基于人源化小鼠的抗人胸腺细胞球蛋白的体内效果和应用风险研究

发布时间：2018-06-20 07:57

本文选题：抗胸腺细胞球蛋白 + 人源化小鼠　；参考：《吉林大学》2016年博士论文

【摘要】：背景:同种异体造血干细胞或器官移植在多种恶性肿瘤、遗传性血液病及器官衰竭治疗等领域应用广泛。但由于供体和宿主间MHC分子配型很难完全一致,移植后经常产生由于供体免疫细胞攻击宿主组织/器官导致的Gv HD症状,或宿主免疫细胞攻击供体器官的Hv G反应。这些移植免疫排斥反应不仅严重影响治疗效果,甚至导致患者死亡。因为T细胞在这些反应过程中起着关键作用,有效清除人T细胞或抑制其功能可有效减缓Gv HD的发生和提高移植器官的生存时间。因此,免疫抑制药物的开发和研究一直被认为是异体移植领域的关键。抗胸腺细胞球蛋白(ATG)具有清除人T细胞的效应,是一种临床常用免疫抑制剂。ATG是通过用人胸腺细胞或T细胞系免疫动物(如兔、马等)后收集血清所获得的一类多克隆抗体。ATG的抗原识别谱广泛,不仅识别在T细胞表面特异性表达的分子(如,CD2、CD3、CD4、CD8),还可结合在其它血液系统细胞表面表达的分子,包括B细胞(CD19和CD20)、自然杀伤(Nature killer,NK)细胞(CD16和CD56)、树突状细胞(CD11b,CD80和CD86),以及在淋巴造血细胞广泛表达的CD45和MHCI类分子等。ATG的这些特征不仅增加了其临床使用效果的不确定性也提高了应用的潜在风险。由于临床用ATG特异性识别人造血免疫细胞的表面抗原而不识别其他种属(如小鼠、大鼠)的免疫细胞,ATG的体内作用效果无法用传统模式动物模型来评价。虽然大量患者外周血的检测信息一定程度反应了ATG对人免疫系统的效果,但ATG对于不同的淋巴组织、器官(如脾脏、淋巴结、胸腺、骨髓)中人体造血免疫细胞的作用却不清楚。为克服这一难题,该研究应用具有人类免疫系统的人源化小鼠对ATG的体内效应进行了系统研究。除了评价ATG对成熟血液淋巴细胞的清除作用,我们还探讨对不同的淋巴组织、器官(如脾脏、淋巴结、胸腺、骨髓)中人体造血免疫细胞的作用。实验室早期工作证明通过给免疫缺陷小鼠移植人胚胎胸腺/造血干细胞可建立具有人类造血免疫系统的人源化小鼠(有学者又称其BLT小鼠模型)。这一模型是当前国际上使用最广泛的、被认为是体内研究人免疫系统功能的最优动物模型之一。为此,我们以此人源化小鼠模型为基础研究临床用ATG的体内作用效果,并评估其应用风险。实验目的:利用人源化小鼠模型系统性研究ATG对不同组织、器官中各种类型人免疫细胞及其前体细胞的影响;通过模拟临床应用ATG的预处理方案评估ATG临床治疗可能导致的风险;根据得到的数据进一步探讨ATG在构建个体化人源化小鼠模型中的应用。实验方法:通过给2Gy全身照射预处理的NOD/SCID或者NSG小鼠移植人胚胎胸腺(肾背膜下)和人胚胎肝脏来源CD34+造血干细胞(静脉注射)建立具有人免疫系统组成的人源化小鼠模型;通过流式细胞术检测人不同亚群免疫细胞的组成;通过H/E染色判断ATG对不同免疫组织的清除作用;通过在免疫缺陷小鼠上移植HLA-A*0201+胚胎胸腺和HLA-A*0201-胚胎CD34+造血干细胞模拟个体化人源化小鼠模型的构建;ATG通过静脉给药。实验结果:体外实验发现临床用ATG能结合几乎所有人外周血淋巴细胞、人骨髓来源造血干细胞及人胎肝来源造血干细胞,说明ATG除清除人T细胞外,还可能清除其他多种人造血免疫细胞(包括人造血干细胞)。体内实验表明ATG处理组人源化小鼠外周血和脾脏中的人CD3+T细胞、CD19+B细胞、CD14+单核细胞都出现了不同程度的下降;我们还首次发现高剂量ATG能有效杀伤人胸腺中的胸腺细胞,包括成熟T细胞和T细胞前体细胞。与外周血、脾脏和胸腺的情况不同,我们发现虽然ATG体内注射后能够结合几乎所有人源化小鼠骨髓中的人造血免疫细胞,但是骨髓内各种人造血免疫细胞的比例在ATG处理前后并无明显变化,提示骨髓的免疫微环境可能导致人免疫细胞对ATG作用的逃逸。在临床上ATG作为预处理药物被用于抑制异体造血细胞移植后Gv HD的发生。我们利用人源化小鼠模型的研究发现,在人造血干细胞移植前后几天内使用ATG预处理宿主可完全清除移植的造血干细胞,导致移植失败;然而,在移植后3周左右(此时人造血干细胞已归巢于骨髓龛)ATG处理宿主对造血干细胞植入无明显影响。该结果首次揭示了ATG作为预处理药物在造血干细胞移植早期应用有降低造血干细胞植入率的风险。通过病人骨髓造血干细胞和HLA部分匹配的人胚胎胸腺共同移植建立的能够复制病人免疫系统的个体化人源化小鼠模型是人源化小鼠模型的发展方向之一。然而,由异体人胚胎胸腺细胞来源的T细胞对人骨髓造血干细胞的排斥作用是模型构建的瓶颈。我们利用ATG有效清除人胸腺中T细胞前体细胞但不清除已归巢的人造血干细胞的特点,提出了新的个体化人源化小鼠模型的构建策略,为实现个体化人源化小鼠广泛应用模型奠定了基础。结论:(1)与体外ATG能够结合几乎所有人免疫细胞及造血干细胞的现象一致,体内使用ATG不但能有效清除外周血中人免疫细胞也能对组织器官(如脾脏、胸腺)中的人血液淋巴细胞进行杀伤;(2)ATG不但有效清除人T细胞,对人B细胞、单核细胞也有不同程度的清除;(3)ATG体内处理虽然能够结合骨髓中的几乎所有人造血免疫细胞,但无明显清除作用,表明骨髓微环境可保护人免疫细胞不被ATG清除,提示临床病人骨髓中T细胞(大多是记忆性T细胞)可能对ATG处理相对不敏感;(4)ATG可有效清除未归巢的人造血干细胞,揭示了临床使用ATG作为异体造血干细胞移植的预处理方案存在降低干细胞植入的潜在风险;(5)ATG具有清除人胸腺细胞的效应,为优化个体化人源化小鼠模型的构建提供了手段。总之,本论文的研究有望进一步加深人们对ATG体内作用效果的理解,为制定更优化的临床ATG使用方案及个体化人源化小鼠模型的构建提供了非常有价值的信息和参照。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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