本文選題：人源化小鼠 + NOD/SCID��； 參考：《中國科學技術(shù)大學》2010年博士論文

【摘要】：人源化小鼠不僅是一個在活體情況下研究人生理學和病理學的強大工具,也是一個前臨床的模型可以用來研究臨床治療的相關(guān)問題。我的論文研究的主要目的是拓展人源化小鼠作為研究人造血和淋巴系統(tǒng)模型的能力和潛力,它由兩部分組成：1)人紅細胞和血小板在人源化小鼠中的發(fā)育2)建立TCR轉(zhuǎn)基因的人源化小鼠模型用于研究抗腫瘤的基因治療。 1.人紅細胞和血小板在人源化小鼠中的發(fā)育 一個能夠支持人紅細胞發(fā)育的動物模型對于研究人紅細胞在生理和病理下的生物學功能具有很高的價值,并且會有助于評估體外條件下從人胚胎干細胞分化人紅細胞的臨床應用策略。雖然NOD背景的免疫缺陷小鼠已經(jīng)被廣泛的應用于研究人造血干細胞的分化,利用這些模型成功的研究人紅細胞生成和紅細胞功能還沒有報道。之前,我們證明通過在NOD/SCID或者NOD/S CID/γc-/-小鼠腎被膜下移植人胚胎胸腺并且轉(zhuǎn)輸CD34+胚胎肝臟細胞能夠發(fā)育出多種人淋巴造血細胞。在本次研究中,我們檢測了人源化小鼠中的人紅細胞發(fā)育。雖然在人源化小鼠的骨髓中發(fā)現(xiàn)了大量的由非成熟有核紅細胞,但是在所有的人源化小鼠,包括那些外周血人白細胞近完全嵌合的人源化小鼠中也無法檢測到人紅細胞。我們發(fā)現(xiàn)受體小鼠的巨噬細胞是這些小鼠缺乏人紅細胞的一個主要原因,因為巨噬細胞清除后就能在人源化小鼠外周血中檢測到人紅細胞,并且這些人紅細胞會隨著停止巨噬細胞清除而逐漸消失。另外,通過補充人的紅細胞生成素(EPO)和人白介素3(IL-3)能夠顯著的提高人紅細胞的組成。如人紅細胞在人源化小鼠中發(fā)育的情況類似,轉(zhuǎn)輸?shù)娜思t細胞會在NOD/SCID小鼠體內(nèi)被迅速清除,并且我們還發(fā)現(xiàn)人紅細胞被清除的速度明顯快于小鼠CD47缺陷的紅細胞,這顯示小鼠巨噬細胞介導的人紅細胞清除可以不依賴于經(jīng)典的CD47—SIRPα信號通路。 和紅細胞的研究類似,現(xiàn)在也缺乏一個小動物模型用以研究人血小板在活體狀態(tài)下的生理和病理學功能。為此,我們也檢測了人源化小鼠中的人血小板組成。雖然這些小鼠的外周血中有包括人T,B細胞在內(nèi)的高水平的人白細胞嵌合,但是即使在那些人白細胞接近完全嵌合的人源化小鼠中人血小板組成非常低。我們發(fā)現(xiàn),受體小鼠中巨噬細胞介導的排斥作用是人血小板重組水平低的重要原因,一經(jīng)巨噬細胞清除,人的血小板的嵌合比例可以達到人白細胞的嵌合水平,并且人的血小板會隨著停止巨噬細胞清除而逐漸消失。類似的,從人血中分離的血小板能夠被NOD/SCID小鼠中巨噬細胞迅速清除,但是這種清除速度卻明顯小于對CD47缺陷小鼠血小板的清除速度。這暗示,小鼠巨噬細胞對于人血小板的排斥可以不依賴于經(jīng)典的CD47- SIRPα信號通路,而某些一種抗原介導了小鼠巨噬細胞的活化。 2.建立用于抗腫瘤研究的TCR轉(zhuǎn)基因人源化小鼠 誘導腫瘤抗原特異性免疫反應被認為是一個潛在的抗腫瘤免疫治療方式。在一些前臨床治療中,通過用含有腫瘤抗原特異性TCR的慢病毒感染病人自身單個核細胞(PBMCs)產(chǎn)生腫瘤特異性T細胞,并將這些T細胞轉(zhuǎn)輸?shù)讲∪梭w內(nèi)被證明能夠明顯的抑制一些病人的癌癥發(fā)生。小鼠上的研究證明利用腫瘤抗原TCR基因,同工程手段改造造血干細胞能夠提供一個更加有效的癌癥治療方式,這些從病毒感的染造血干細胞發(fā)育來源的腫瘤抗原特異性T細胞能夠產(chǎn)生很強的抗腫瘤作用,并介導對已經(jīng)形成腫瘤的清除。在本次研究中我們拓展了該治療策略,使得人源化小鼠中的人免疫系統(tǒng)具備抗腫瘤作用。我們在免疫缺陷小鼠上通過移植人胚胎胸腺(腎被膜下)和尾靜脈注射經(jīng)過含有TCR基因的慢病毒感染過的CD34+造血干細胞,建立起HLA-A*0201限制性Mart-1(黑色素瘤抗原T細胞識別抗原)特異性TCR轉(zhuǎn)基因的人源化小鼠。通過Mart-1特異性Tetramer作流失細胞術(shù)檢測證實了這些人源化小鼠中存在表達：Mart-1特異性TCR的人CD8+T細胞,并且Mart-1 TCR+的T細胞的比例可以通過清除胸腺細胞進一步提高。重要的是,人源化小鼠中Mart-1 TCR+的T細胞能夠?qū)art-1抗原反應,在經(jīng)過Mart-1多肽免疫后能夠分泌IFN-γ。據(jù)我們所知,這是第一個具有功能性的抗原特異性T細胞產(chǎn)生的TCR轉(zhuǎn)基因人源化小鼠,它為通過利用腫瘤抗原特異TCR工程改造人造血干細胞進行抗腫瘤的免疫治療提供有效平臺。 綜合以上研究,人源化小鼠為研究人的造血和淋巴系統(tǒng)提供了一個非常有效的小動物模型。通過克服由巨噬細胞介導的排斥作用,該模型可以進一步用于研究人紅系細胞和巨核細胞在人源化小鼠中的發(fā)育、分化和功能。并且,這種人源化小鼠不光具有功能性的人免疫系統(tǒng),通過重建具有功能性的腫瘤抗原特異性T細胞,使得該小鼠成為一個能夠用于拓展人癌癥免疫治療的有價值的新模型。
[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.
【學位授予單位】：中國科學技術(shù)大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R392

【共引文獻】

相關(guān)期刊論文 前3條

1 費小明;汪承亞;繆扣榮;吳雨潔;楊慧;周小玉;潘芹芹;;建立非肥胖型糖尿病/重癥聯(lián)合免疫缺陷(NOD/SCID)小鼠人白血病模型的實驗研究[J];南京醫(yī)科大學學報(自然科學版);2007年10期

2 達萬明;馮四洲;;清腫瘤性異基因造血干細胞移植[J];生物醫(yī)學工程與臨床;2012年01期

3 廖樂樂;婁世鋒;曾翰慶;白鳳霞;;高純度白介素-3與假單胞菌外毒素融合蛋白的表達、純化及質(zhì)譜鑒定[J];生物技術(shù)通報;2010年06期

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1 董漢華;肝卵圓細胞在局部微環(huán)境中通過EMT表現(xiàn)上皮/間葉可塑性的研究[D];華中科技大學;2011年

2 楊智勇;人腦膠質(zhì)瘤干細胞的分離、培養(yǎng)、初步鑒定以及腦膠質(zhì)瘤治療的新探討[D];昆明醫(yī)學院;2008年

3 馬力;針對Hh信號通路的人惡性膠質(zhì)瘤靶向治療的實驗研究[D];浙江大學;2010年

4 毛錦龍;Ⅱ-Ⅲ級星型細胞瘤體外誘導耐藥細胞—干細胞基因及耐藥基因研究[D];北京協(xié)和醫(yī)學院;2010年

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1 黃金;逆轉(zhuǎn)錄酶抑制劑對膠質(zhì)瘤干細胞端粒酶活性和細胞增殖影響的實驗研究[D];昆明醫(yī)學院;2011年

2 楊轉(zhuǎn)移;腦腫瘤干細胞化療藥物敏感性的實驗研究[D];中南大學;2007年

3 黃曉斌;BCNU對人腦腫瘤干細胞增殖及細胞周期的影響[D];昆明醫(yī)學院;2008年

4 梅樹婷;~（125）IUDR對C6腦膠質(zhì)瘤中CSCs殺傷作用研究[D];昆明醫(yī)學院;2008年

5 宋峻巖;腎癌中CD133~+細胞和CD133~-細胞生物學特性差異的研究[D];蘭州大學;2008年

6 楊保旺;ABCG2在腎癌中的表達及其與腎癌干細胞關(guān)系的初步研究[D];蘭州大學;2009年

7 張振興;三氧化二砷（AS2O3）對腦腫瘤干細胞的治療研究[D];中南大學;2009年

8 廖樂樂;高純度白介素-3與假單胞菌外毒素融合蛋白的表達、純化及質(zhì)譜鑒定[D];重慶醫(yī)科大學;2010年

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本文編號：1997030