本文選題：抗胸腺細胞球蛋白 + 人源化小鼠　； 參考：《吉林大學》2016年博士論文

【摘要】：背景:同種異體造血干細胞或器官移植在多種惡性腫瘤、遺傳性血液病及器官衰竭治療等領域應用廣泛。但由于供體和宿主間MHC分子配型很難完全一致,移植后經(jīng)常產生由于供體免疫細胞攻擊宿主組織/器官導致的Gv HD癥狀,或宿主免疫細胞攻擊供體器官的Hv G反應。這些移植免疫排斥反應不僅嚴重影響治療效果,甚至導致患者死亡。因為T細胞在這些反應過程中起著關鍵作用,有效清除人T細胞或抑制其功能可有效減緩Gv HD的發(fā)生和提高移植器官的生存時間。因此,免疫抑制藥物的開發(fā)和研究一直被認為是異體移植領域的關鍵�？剐叵偌毎虻鞍�(ATG)具有清除人T細胞的效應,是一種臨床常用免疫抑制劑。ATG是通過用人胸腺細胞或T細胞系免疫動物(如兔、馬等)后收集血清所獲得的一類多克隆抗體。ATG的抗原識別譜廣泛,不僅識別在T細胞表面特異性表達的分子(如,CD2、CD3、CD4、CD8),還可結合在其它血液系統(tǒng)細胞表面表達的分子,包括B細胞(CD19和CD20)、自然殺傷(Nature killer,NK)細胞(CD16和CD56)、樹突狀細胞(CD11b,CD80和CD86),以及在淋巴造血細胞廣泛表達的CD45和MHCI類分子等。ATG的這些特征不僅增加了其臨床使用效果的不確定性也提高了應用的潛在風險。由于臨床用ATG特異性識別人造血免疫細胞的表面抗原而不識別其他種屬(如小鼠、大鼠)的免疫細胞,ATG的體內作用效果無法用傳統(tǒng)模式動物模型來評價。雖然大量患者外周血的檢測信息一定程度反應了ATG對人免疫系統(tǒng)的效果,但ATG對于不同的淋巴組織、器官(如脾臟、淋巴結、胸腺、骨髓)中人體造血免疫細胞的作用卻不清楚。為克服這一難題,該研究應用具有人類免疫系統(tǒng)的人源化小鼠對ATG的體內效應進行了系統(tǒng)研究。除了評價ATG對成熟血液淋巴細胞的清除作用,我們還探討對不同的淋巴組織、器官(如脾臟、淋巴結、胸腺、骨髓)中人體造血免疫細胞的作用。實驗室早期工作證明通過給免疫缺陷小鼠移植人胚胎胸腺/造血干細胞可建立具有人類造血免疫系統(tǒng)的人源化小鼠(有學者又稱其BLT小鼠模型)。這一模型是當前國際上使用最廣泛的、被認為是體內研究人免疫系統(tǒng)功能的最優(yōu)動物模型之一。為此,我們以此人源化小鼠模型為基礎研究臨床用ATG的體內作用效果,并評估其應用風險。實驗目的:利用人源化小鼠模型系統(tǒng)性研究ATG對不同組織、器官中各種類型人免疫細胞及其前體細胞的影響;通過模擬臨床應用ATG的預處理方案評估ATG臨床治療可能導致的風險;根據(jù)得到的數(shù)據(jù)進一步探討ATG在構建個體化人源化小鼠模型中的應用。實驗方法:通過給2Gy全身照射預處理的NOD/SCID或者NSG小鼠移植人胚胎胸腺(腎背膜下)和人胚胎肝臟來源CD34+造血干細胞(靜脈注射)建立具有人免疫系統(tǒng)組成的人源化小鼠模型;通過流式細胞術檢測人不同亞群免疫細胞的組成;通過H/E染色判斷ATG對不同免疫組織的清除作用;通過在免疫缺陷小鼠上移植HLA-A*0201+胚胎胸腺和HLA-A*0201-胚胎CD34+造血干細胞模擬個體化人源化小鼠模型的構建;ATG通過靜脈給藥。實驗結果:體外實驗發(fā)現(xiàn)臨床用ATG能結合幾乎所有人外周血淋巴細胞、人骨髓來源造血干細胞及人胎肝來源造血干細胞,說明ATG除清除人T細胞外,還可能清除其他多種人造血免疫細胞(包括人造血干細胞)。體內實驗表明ATG處理組人源化小鼠外周血和脾臟中的人CD3+T細胞、CD19+B細胞、CD14+單核細胞都出現(xiàn)了不同程度的下降;我們還首次發(fā)現(xiàn)高劑量ATG能有效殺傷人胸腺中的胸腺細胞,包括成熟T細胞和T細胞前體細胞。與外周血、脾臟和胸腺的情況不同,我們發(fā)現(xiàn)雖然ATG體內注射后能夠結合幾乎所有人源化小鼠骨髓中的人造血免疫細胞,但是骨髓內各種人造血免疫細胞的比例在ATG處理前后并無明顯變化,提示骨髓的免疫微環(huán)境可能導致人免疫細胞對ATG作用的逃逸。在臨床上ATG作為預處理藥物被用于抑制異體造血細胞移植后Gv HD的發(fā)生。我們利用人源化小鼠模型的研究發(fā)現(xiàn),在人造血干細胞移植前后幾天內使用ATG預處理宿主可完全清除移植的造血干細胞,導致移植失敗;然而,在移植后3周左右(此時人造血干細胞已歸巢于骨髓龕)ATG處理宿主對造血干細胞植入無明顯影響。該結果首次揭示了ATG作為預處理藥物在造血干細胞移植早期應用有降低造血干細胞植入率的風險。通過病人骨髓造血干細胞和HLA部分匹配的人胚胎胸腺共同移植建立的能夠復制病人免疫系統(tǒng)的個體化人源化小鼠模型是人源化小鼠模型的發(fā)展方向之一。然而,由異體人胚胎胸腺細胞來源的T細胞對人骨髓造血干細胞的排斥作用是模型構建的瓶頸。我們利用ATG有效清除人胸腺中T細胞前體細胞但不清除已歸巢的人造血干細胞的特點,提出了新的個體化人源化小鼠模型的構建策略,為實現(xiàn)個體化人源化小鼠廣泛應用模型奠定了基礎。結論:(1)與體外ATG能夠結合幾乎所有人免疫細胞及造血干細胞的現(xiàn)象一致,體內使用ATG不但能有效清除外周血中人免疫細胞也能對組織器官(如脾臟、胸腺)中的人血液淋巴細胞進行殺傷;(2)ATG不但有效清除人T細胞,對人B細胞、單核細胞也有不同程度的清除;(3)ATG體內處理雖然能夠結合骨髓中的幾乎所有人造血免疫細胞,但無明顯清除作用,表明骨髓微環(huán)境可保護人免疫細胞不被ATG清除,提示臨床病人骨髓中T細胞(大多是記憶性T細胞)可能對ATG處理相對不敏感;(4)ATG可有效清除未歸巢的人造血干細胞,揭示了臨床使用ATG作為異體造血干細胞移植的預處理方案存在降低干細胞植入的潛在風險;(5)ATG具有清除人胸腺細胞的效應,為優(yōu)化個體化人源化小鼠模型的構建提供了手段。總之,本論文的研究有望進一步加深人們對ATG體內作用效果的理解,為制定更優(yōu)化的臨床ATG使用方案及個體化人源化小鼠模型的構建提供了非常有價值的信息和參照。
[Abstract]:Background: allogeneic hematopoietic stem cells or organ transplantation are widely used in a variety of malignant tumors, hereditary hematopathy and organ failure treatment. However, the MHC molecular configuration between donor and host is difficult to be completely identical. After transplantation, Gv HD symptoms caused by donor immune cells attacking host tissues / organs, or host immunity, are often produced. Cells attack the Hv G reaction of donor organs. These graft rejection reactions not only seriously affect the effect of treatment, but also cause death of the patients. Because T cells play a key role in these reactions, effective clearance of human T cells or inhibition of their function can effectively slow the occurrence of Gv HD and improve the survival time of transplanted organs. Therefore, immunization The development and research of inhibitory drugs have been considered as the key to the field of allograft. Anti thymosin (ATG) has the effect of scavenging human T cells. It is a commonly used immunosuppressant,.ATG, to collect a class of polyclonal antibody.ATG obtained by using human thymus or T cell lines, such as rabbits, horses, etc., to collect a kind of polyclonal antibody. The antigen recognition spectrum is widely used not only to identify molecules specifically expressed on the surface of T cells (such as CD2, CD3, CD4, CD8), but also to combine molecules expressed on the surface of other blood system cells, including B cells (CD19 and CD20), natural killer (Nature killer, NK) cells, dendritic cells, and lymphohemopoietic cells. These features, such as the ubiquitous CD45 and MHCI class molecules, not only increase the uncertainty of the effect of the clinical use, but also increase the potential risk of the application. Because the clinical use of ATG specifically recognizes the surface antigen of artificial blood immune cells and does not identify the immune cells of other species (such as mice, rats), the effect of ATG can not be used in vivo. Although a large number of patients' peripheral blood detection information has reacted to the effect of ATG on human immune system to a certain extent, the effect of ATG on human immune cells in different lymphoid tissues, such as spleen, lymph node, thymus and bone marrow, is not clear. In addition to evaluating the scavenging effect of ATG on mature blood lymphocytes, we also explore the role of human hematopoietic cells in different lymphoid tissues and organs (such as spleen, lymph node, thymus, bone marrow). The early work in the laboratory proved to be immune to immune deficiency, in addition to evaluating the scavenging effect of ATG on mature blood lymphocytes. Mouse transplanted human embryo thymus / hematopoietic stem cells can establish human derived mice with human hematopoietic immune system (some scholars also called BLT mice model). This model is the most widely used in the world and is considered to be one of the best animal models to study the function of human immune system in vivo. Therefore, we use this human model as a human model. To study the effect of ATG in vivo and evaluate its application risk. Objective: to systematically study the effects of ATG on various types of human immune cells and their precursor cells in different tissues and organs by using a humanized mouse model, and to evaluate the possible wind induced by the pretreatment of the clinical application of ATG by simulating clinical application of the clinical application of ATG. Risk; further explore the application of ATG in the construction of a personalized humanized mouse model based on the data obtained. Experimental methods: a person with human immune system composition is established by the NOD/SCID or NSG mice transplanted into the human embryonic thymus (under the renal dorsal membrane) and the human fetal liver derived CD34+ hematopoietic stem cells (intravenous injection). To determine the composition of different subsets of human immune cells by flow cytometry; to determine the scavenging effect of ATG on different immune tissues by H/E staining; the construction of a individualized humanized mouse model by transplantation of HLA-A*0201+ embryonic thymus and HLA-A*0201- embryo CD34+ hematopoietic stem cells on immunodeficient mice; ATG pass. Intravenous administration. Experimental results: in vitro, ATG can combine almost all human peripheral blood lymphocytes, human bone marrow derived hematopoietic stem cells and human fetal liver stem cells. It is suggested that ATG may remove a variety of other artificial blood immune cells (including artificial blood stem cells) except for human T cells. In vivo experiments show that ATG is in the body. The human CD3+T cells, CD19+B cells and CD14+ mononuclear cells in the peripheral blood and spleen of the mice were reduced to varying degrees. We also found that the high dose ATG could effectively kill the thymus cells in the human thymus, including the mature T cells and the T cell precursor cells. We found that the cases were different from the peripheral blood, the spleen and the thymus. Although the intramedullary injection of ATG can combine almost everyone to the human immune cells in the bone marrow of mice, the proportion of various artificial blood immune cells in the bone marrow does not change significantly before and after ATG treatment, suggesting that the immune microenvironment of the bone marrow may lead to the escape of human immune cells to ATG. In clinical, ATG is used as a preconditioning agent. The substance was used to inhibit the occurrence of Gv HD after allogeneic hematopoietic cells transplantation. We found that the use of ATG preconditioning hosts within a few days before and after artificial blood stem cell transplantation can completely remove the transplanted hematopoietic stem cells in a few days before and after the artificial blood stem cell transplantation, leading to the failure of transplantation; however, 3 weeks after transplantation (at this time artificial blood stem cells have been returned to the nest. " The ATG treatment host has no significant effect on hematopoietic stem cell implantation. This results first revealed the risk of reducing the rate of hematopoietic stem cell implantation by ATG as a preconditioning drug in the early use of hematopoietic stem cells. The co transplantation of human embryonic thymus with the patient's bone marrow hematopoietic stem cells and HLA parts can be replicated. The individualized humanized mouse model of the patient's immune system is one of the developing directions of the humanized mouse model. However, the rejection of T cells derived from human embryonic thymus cells to human bone marrow hematopoietic stem cells is the bottleneck of the model construction. We use ATG to effectively remove the T cell precursor cells in the human thymus, but do not clear the homing. The characteristics of artificial blood stem cells put forward a new individualized humanized mouse model construction strategy, which laid the foundation for the realization of the extensive application model of individualized humanized mice. Conclusion: (1) in vitro ATG can combine almost all human immune cells and hematopoietic stem cells, and the use of ATG in vivo can not only effectively remove the peripheral blood in the blood. Human immune cells can also kill human lymphocytes in tissues and organs such as spleen and thymus. (2) ATG not only effectively scavenging human T cells, but also to human B cells and monocytes to varying degrees. (3) although the treatment of ATG in vivo can be combined with almost all artificial blood cells in the bone marrow, there is no clear scavenging effect, indicating bone. Intramedullary microenvironment can protect human immune cells from ATG clearance, suggesting that T cells in bone marrow (mostly memory T cells) in clinical patients may be relatively insensitive to ATG treatment. (4) ATG can effectively remove unhomed artificial blood stem cells. It is revealed that the clinical use of ATG as a preconditioning scheme for allogeneic stem cell transplantation reduces stem cell transplantation. The potential risk of entry; (5) ATG has the effect of scavenging human thymus cells, which provides a means to optimize the construction of individual humanized mouse models. In a word, this study is expected to further deepen people's understanding of the effect of ATG in vivo, and to formulate a more optimized clinical ATG use scheme and individual humanized mouse model. It provides very valuable information and reference.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R96

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