受體編輯在天然多反應(yīng)性B細(xì)胞發(fā)育中的作用及機(jī)制研究
發(fā)布時間：2018-10-13 09:26
【摘要】： 天然自身抗體(natural autoantibody,NAA)是指在沒有任何抗原免疫的情況下,正常機(jī)體中針對一種或多種自身抗原的自身抗體;產(chǎn)生NAA的B細(xì)胞則被稱為天然自身反應(yīng)性B細(xì)胞。NAA及天然自身反應(yīng)性B細(xì)胞廣泛存在并發(fā)揮重要功能,大量研究提示,自身反應(yīng)性B細(xì)胞存在的耐受機(jī)制與自身免疫病的發(fā)病機(jī)理密切相關(guān)。 受體編輯現(xiàn)象是自身反應(yīng)性B細(xì)胞的發(fā)育耐受的主導(dǎo)機(jī)制。受體編輯通過多種作用方式,一方面保證了人體外周中具有自身反應(yīng)性的B細(xì)胞比例與骨髓相比大大降低;另一方面,發(fā)生了受體編輯從而改變特異性的自身反應(yīng)性B細(xì)胞可以逃脫克隆清除或失活的命運(yùn)而發(fā)育成熟。但是,受體編輯現(xiàn)象作為B細(xì)胞發(fā)育耐受的核心機(jī)制,其參與病理性自身免疫即自身免疫病的方式存在巨大爭論。因此,研究生理性天然多反應(yīng)性B細(xì)胞發(fā)育中受體編輯的作用必將有助于全面揭示自身免疫失耐受的真正原因。 本課題組以往的研究進(jìn)展,為深入研究受體編輯現(xiàn)象在自身反應(yīng)性B細(xì)胞發(fā)育中的作用機(jī)制奠定了良好的基礎(chǔ)。應(yīng)用未免疫小鼠B細(xì)胞與骨髓瘤細(xì)胞融合,我們成功篩選到了分泌抗角蛋白、肌動蛋白等多種自身抗原的自身抗體的3B4等雜交瘤,其中由未免疫小鼠獲得的3B4天然多反應(yīng)性抗體是一種完全意義上的NAA,而表達(dá)3B4抗體的B細(xì)胞可以代表生理廣泛存在的天然自身反應(yīng)性B細(xì)胞。通過克隆3B4可變區(qū)基因,我們成功構(gòu)建了可以表達(dá)天然多反應(yīng)性BCR的3B4重鏈(TgVH3B4)和輕鏈(TgVL3B4)轉(zhuǎn)基因小鼠�？贵w轉(zhuǎn)基因小鼠的應(yīng)用,為深入研究自身反應(yīng)性B細(xì)胞發(fā)育耐受提供了理想的平臺。而來源于未免疫小鼠天然自身反應(yīng)性IgM的3B4抗體轉(zhuǎn)基因小鼠,為深入研究受體編輯在天然自身反應(yīng)B細(xì)胞發(fā)育中的作用機(jī)制等問題提供了絕佳模型。 本研究通過交配重鏈與輕鏈小鼠,在其子代中篩選到了完整表達(dá)天然多反應(yīng)性3B4抗體的轉(zhuǎn)基因重輕鏈小鼠(TgVH/L3B4)。TgVH/L3B4小鼠中表達(dá)轉(zhuǎn)基因完整BCR的B細(xì)胞復(fù)制了3B4抗體分泌細(xì)胞在野生小鼠中的發(fā)育過程;而表達(dá)內(nèi)源輕鏈的TgVH3B4小鼠由于表達(dá)轉(zhuǎn)基因重鏈與不同內(nèi)源輕鏈所組成的BCR,更有利于觀察不同特異性自身反應(yīng)性B細(xì)胞發(fā)育耐受的普遍規(guī)律。利用這個平臺,通過比較研究TgVH3B4小鼠和TgVH/L3B4小鼠,我們就受體編輯中的等位相容現(xiàn)象對自身反應(yīng)性B細(xì)胞發(fā)育分化及其功能的影響進(jìn)行了分析。 一、受體編輯在天然自身反應(yīng)性B細(xì)胞發(fā)育中的檢測 利用流式分析轉(zhuǎn)基因重鏈與內(nèi)源性重鏈表達(dá),并且檢測不同來源B細(xì)胞輕鏈的表達(dá)情況,發(fā)現(xiàn)H鏈?zhǔn)笸庵蹷細(xì)胞發(fā)生明顯的重鏈等位相容現(xiàn)象,并且伴隨活躍的輕鏈?zhǔn)荏w編輯,而HL鏈?zhǔn)驜細(xì)胞發(fā)育中并沒有明顯的受體編輯和等位相容現(xiàn)象,始終完整表達(dá)3B4抗體基因。 二、轉(zhuǎn)基因小鼠中天然自身反應(yīng)性B細(xì)胞的發(fā)育和分化 通過對骨髓中不同發(fā)育階段的B細(xì)胞的表面標(biāo)記進(jìn)行抗體染色和流式分析,發(fā)現(xiàn)HL鏈?zhǔn)驜細(xì)胞在骨髓中得到陽性選擇;而H鏈小鼠B細(xì)胞在中樞發(fā)育中受到嚴(yán)格的陰性選擇,相應(yīng)階段細(xì)胞發(fā)育受阻并誘導(dǎo)了受體編輯的發(fā)生。同樣利用B細(xì)胞外周不同發(fā)育階段和成熟亞群的不同表型,通過流式和免疫熒光染色,明確了自身反應(yīng)性B細(xì)胞在H鏈?zhǔn)蟮钠⑴K和腹腔分別向邊緣帶(MZ)和B-1方向分化;在HL鏈小鼠表達(dá)3B4的B細(xì)胞則主要發(fā)育為T2’的特殊表型。 三、等位相容在自身反應(yīng)性B細(xì)胞發(fā)育中的作用分析 同樣利用流式和激光共聚焦等方法,明確了發(fā)生顯著重鏈等位相容的H鏈小鼠中,等位相容可以誘導(dǎo)B細(xì)胞向MZ和B-1a細(xì)胞發(fā)育分化。四、自身反應(yīng)性B細(xì)胞功能狀態(tài)分析 體內(nèi)分析:利用ELISA檢測到H鏈和HL鏈小鼠血清中同時存在高表達(dá)的天然多反應(yīng)性自身抗體;流式分析發(fā)現(xiàn)H鏈?zhǔn)笾邪l(fā)生等位相容的B細(xì)胞表面活化分子表達(dá)水平升高;凋亡檢測明確了B細(xì)胞向不同亞群分化由陽性選擇決定。體外分析:通過體外培養(yǎng)不同組織或流式分選的不同亞群B細(xì)胞,明確了天然多反應(yīng)性自身抗體可以同時來自脾臟和腹腔,并且在H鏈小鼠中,發(fā)生重鏈等位相容的B細(xì)胞可以被抗原刺激而引起胞內(nèi)Ca2+濃度改變,提示BCR信號通路的活化;并且其可以在體外分泌天然多反應(yīng)性自身抗體。 總結(jié)這些結(jié)果,我們可以得出如下結(jié)論:受體編輯現(xiàn)象很可能并未參與表達(dá)胚系基因編碼的3B4抗體的B細(xì)胞發(fā)育過程;但是重鏈等位相容可以作為特殊的耐受機(jī)制影響B(tài)細(xì)胞在外周的分化方向并且保持其天然多反應(yīng)性。這些發(fā)現(xiàn)不僅有助于理解受體編輯在生理性自身反應(yīng)性B細(xì)胞發(fā)育耐受中的作用,并且對于盡早揭示其在自身免疫病發(fā)病中的作用提供了積極的線索。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392
【共引文獻(xiàn)】
相關(guān)期刊論文 前1條
1 陳新輝;趙明玄;安金剛;李巍;劉玉峰;;單克隆天然抗角蛋白抗體IgM 3B4體外抑制白念珠菌芽管形成及粘附作用[J];第四軍醫(yī)大學(xué)學(xué)報;2008年01期
相關(guān)碩士學(xué)位論文 前4條
1 鄭力強(qiáng);天然抗角蛋白自身抗體在病理性自身免疫中的作用及機(jī)制的研究[D];第四軍醫(yī)大學(xué);2007年
2 陳新輝;天然抗體IgM 3B4體外抑制白念珠菌與宿主細(xì)胞相互作用的實驗研究[D];第四軍醫(yī)大學(xué);2008年
3 周瀾華;抗白念珠菌人鼠嵌合抗體真核表達(dá)載體的構(gòu)建與表達(dá)[D];第四軍醫(yī)大學(xué);2008年
4 馬小瑩;B-1 B細(xì)胞表面吞噬相關(guān)分子分析[D];第四軍醫(yī)大學(xué);2009年
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