本文選題：造血干細胞移植　切入點：移植物抗宿主病　出處：《山西醫(yī)科大學》2008年碩士論文

【摘要】： 目前異基因造血干細胞移植(Allogeneic Hematopoietic Stem Cell Transplantation,Allo-HSCT)是根治多種惡性血液病的唯一手段。但移植后的并發(fā)癥,尤其是移植物抗宿主病(Graft Versus Host Disease,GVHD)仍然是影響移植療效的重要因素,是目前移植領域亟待解決的課題。GVHD是由供者移植物中的T淋巴細胞識別宿主主要和次要組織相容性抗原引起的免疫反應。這些抗原由抗原遞呈細胞(APC)呈遞后,通過MHC-Ⅰ、Ⅱ分子結合于供者T細胞,從而激活T細胞,發(fā)生GVHD。在防治移植排斥的研究和臨床實踐中,強有力的免疫抑制劑及去除供者移植物中T淋巴細胞的策略最常采用。但是,大量免疫抑制劑的使用,除本身諸如肝腎毒性等毒副作用外,隨用藥時間的延長,免疫抑制劑所引起的嚴重的機會感染等矛盾日益突出。同樣去除移植物中的T細胞雖然可以顯著降低GVHD的發(fā)生率并可減輕其嚴重程度,但是移植物中由于缺乏T細胞,其增強免疫和移植物抗白血病(GVL)作用也顯著降低。統(tǒng)計結果表明,用去除供者T細胞的骨髓移植治療白血病,病人的復發(fā)率明顯升高。實際上在移植物中導致GVHD發(fā)生的T細胞僅僅是T細胞中很少的一部分,是針對主要及次要組織相容性抗原特異性的Th1細胞,而與GVL相關的是Th2細胞。所以,最理想的方法是能夠選擇性地清除引起GVHD的淋巴細胞,同時保留具有GVL作用和其它功能的細胞。 樹突狀細胞(Dendritic Cell,DC)是體內最重要的抗原提呈細胞(Antigen Presenting Cell,APC),其免疫學功能具有兩重性。在一定條件下,能處理提呈抗原,表達共刺激分子,分泌細胞因子,活化T淋巴細胞,產生免疫應答,但在另一些條件下卻可使T細胞產生耐受,即介導免疫耐受。近來有應用腺病毒或逆轉錄病毒為載體,將具有誘導耐受作用的免疫抑制分子靶基因轉染給供體DC并使其表達。已不斷有實驗證實,單獨轉導以上各靶基因可降低移植物排斥機率,延長器官植活時間。 免疫球蛋白樣轉錄物(Immunoglobulin-like Transcript,ILT)3和ILT4是表達于DC表面的抑制性受體,對于耐受性DC的形成起著非常重要的作用。研究發(fā)現(xiàn),維生素D3或細胞因子(如IL-10,IFN-α等)能誘導未成熟DC轉化為耐受性DC,而IL-10還可使ILT4表達增高,同時下調共刺激信號分子CD86等的表達。這些都說明ILT3、ILT4表達增加可能是耐受性DC的共同特征,高表達或強制性表達的ILT3、ILT4可誘導DC耐受,從而使機體產生免疫耐受。 基于以上的研究,我們認為用ILT基因轉染DC后,體外與供體造血干細胞移植物混合培養(yǎng),有望能選擇性清除引起GVHD反應的T淋巴細胞,而同時保留抗腫瘤、抗感染的淋巴細胞。第一部分:首先從大鼠骨髓中分離并誘導擴增DC進行功能鑒定,將重組腺病毒載體構建ILT轉染DC并進一步檢測轉染后DC生物學特性,觀察ILT基因轉染對DC的影響。結果:基因轉染后RT-PCR檢測高表達ILT,表明腺病毒載體能有效的將ILT轉染DC上;用流式細胞術對基因轉染前后DC細胞表面的CD80、CD86、MHC-Ⅱ等表型進行分析,沒有發(fā)現(xiàn)明顯改變,這些說明ILT基因轉染后沒有改變DC原有的活性和基本功能,作為本目的基因的表達載體具有可使用性。第二部分:探討ILT-DC對異基因T細胞的作用。通過MTT法檢測異基因混合淋巴細胞增殖情況表明,經過ILT-DC與無病毒轉染DC(對照組)相比顯著抑制了淋巴細胞增殖。進一步用Annexin V/PI雙檢法檢測ILT-DC與異基因反應性T細胞作用后T細胞的凋亡情況,結果發(fā)現(xiàn)ILT-DC有誘導同種異基因T細胞凋亡的作用。 通過同種異基因體外反應模型,重組腺病毒載體構建ILT轉染DC可通過使T細胞低反應性和細胞凋亡在移植中誘導一定程度的免疫耐受,移植經過這種處理的骨髓有望能夠抑制GVHD的發(fā)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 鍏鋒湁鍙嬌鐢ㄦ，

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