ILT基因修饰的树突状细胞诱导移植物免疫耐受的研究

发布时间：2018-03-23 23:03

  本文选题：造血干细胞移植　切入点：移植物抗宿主病　出处：《山西医科大学》2008年硕士论文

【摘要】： 目前异基因造血干细胞移植(Allogeneic Hematopoietic Stem Cell Transplantation,Allo-HSCT)是根治多种恶性血液病的唯一手段。但移植后的并发症,尤其是移植物抗宿主病(Graft Versus Host Disease,GVHD)仍然是影响移植疗效的重要因素,是目前移植领域亟待解决的课题。GVHD是由供者移植物中的T淋巴细胞识别宿主主要和次要组织相容性抗原引起的免疫反应。这些抗原由抗原递呈细胞(APC)呈递后,通过MHC-Ⅰ、Ⅱ分子结合于供者T细胞,从而激活T细胞,发生GVHD。在防治移植排斥的研究和临床实践中,强有力的免疫抑制剂及去除供者移植物中T淋巴细胞的策略最常采用。但是,大量免疫抑制剂的使用,除本身诸如肝肾毒性等毒副作用外,随用药时间的延长,免疫抑制剂所引起的严重的机会感染等矛盾日益突出。同样去除移植物中的T细胞虽然可以显著降低GVHD的发生率并可减轻其严重程度,但是移植物中由于缺乏T细胞,其增强免疫和移植物抗白血病(GVL)作用也显著降低。统计结果表明,用去除供者T细胞的骨髓移植治疗白血病,病人的复发率明显升高。实际上在移植物中导致GVHD发生的T细胞仅仅是T细胞中很少的一部分,是针对主要及次要组织相容性抗原特异性的Th1细胞,而与GVL相关的是Th2细胞。所以,最理想的方法是能够选择性地清除引起GVHD的淋巴细胞,同时保留具有GVL作用和其它功能的细胞。 树突状细胞(Dendritic Cell,DC)是体内最重要的抗原提呈细胞(Antigen Presenting Cell,APC),其免疫学功能具有两重性。在一定条件下,能处理提呈抗原,表达共刺激分子,分泌细胞因子,活化T淋巴细胞,产生免疫应答,但在另一些条件下却可使T细胞产生耐受,即介导免疫耐受。近来有应用腺病毒或逆转录病毒为载体,将具有诱导耐受作用的免疫抑制分子靶基因转染给供体DC并使其表达。已不断有实验证实,单独转导以上各靶基因可降低移植物排斥机率,延长器官植活时间。 免疫球蛋白样转录物(Immunoglobulin-like Transcript,ILT)3和ILT4是表达于DC表面的抑制性受体,对于耐受性DC的形成起着非常重要的作用。研究发现,维生素D3或细胞因子(如IL-10,IFN-α等)能诱导未成熟DC转化为耐受性DC,而IL-10还可使ILT4表达增高,同时下调共刺激信号分子CD86等的表达。这些都说明ILT3、ILT4表达增加可能是耐受性DC的共同特征,高表达或强制性表达的ILT3、ILT4可诱导DC耐受,从而使机体产生免疫耐受。 基于以上的研究,我们认为用ILT基因转染DC后,体外与供体造血干细胞移植物混合培养,有望能选择性清除引起GVHD反应的T淋巴细胞,而同时保留抗肿瘤、抗感染的淋巴细胞。第一部分:首先从大鼠骨髓中分离并诱导扩增DC进行功能鉴定,将重组腺病毒载体构建ILT转染DC并进一步检测转染后DC生物学特性,观察ILT基因转染对DC的影响。结果:基因转染后RT-PCR检测高表达ILT,表明腺病毒载体能有效的将ILT转染DC上;用流式细胞术对基因转染前后DC细胞表面的CD80、CD86、MHC-Ⅱ等表型进行分析,没有发现明显改变,这些说明ILT基因转染后没有改变DC原有的活性和基本功能,作为本目的基因的表达载体具有可使用性。第二部分:探讨ILT-DC对异基因T细胞的作用。通过MTT法检测异基因混合淋巴细胞增殖情况表明,经过ILT-DC与无病毒转染DC(对照组)相比显著抑制了淋巴细胞增殖。进一步用Annexin V/PI双检法检测ILT-DC与异基因反应性T细胞作用后T细胞的凋亡情况,结果发现ILT-DC有诱导同种异基因T细胞凋亡的作用。 通过同种异基因体外反应模型,重组腺病毒载体构建ILT转染DC可通过使T细胞低反应性和细胞凋亡在移植中诱导一定程度的免疫耐受,移植经过这种处理的骨髓有望能够抑制GVHD的发生。
[Abstract]:At present, allogeneic hematopoietic stem cell transplantation (Allogeneic Hematopoietic Stem Cell Transplantation, Allo-HSCT) is the only curative treatment for many hematologic malignancies. But complications after transplantation, especially graft-versus-host disease (Graft Versus Host Disease, GVHD) is still the important factors affecting the efficacy of transplantation,.GVHD is currently the urgent subject field of transplantation the immune response by donor grafts in T lymphocytes recognize host major and minor histocompatibility antigens. These antigens induced by antigen presenting cells (APC) after the presentation, by MHC- I, II molecules on donor T cells, leading to activation of T cells, the occurrence of GVHD. in the study on prevention of allograft rejection and in the clinical practice, the removal of potent immunosuppressive agents and donor lymphocytes in plants T shift strategy is most commonly used. However, a large number of immunosuppressive agents used in itself such as Side effects such as liver and kidney toxicity, with prolonged treatment, serious opportunistic infections and other immunosuppression caused by contradictions have become increasingly prominent. The same removal of T cells in the allograft can significantly decrease the incidence rate of GVHD and reduce its severity, but due to the lack of graft in T cells, the immune enhancement and graft versus leukemia (GVL) effect is also significantly reduced. The statistical results showed that bone marrow transplantation for treatment of leukemia removal of donor T cells, the recurrence rate was significantly increased in the graft. In fact lead to the occurrence of GVHD T cells is only a small part of T cells, is the major and minor histocompatibility antigen specific Th1 cells, and GVL is related to the Th2 cells. Therefore, the best method is to selectively remove GVHD lymphocytes, while preserving the GVL function and other functions is fine Cell.
Dendritic cells (Dendritic Cell, DC) are the most potent antigen-presenting cells (Antigen Presenting Cell, APC), its immune function has double sex. Under certain conditions, can present antigen, expression of costimulatory molecules, cytokine secretion, activation of T lymphocytes, immune response, but on the other some conditions can make the T cell tolerance, which is mediated by immune tolerance. The recent application of adenoviral or retroviral vector, with immune tolerance induced by inhibition of the molecular target of gene transfection to donor DC and express it. There have been confirmed, the target gene transduction alone can reduce graft the probability of rejection, prolong organ engraftment.
Immunoglobulin like transcripts (Immunoglobulin-like, Transcript, ILT) 3 and ILT4 is expressed on the surface of DC inhibitory receptor, for the formation of the tolerance of DC plays a very important role. The study found that vitamin D3 or cytokines (such as IL-10, IFN-, alpha etc.) can induce not mature DC into tolerance DC and IL-10 can make the expression of ILT4 and decreased the expression of costimulatory signal molecule CD86. These are examples of ILT3, increase the expression of ILT4 may be a common feature of the tolerance of DC, high expression or mandatory ILT3, ILT4 can induce DC tolerance, immune tolerance from the body.
Based on the above research, we believe that the ILT gene after DC transfection in vitro and donor hematopoietic stem cell graft mixed culture, is expected to cause the selective removal of GVHD reaction of T lymphocytes, while retaining the anti-tumor, anti infection of lymphocytes. The first part: first isolated from rat bone marrow and induced by DC to identify the function of amplification that will be the construction of a recombinant adenovirus vector ILT was transfected into DC and DC biological characteristics further detection after transfection, to observe the effect of ILT gene transfection on DC. Results: after transfection of RT-PCR detection showed that high expression of ILT adenovirus vector can effectively transfect ILT DC; on CD80, the surface of DC cells before and after transfection with CD86 gene flow cytometry, MHC- II phenotype analysis found no significant changes. These indicated that ILT gene transfection of DC does not change the original activity and basic function, as the target gene expression vector 鍏锋湁鍙�浣跨敤妫�

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