受体编辑在天然多反应性B细胞发育中的作用及机制研究
发布时间：2018-10-13 09:26
【摘要】： 天然自身抗体(natural autoantibody,NAA)是指在没有任何抗原免疫的情况下,正常机体中针对一种或多种自身抗原的自身抗体;产生NAA的B细胞则被称为天然自身反应性B细胞。NAA及天然自身反应性B细胞广泛存在并发挥重要功能,大量研究提示,自身反应性B细胞存在的耐受机制与自身免疫病的发病机理密切相关。 受体编辑现象是自身反应性B细胞的发育耐受的主导机制。受体编辑通过多种作用方式,一方面保证了人体外周中具有自身反应性的B细胞比例与骨髓相比大大降低;另一方面,发生了受体编辑从而改变特异性的自身反应性B细胞可以逃脱克隆清除或失活的命运而发育成熟。但是,受体编辑现象作为B细胞发育耐受的核心机制,其参与病理性自身免疫即自身免疫病的方式存在巨大争论。因此,研究生理性天然多反应性B细胞发育中受体编辑的作用必将有助于全面揭示自身免疫失耐受的真正原因。 本课题组以往的研究进展,为深入研究受体编辑现象在自身反应性B细胞发育中的作用机制奠定了良好的基础。应用未免疫小鼠B细胞与骨髓瘤细胞融合,我们成功筛选到了分泌抗角蛋白、肌动蛋白等多种自身抗原的自身抗体的3B4等杂交瘤,其中由未免疫小鼠获得的3B4天然多反应性抗体是一种完全意义上的NAA,而表达3B4抗体的B细胞可以代表生理广泛存在的天然自身反应性B细胞。通过克隆3B4可变区基因,我们成功构建了可以表达天然多反应性BCR的3B4重链(TgVH3B4)和轻链(TgVL3B4)转基因小鼠。抗体转基因小鼠的应用,为深入研究自身反应性B细胞发育耐受提供了理想的平台。而来源于未免疫小鼠天然自身反应性IgM的3B4抗体转基因小鼠,为深入研究受体编辑在天然自身反应B细胞发育中的作用机制等问题提供了绝佳模型。 本研究通过交配重链与轻链小鼠,在其子代中筛选到了完整表达天然多反应性3B4抗体的转基因重轻链小鼠(TgVH/L3B4)。TgVH/L3B4小鼠中表达转基因完整BCR的B细胞复制了3B4抗体分泌细胞在野生小鼠中的发育过程;而表达内源轻链的TgVH3B4小鼠由于表达转基因重链与不同内源轻链所组成的BCR,更有利于观察不同特异性自身反应性B细胞发育耐受的普遍规律。利用这个平台,通过比较研究TgVH3B4小鼠和TgVH/L3B4小鼠,我们就受体编辑中的等位相容现象对自身反应性B细胞发育分化及其功能的影响进行了分析。 一、受体编辑在天然自身反应性B细胞发育中的检测 利用流式分析转基因重链与内源性重链表达,并且检测不同来源B细胞轻链的表达情况,发现H链鼠外周B细胞发生明显的重链等位相容现象,并且伴随活跃的轻链受体编辑,而HL链鼠B细胞发育中并没有明显的受体编辑和等位相容现象,始终完整表达3B4抗体基因。 二、转基因小鼠中天然自身反应性B细胞的发育和分化 通过对骨髓中不同发育阶段的B细胞的表面标记进行抗体染色和流式分析,发现HL链鼠B细胞在骨髓中得到阳性选择;而H链小鼠B细胞在中枢发育中受到严格的阴性选择,相应阶段细胞发育受阻并诱导了受体编辑的发生。同样利用B细胞外周不同发育阶段和成熟亚群的不同表型,通过流式和免疫荧光染色,明确了自身反应性B细胞在H链鼠的脾脏和腹腔分别向边缘带(MZ)和B-1方向分化;在HL链小鼠表达3B4的B细胞则主要发育为T2’的特殊表型。 三、等位相容在自身反应性B细胞发育中的作用分析 同样利用流式和激光共聚焦等方法,明确了发生显著重链等位相容的H链小鼠中,等位相容可以诱导B细胞向MZ和B-1a细胞发育分化。四、自身反应性B细胞功能状态分析 体内分析:利用ELISA检测到H链和HL链小鼠血清中同时存在高表达的天然多反应性自身抗体;流式分析发现H链鼠中发生等位相容的B细胞表面活化分子表达水平升高;凋亡检测明确了B细胞向不同亚群分化由阳性选择决定。体外分析:通过体外培养不同组织或流式分选的不同亚群B细胞,明确了天然多反应性自身抗体可以同时来自脾脏和腹腔,并且在H链小鼠中,发生重链等位相容的B细胞可以被抗原刺激而引起胞内Ca2+浓度改变,提示BCR信号通路的活化;并且其可以在体外分泌天然多反应性自身抗体。 总结这些结果,我们可以得出如下结论:受体编辑现象很可能并未参与表达胚系基因编码的3B4抗体的B细胞发育过程;但是重链等位相容可以作为特殊的耐受机制影响B细胞在外周的分化方向并且保持其天然多反应性。这些发现不仅有助于理解受体编辑在生理性自身反应性B细胞发育耐受中的作用,并且对于尽早揭示其在自身免疫病发病中的作用提供了积极的线索。
[Abstract]:Natural autoantibody (NAA) refers to autoantibody against one or more autoantigens in normal organisms without any antigen immunity; and B cells producing NAA are referred to as natural self-reactive B cells. NAA and natural self-reactive B cells are widely present and play an important role, and a large number of studies suggest that the mechanism of tolerance of self-reactive B cells is closely related to the pathogenesis of autoimmune diseases. The receptor editing phenomenon is the main development tolerance of self-reactive B cells. in one aspect, that receptor edit ensures that the proportion of B cells with self reactivity in the periphery of the human body is greatly reduced compared with the bone marrow; On the one hand, receptor editing has occurred to alter specific self-reactive B cells that can escape the fate of cloning or inactivation However, receptor editing is the core mechanism of B cell development tolerance, and it is involved in pathological autoimmune disease. Therefore, the role of receptor editing in the development of rational natural multi-reactive B cells in postgraduates will help to fully reveal the true self-immune tolerance. The current research progress of our research group is to study the mechanism of receptor editing in the development of self-reactive B cells. We successfully screened 3B4 and other hybridomas secreting anti-keratin, actin, etc., in which the 3B4 natural multi-reactive antibody obtained from unimmunized mice was a kind of complete hybridoma. NAA in sense, while B cell expressing 3B4 antibody can represent a naturally occurring natural self body reactive B cells. By cloning the 3B4 variable region gene, we successfully constructed the 3B4 heavy chain (TgVH3B4) and light chain (TgVL3B) which can express the natural multi-reactive protein. 4) The application of transgenic mice and antibody transgenic mice, in order to study the development tolerance of self-reactive B cells. For the sake of ideal platform, 3B4 antibody transgenic mice derived from the natural autoreactive IgM of non-immunized mice were studied in order to study the role mechanism of receptor editing in the development of natural autoreactive B cells. An excellent model was provided. In this study, transgenic heavy light chain mice (TgVH/ CDB4) expressing a natural polyreactive 3B4 antibody were screened in their progeny by mating heavy chain and light chain mice, and B cells expressing the intact CDR3 in TgVH/ OKB4 mice replicated 3B4 antibody secreting cells. During the development of wild mice, TgVH3B4 mice expressing the endogenous light chain are more favorable for observing the different specific self-reactivity due to the difference between the expression of the transgenic heavy chain and the different endogenous light chains. Using this platform, we investigated the development and differentiation of self-reactive B cells by comparing the allelic compatibility of TgVH3B4 mice and TgVH/ LacB4 mice. The effect of its function is analyzed. Detection of natural self-reactive B cell development utilizes flow cytometry to analyze the expression of transgenic heavy chain and endogenous heavy chain, and detects the expression of light chain in different source B cells, and finds that H chain The peripheral B cells of the mouse had obvious allelic compatibility, and accompanied with the active light chain receptor editing, but there was no obvious receptor editing in the development of the HL chain mouse B cells. and the 3B4 antibody gene is always fully expressed. 2. The development and differentiation of natural self-reactive B cells in transgenic mice are carried out by labeling the surface markers of B cells at different developmental stages in the bone marrow The results showed that HL-chain mouse B cells were positively selected in bone marrow, while H-chain mouse B cells were strictly observed in central development. The negative selection of B-cells and the different phenotypes of mature subpopulations were also used, and the self-reactive B-cells were identified by flow-flow and immunofluorescence staining. The spleen and the abdominal cavity of the H-chain mice are respectively directed to the edge bands (B9) and B-1 Directional differentiation; in H The B cells expressing 3B4 in L-chain mice were mainly developed as T2 '. 3. The role of allelic compatibility in the development of self-reactive B cells is also analyzed by means of streaming and laser co-focusing, which makes clear the occurrence of significant heavy chain and so on. In a compatible H-chain mouse, allelic compatibility may The growth and differentiation of B-cells into B-1a and B-1a cells were induced. Four, self-reactive B-cell function states were analyzed in vivo: high expression of natural polyclonal antibodies were detected in serum of H-chain and HL-chain mice by ELISA. Sex autoantibody; Flow analysis found allelic compatible B in H-chain mice The expression level of activated molecules on the surface of cells was elevated. Apoptosis was determined by apoptosis and the differentiation of B cells to different subgroups was determined by positive selection. Subgroup B cells clearly show that natural polyreactive autoantibodies can be simultaneously derived from spleen and abdominal cavity, and in H-chain mice, B-cells that generate heavy chain allelic compatibility can be stimulated to cause intracellular Ca2 +. + concentration changes suggesting activation of the extracellular signal pathway; and it can secrete natural polyreactive autoantibodies in vitro. summary of these results, we can It is concluded that the receptor-editing phenomenon is probably not involved in the B-cell development of the 3B4 antibody which expresses embryo-based gene coding; However, the allelic compatibility of heavy chain can act as a special tolerance mechanism to influence the differentiation of B cells in the peripheral direction and maintain its natural multi-reactivity. These findings not only help to understand the receptor editing in physiological itself.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392
本文编号：2268100
[Abstract]:Natural autoantibody (NAA) refers to autoantibody against one or more autoantigens in normal organisms without any antigen immunity; and B cells producing NAA are referred to as natural self-reactive B cells. NAA and natural self-reactive B cells are widely present and play an important role, and a large number of studies suggest that the mechanism of tolerance of self-reactive B cells is closely related to the pathogenesis of autoimmune diseases. The receptor editing phenomenon is the main development tolerance of self-reactive B cells. in one aspect, that receptor edit ensures that the proportion of B cells with self reactivity in the periphery of the human body is greatly reduced compared with the bone marrow; On the one hand, receptor editing has occurred to alter specific self-reactive B cells that can escape the fate of cloning or inactivation However, receptor editing is the core mechanism of B cell development tolerance, and it is involved in pathological autoimmune disease. Therefore, the role of receptor editing in the development of rational natural multi-reactive B cells in postgraduates will help to fully reveal the true self-immune tolerance. The current research progress of our research group is to study the mechanism of receptor editing in the development of self-reactive B cells. We successfully screened 3B4 and other hybridomas secreting anti-keratin, actin, etc., in which the 3B4 natural multi-reactive antibody obtained from unimmunized mice was a kind of complete hybridoma. NAA in sense, while B cell expressing 3B4 antibody can represent a naturally occurring natural self body reactive B cells. By cloning the 3B4 variable region gene, we successfully constructed the 3B4 heavy chain (TgVH3B4) and light chain (TgVL3B) which can express the natural multi-reactive protein. 4) The application of transgenic mice and antibody transgenic mice, in order to study the development tolerance of self-reactive B cells. For the sake of ideal platform, 3B4 antibody transgenic mice derived from the natural autoreactive IgM of non-immunized mice were studied in order to study the role mechanism of receptor editing in the development of natural autoreactive B cells. An excellent model was provided. In this study, transgenic heavy light chain mice (TgVH/ CDB4) expressing a natural polyreactive 3B4 antibody were screened in their progeny by mating heavy chain and light chain mice, and B cells expressing the intact CDR3 in TgVH/ OKB4 mice replicated 3B4 antibody secreting cells. During the development of wild mice, TgVH3B4 mice expressing the endogenous light chain are more favorable for observing the different specific self-reactivity due to the difference between the expression of the transgenic heavy chain and the different endogenous light chains. Using this platform, we investigated the development and differentiation of self-reactive B cells by comparing the allelic compatibility of TgVH3B4 mice and TgVH/ LacB4 mice. The effect of its function is analyzed. Detection of natural self-reactive B cell development utilizes flow cytometry to analyze the expression of transgenic heavy chain and endogenous heavy chain, and detects the expression of light chain in different source B cells, and finds that H chain The peripheral B cells of the mouse had obvious allelic compatibility, and accompanied with the active light chain receptor editing, but there was no obvious receptor editing in the development of the HL chain mouse B cells. and the 3B4 antibody gene is always fully expressed. 2. The development and differentiation of natural self-reactive B cells in transgenic mice are carried out by labeling the surface markers of B cells at different developmental stages in the bone marrow The results showed that HL-chain mouse B cells were positively selected in bone marrow, while H-chain mouse B cells were strictly observed in central development. The negative selection of B-cells and the different phenotypes of mature subpopulations were also used, and the self-reactive B-cells were identified by flow-flow and immunofluorescence staining. The spleen and the abdominal cavity of the H-chain mice are respectively directed to the edge bands (B9) and B-1 Directional differentiation; in H The B cells expressing 3B4 in L-chain mice were mainly developed as T2 '. 3. The role of allelic compatibility in the development of self-reactive B cells is also analyzed by means of streaming and laser co-focusing, which makes clear the occurrence of significant heavy chain and so on. In a compatible H-chain mouse, allelic compatibility may The growth and differentiation of B-cells into B-1a and B-1a cells were induced. Four, self-reactive B-cell function states were analyzed in vivo: high expression of natural polyclonal antibodies were detected in serum of H-chain and HL-chain mice by ELISA. Sex autoantibody; Flow analysis found allelic compatible B in H-chain mice The expression level of activated molecules on the surface of cells was elevated. Apoptosis was determined by apoptosis and the differentiation of B cells to different subgroups was determined by positive selection. Subgroup B cells clearly show that natural polyreactive autoantibodies can be simultaneously derived from spleen and abdominal cavity, and in H-chain mice, B-cells that generate heavy chain allelic compatibility can be stimulated to cause intracellular Ca2 +. + concentration changes suggesting activation of the extracellular signal pathway; and it can secrete natural polyreactive autoantibodies in vitro. summary of these results, we can It is concluded that the receptor-editing phenomenon is probably not involved in the B-cell development of the 3B4 antibody which expresses embryo-based gene coding; However, the allelic compatibility of heavy chain can act as a special tolerance mechanism to influence the differentiation of B cells in the peripheral direction and maintain its natural multi-reactivity. These findings not only help to understand the receptor editing in physiological itself.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392
【共引文献】
相关期刊论文 前1条
1 陈新辉;赵明玄;安金刚;李巍;刘玉峰;;单克隆天然抗角蛋白抗体IgM 3B4体外抑制白念珠菌芽管形成及粘附作用[J];第四军医大学学报;2008年01期
相关硕士学位论文 前4条
1 郑力强;天然抗角蛋白自身抗体在病理性自身免疫中的作用及机制的研究[D];第四军医大学;2007年
2 陈新辉;天然抗体IgM 3B4体外抑制白念珠菌与宿主细胞相互作用的实验研究[D];第四军医大学;2008年
3 周澜华;抗白念珠菌人鼠嵌合抗体真核表达载体的构建与表达[D];第四军医大学;2008年
4 马小莹;B-1 B细胞表面吞噬相关分子分析[D];第四军医大学;2009年
，本文编号：2268100
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2268100.html
最近更新
教材专著
