人源化小鼠模型在人造血和淋巴系统研究中的拓展

发布时间：2018-06-08 20:05

本文选题：人源化小鼠 + NOD/SCID　；参考：《中国科学技术大学》2010年博士论文

【摘要】：人源化小鼠不仅是一个在活体情况下研究人生理学和病理学的强大工具,也是一个前临床的模型可以用来研究临床治疗的相关问题。我的论文研究的主要目的是拓展人源化小鼠作为研究人造血和淋巴系统模型的能力和潜力,它由两部分组成：1)人红细胞和血小板在人源化小鼠中的发育2)建立TCR转基因的人源化小鼠模型用于研究抗肿瘤的基因治疗。 1.人红细胞和血小板在人源化小鼠中的发育一个能够支持人红细胞发育的动物模型对于研究人红细胞在生理和病理下的生物学功能具有很高的价值,并且会有助于评估体外条件下从人胚胎干细胞分化人红细胞的临床应用策略。虽然NOD背景的免疫缺陷小鼠已经被广泛的应用于研究人造血干细胞的分化,利用这些模型成功的研究人红细胞生成和红细胞功能还没有报道。之前,我们证明通过在NOD/SCID或者NOD/S CID/γc-/-小鼠肾被膜下移植人胚胎胸腺并且转输CD34+胚胎肝脏细胞能够发育出多种人淋巴造血细胞。在本次研究中,我们检测了人源化小鼠中的人红细胞发育。虽然在人源化小鼠的骨髓中发现了大量的由非成熟有核红细胞,但是在所有的人源化小鼠,包括那些外周血人白细胞近完全嵌合的人源化小鼠中也无法检测到人红细胞。我们发现受体小鼠的巨噬细胞是这些小鼠缺乏人红细胞的一个主要原因,因为巨噬细胞清除后就能在人源化小鼠外周血中检测到人红细胞,并且这些人红细胞会随着停止巨噬细胞清除而逐渐消失。另外,通过补充人的红细胞生成素(EPO)和人白介素3(IL-3)能够显著的提高人红细胞的组成。如人红细胞在人源化小鼠中发育的情况类似,转输的人红细胞会在NOD/SCID小鼠体内被迅速清除,并且我们还发现人红细胞被清除的速度明显快于小鼠CD47缺陷的红细胞,这显示小鼠巨噬细胞介导的人红细胞清除可以不依赖于经典的CD47—SIRPα信号通路。和红细胞的研究类似,现在也缺乏一个小动物模型用以研究人血小板在活体状态下的生理和病理学功能。为此,我们也检测了人源化小鼠中的人血小板组成。虽然这些小鼠的外周血中有包括人T,B细胞在内的高水平的人白细胞嵌合,但是即使在那些人白细胞接近完全嵌合的人源化小鼠中人血小板组成非常低。我们发现,受体小鼠中巨噬细胞介导的排斥作用是人血小板重组水平低的重要原因,一经巨噬细胞清除,人的血小板的嵌合比例可以达到人白细胞的嵌合水平,并且人的血小板会随着停止巨噬细胞清除而逐渐消失。类似的,从人血中分离的血小板能够被NOD/SCID小鼠中巨噬细胞迅速清除,但是这种清除速度却明显小于对CD47缺陷小鼠血小板的清除速度。这暗示,小鼠巨噬细胞对于人血小板的排斥可以不依赖于经典的CD47- SIRPα信号通路,而某些一种抗原介导了小鼠巨噬细胞的活化。 2.建立用于抗肿瘤研究的TCR转基因人源化小鼠诱导肿瘤抗原特异性免疫反应被认为是一个潜在的抗肿瘤免疫治疗方式。在一些前临床治疗中,通过用含有肿瘤抗原特异性TCR的慢病毒感染病人自身单个核细胞(PBMCs)产生肿瘤特异性T细胞,并将这些T细胞转输到病人体内被证明能够明显的抑制一些病人的癌症发生。小鼠上的研究证明利用肿瘤抗原TCR基因,同工程手段改造造血干细胞能够提供一个更加有效的癌症治疗方式,这些从病毒感的染造血干细胞发育来源的肿瘤抗原特异性T细胞能够产生很强的抗肿瘤作用,并介导对已经形成肿瘤的清除。在本次研究中我们拓展了该治疗策略,使得人源化小鼠中的人免疫系统具备抗肿瘤作用。我们在免疫缺陷小鼠上通过移植人胚胎胸腺(肾被膜下)和尾静脉注射经过含有TCR基因的慢病毒感染过的CD34+造血干细胞,建立起HLA-A*0201限制性Mart-1(黑色素瘤抗原T细胞识别抗原)特异性TCR转基因的人源化小鼠。通过Mart-1特异性Tetramer作流失细胞术检测证实了这些人源化小鼠中存在表达：Mart-1特异性TCR的人CD8+T细胞,并且Mart-1 TCR+的T细胞的比例可以通过清除胸腺细胞进一步提高。重要的是,人源化小鼠中Mart-1 TCR+的T细胞能够对Mart-1抗原反应,在经过Mart-1多肽免疫后能够分泌IFN-γ。据我们所知,这是第一个具有功能性的抗原特异性T细胞产生的TCR转基因人源化小鼠,它为通过利用肿瘤抗原特异TCR工程改造人造血干细胞进行抗肿瘤的免疫治疗提供有效平台。综合以上研究,人源化小鼠为研究人的造血和淋巴系统提供了一个非常有效的小动物模型。通过克服由巨噬细胞介导的排斥作用,该模型可以进一步用于研究人红系细胞和巨核细胞在人源化小鼠中的发育、分化和功能。并且,这种人源化小鼠不光具有功能性的人免疫系统,通过重建具有功能性的肿瘤抗原特异性T细胞,使得该小鼠成为一个能够用于拓展人癌症免疫治疗的有价值的新模型。
[Abstract]:Humanized mice are not only a powerful tool for studying life science and pathology in living conditions, but also a pre clinical model that can be used to study clinical treatment. The main purpose of my thesis is to expand the ability and potential of humanized mice as a model of artificial blood and lymphatic systems, which are two Some components: 1) human erythrocytes and platelets in the development of humanized mice 2) the establishment of a TCR transgenic mouse model was used to study the antitumor gene therapy.
Development of 1. human red blood cells and platelets in humanized mice
An animal model that supports human erythrocyte development is of great value to study the biological function of human erythrocytes in physiology and pathology, and will help to evaluate the clinical application of human erythrocytes from human embryonic stem cells in vitro. Although NOD background immunodeficiency mice have been widely used. Studies on the differentiation of artificial blood stem cells have not been reported successfully using these models. Previously, we have shown that a variety of human lymphohemopoietic cells can be developed by transplanting human embryonic thymus under the NOD/SCID or NOD/S CID/ gamma c-/- mouse's renal capsule and transferring the CD34+ embryo liver cells. In this study, we detected human erythrocyte development in humanized mice. Although a large number of non mature nucleated red cells were found in the bone marrow of humanized mice, human erythrocytes were not detected in all human mice, including those in humanized mice that were nearly completely chimeric in the peripheral blood leucocytes. Macrophages in receptor mice are one of the main reasons for the lack of human red blood cells in these mice, because macrophages can detect human red blood cells in human peripheral blood after removal of macrophages, and these human red cells gradually disappear with the removal of macrophages. In addition, by supplementing human erythropoietin (EPO) and human white. Mediator 3 (IL-3) can significantly improve the composition of human erythrocytes. For example, human erythrocytes are developed in human mice, and the transferred human red cells are quickly removed in NOD/SCID mice, and we also found that human red cells are cleared faster than the red cells of CD47 deficient mice. This shows that mouse macrophages are mediated by macrophages. The removal of human red blood cells can not depend on the classic CD47 - SIRP - alpha signaling pathway.
Similar to the study of red blood cells, there is now a lack of a small animal model to study the physiological and pathological functions of human platelets in living conditions. To this end, we have also detected human platelets in human mice, although there are high levels of human leukocyte chimerism including human T, B cells in the peripheral blood of these mice. We found that macrophage mediated rejection in receptor mice is an important cause of low platelet recombination, and that the inlay ratio of human platelets can reach the level of human leukocyte chimerism when macrophages are removed by macrophages. And human platelets gradually disappear with the removal of macrophages. Similarly, platelets separated from human blood can be quickly removed by macrophages in NOD/SCID mice, but the clearance rate is significantly smaller than the clearance rate of platelets in CD47 deficient mice. This suggests that mouse macrophages are repelled to human platelets. It does not depend on the classic CD47- SIRP alpha signaling pathway, and some antigens mediate the activation of macrophages in mice.
2. to establish TCR transgenic humanized mice for anti-tumor research.
Inducing tumor antigen specific immunoreaction is considered to be a potential antitumor immunotherapy. In some pre clinical treatments, tumor specific T cells are produced by using the lentivirus infected patients' own mononuclear cells (PBMCs) containing tumor antigen specific TCR, and the transfer of these T cells into the patient is proved to be possible. The study in mice shows that the use of the tumor antigen TCR gene to transform the hematopoietic stem cells with the engineered methods can provide a more effective way for cancer treatment, which can produce a strong anti-tumor activity from the tumor antigen specific T cells derived from the virus infected hematopoietic stem cells. In this study we expanded the treatment strategy to make the human immune system in humanized mice antitumor. We injected the human embryonic thymus (under the renal capsule) and the tail vein in the immunodeficient mice by injecting the CD34+ infected with the TCR gene. Human derived mice were established with HLA-A*0201 restrictive Mart-1 (melanoma antigen T cell recognition antigen) specific TCR transgenic mice. Through Mart-1 specific Tetramer as the loss cell technique, the expression of these human derived mice was confirmed: the CD8+T cells of Mart-1 specific TCR, and the proportion of Mart-1 TCR+ T cells It is possible to further improve the thymus cells. It is important that the T cells of Mart-1 TCR+ in humanized mice react to the Mart-1 antigen, and can secrete IFN- gamma after being immunized with Mart-1 peptides. As we know, this is the first functional antigen specific T cell produced by TCR transgenic human derived mice, and it is through the benefit of it. The use of tumor antigen specific TCR in engineered hematopoietic stem cells can provide an effective platform for anti-tumor immunotherapy.
This model can be used to further study the development, differentiation and function of human erythroid cells and megakaryocytes in humanized mice. The mice are not only functional human immune systems, by reconstructing functional tumor antigen specific T cells, making the mouse a valuable new model that can be used to expand human cancer immunotherapy.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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