, KIR)是表達在NK細胞及部分T細胞表面的一類受體,在異基因造血干細胞移植中起著重要的作用。KIR基因的分布在不同人種不同個體間存在很大的差異,目前尚缺乏對中國漢族人群KIR基因分布規(guī)律的了解,因而,研究KIR基因遺傳學背景的差異對進一步探討其對疾病的影響具有重要意義。KIR基因有兩種單體型:單體型A和單體型B。單體型A存在唯一的活化型受體基因2DS4,其中只有2DS4*001 (包括2DS4*0010101、*0010102、*0010103、*00102、*00103)編碼NK細胞表面有功能的受體,其他等位基因(如2DS4*003, *004, *006, *007, *008,*009)在第5外顯子有22個堿基的缺失,導致移框突變使其所編碼的蛋白缺失了跨膜區(qū)和胞內區(qū),從而無法錨定在細胞膜上,以功能尚不明確的游離蛋白的形式存在。但是,到目前為止,國內外尚無針對KIR2DS4基因與造血干細胞移植相關的研究。隨著對KIR受體識別和殺傷腫瘤細胞重要作用的不斷了解,人們逐漸認識到供者KIR與受者HLA遺傳背景影響了HSCT預后,由抑制型KIR (inhibitory KIR receptors, iKIR) -HLA“錯配模式”引起的NK細胞異源反應性在HSCT中促進移植物植入以及增強移植物抗白血病效應(graft-versus-leukemia, GVL)的作用,已經成為國內外學者研究的熱點。同時,供者來源的活化型KIR受體(activating KIR receptors, aKIR)傳導的活化信號對NK細胞活性的調節(jié)可能起到在臨床治療中如何控制移植物抗宿主病(graft-versus-host disease, GVHD)和調節(jié)移植物抗白血病效應的作用。 【方法】研究對象為150例中國漢族人,75對接受異基因造血干細胞移植的供者及受者,患者白血病分型為ALL29例、CML24例、AML19例、其他惡性血液病3例。 此項研究共分為三部分:⑴采用基因測序(Sequence-based typing, SBT)對HLA-A, -B, -C, -DRB1和-DQB1位點進行高分辨分型;采用序列特異性引物聚合酶鏈反應(PCR-sequence-specific primers PCR-SSP)進行KIR基因分型,研究了KIR基因以及KIR單體型在中國漢族人群中的分布情況以及與其他人種進行了比較,并且在此基礎上分析了供者KIR和受者HLA的配受體關系。⑵建立了SBT結合TOPO TA克隆的方法對2DS4等位基因進行全長測序,證實了2DS4完整型與缺失型等位基因的存在,研究KIR2DS4等位基因在中國人群中的分布,分析了中國人群KIR2DS4等位基因的分布特點并發(fā)現(xiàn)了三個新等位基因。⑶建立了供者KIR-受者HLA的配受體模式,分析了“缺失配體”模式對異基因HSCT預后的影響;采用實時定量PCR (Real-time PCR),對30例存在“缺失配體”的移植動態(tài)隨訪了受者體內KIR2DL1、2DS1、3DL1和3DS1基因在移植后的mRNA表達;并結合臨床資料,分析KIR單體型對移植預后的影響,同時進一步分析了其中35例供者為KIR單體型A純合的移植中供者來源2DS4等位基因與移植后急性GVHD發(fā)生的關系,并且從轉錄水平研究了KIR2DL1/3DL1基因與異基因HSCT預后的相關性。 【結果】⑴17種KIR基因均能夠被檢測出,比例從21.3%至100%不等,所有樣本都存在框架基因3DL3、3DP1、2DL4和3DL2,除框架基因以外,2DL1和2DP1是在中國漢族人群中分布最廣的基因,而比例最低的是2DL2和2DS3;44.0%的個體KIR分型為A/A,56.0%的個體KIR分型為B/x。KIR基因與KIR單體型在中國漢族人群中的分布比例與日本人群相似,而與高加索人群和非洲裔美國人群存在較大差異。⑵分析供者抑制型KIR受體與受者HLA配體分子,75例移植中有53例供者存在KIR2DL1基因而受者沒有HLA-C2組位點;2例供者存在KIR2DL12/L3基因而受者沒有HLA-C1組位點;24例供者存在KIR3DL1基因而受者沒有HLA-Bw4位點。⑶中國漢族人群在KIR基因及2DS4等位基因分布上具有其獨特性。150例樣本中139例(92.7%)存在KIR2DS4基因,對2DS4基因全長測序后發(fā)現(xiàn)4個2DS4等位基因:KIR2DS4*00101、*003、*004和*007;78.4%(109/139)的個體存在2DS4*00101;2DS4缺失型(個體存在1個或缺失2DS4*00101基因)與完整型(個體存在兩個2DS4*00101基因)的存在比率為1:2。在139例2DS4陽性的個體中,66例KIR分型為A/A,73例KIR分型為B/x型。在A/A組中,42.4% (28/66)的個體為完整型/缺失型2DS4雜合,明顯高于B/x組(11.0%, 8/73);12.1% (8/66)的個體為缺失型2DS4純合,這個比例在B/x組中為30.1% (22/73)。發(fā)現(xiàn)了3個KIR2DS4新基因。⑷研究KIR基因對75例進行HSCT預后的影響,60例移植供受者均為HLA-A, -B, -C, -DRB1, -DQB1位點高分辨全相合,15例有單個HLA-C等位基因分型不相合。存在“缺失配體”模式的移植在A/A組和B/x組中所占的比例分別為88.6%和87.5%。采用多因素分析研究“缺失配體”模式移植的比例對移植預后的影響沒有發(fā)現(xiàn)統(tǒng)計學差異。75例移植中,當供者KIR分型為B/x (n=40)時,移植后總體生存率(Overall survival, OS)要明顯高于供者為A/A組(n=35) (RR 3.1 [95% CI 1.1-8.6], P= 0.007)。在其中35例供者為KIR單體型A/A純合的移植中,當供者2DS4等位基因為完整型(n=14)時,移植后急性GVHD的發(fā)生率要明顯高于供者2DS4等位基因為缺失型(n=21)的情況(RR 9.0 [95% CI 1.2-66.9], P= 0.010),其中以Ⅲ-Ⅳ度aGVHD尤為明顯(P=0.006)。Real-time PCR動態(tài)隨訪患者移植后2DL1、2DS1、3DL1和3DS1基因mRNA表達水平發(fā)現(xiàn):發(fā)生aGVHD的患者組在移植后30-90天,2DL1和3DL1和的mRNA拷貝數(shù)相比無aGVHD組明顯升高(P值分別為0.021和0.005);當供者與受者均為KIR單體型A純合時,這一差異更為顯著:在發(fā)生aGVHD的患者組與不發(fā)生aGVHD的患者組之間,比較移植后90天時KIR2DL1和3DL1基因的mRNA表達水平,具有統(tǒng)計學上的差異(z=2.558, P=0.011)。結果提示,移植早期(90天) KIR2DL1和3DL1基因的mRNA高表達與aGVHD的發(fā)生呈正相關。 【結論】中國漢族人群在KIR基因及2DS4等位基因分布上具有獨特性;供者為KIR單體型(B/x)的移植預后較好;供者來源完整型2DS4等位基因以及患者移植早期2DL1和3DL1基因mRNA高表達增加了移植后aGVHD的發(fā)生風險。因此,移植前在HLA分型的基礎上結合KIR基因分型和KIR2DS4等位基因分型,可能對臨床上選擇合適的供者提供有利依據(jù);移植后動態(tài)隨訪患者KIR2DL1/3DL1基因mRNA表達有利于預測aGVHD的發(fā)生;探索供者KIR和受者HLA遺傳背景與異基因造血干細胞移植預后的關系,為遺傳學指標指導下供者的優(yōu)化選擇提供分子依據(jù),也為臨床制定治療方案提供參考。
[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絳変綅鍩哄洜鍒嗗竷涓婂叿鏈夊叾鐙壒鎬，

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