IOCAS-IR
鲆鲽鱼IgM+ B细胞内吞作用及其调控机制研究
李逸群
第一作者单位中国科学院海洋研究所
学位类型博士
导师孙黎
2018-05-11
学位授予单位中国科学院大学
学位授予地点中国科学院大学 中国科学院海洋研究所
学位名称博士
学位专业海洋生物学
关键词大菱鲆 牙鲆 半滑舌鳎 Igm Plus B细胞 内吞机制 Cd22
摘要

    越来越多的证据表明硬骨鱼类的B淋巴细胞(简称B细胞)具有内吞作用。2006年,在虹鳟鱼中首先发现了B细胞可以摄取胞外颗粒,形成吞噬溶酶体并启动下游的溶酶体降解通路。随后在多种硬骨鱼类以及两栖动物、爬行动物和哺乳动物中都发现了具有内吞功能的B细胞。尽管B细胞内吞现象已经得到广泛的认可,然而其内在的分子机制及调控仍不清楚。本研究以鲆类硬骨鱼为研究对象,通过免疫学、细胞生物学以及分子生物学等研究手段,分析了IgM+ B细胞对不同大小非己颗粒的内吞效率和内吞途径,明确了不同免疫器官中IgM+ B细胞内吞活性和内吞方式的异同,探究了B细胞共受体分子CD22B细胞以及外周血白细胞免疫激活的调控机制。

    利用流式细胞技术和免疫荧光显微镜观察等方法,鉴定了大菱鲆外周血白细胞中具有内吞作用的IgM+ B细胞及其形态特征,探究了外周血白细胞中IgM+ B细胞对不同大小非己颗粒的内吞效率和内吞途径。结果表明,具有内吞作用的IgM+ B细胞核质比较大、体积较小且胞内颗粒度较少,IgM+ B细胞对不同大小的荧光乳胶微球(0.10.512 μm)的内吞效率不同,其中对0.5 μm微球的内吞能力最强,对1 μm微球的内吞能力次之,对0.1 μm2 μm微球的内吞能力最弱。对于0.5 μm荧光乳胶微球,IgM+ B细胞的内吞活性依赖于巨胞饮途径,而IgM细胞的内吞活性呈现出网格蛋白依赖性和小窝蛋白依赖性;对于1 μm荧光乳胶微球,IgM+ B细胞主要依赖于巨胞饮途径,部分依赖于小窝蛋白介导的内吞途径,而IgM细胞对于1 μm微球的内吞与其对0.5 μm微球的内吞相似。此外,IgM+ B细胞能够同时内吞乳胶微球以及巨胞饮标志物——高分子量的右旋糖苷,并且能够通过巨胞饮途径和小窝蛋白介导内吞作用摄取灭活的大肠杆菌。上述结果表明IgM+ B细胞主要通过巨胞饮途径实现对颗粒的摄取。

    在牙鲆中,IgM+ B细胞在脾脏白细胞中所占的比例高于其在外周血白细胞的比例,但是具有内吞能力的脾脏IgM+ B细胞的比例低于具有内吞能力的外周血IgM+ B细胞的比例。虽然外周血白细胞和脾脏白细胞中IgM+ B细胞能够摄取非己颗粒,但对不同大小颗粒的摄取效率不同:外周血IgM+ B细胞对0.5 μm荧光乳胶微球的内吞活性高于其对1 μm荧光乳胶微球的内吞活性,而脾脏IgM+ B细胞对1 μm荧光乳胶微球的内吞活性高于其对0.5 μm荧光乳胶微球的内吞活性。巨胞饮抑制剂能够显著抑制牙鲆外周血白细胞和脾脏白细胞中 IgM+ B细胞对0.5 mm1 mm微球的内吞活性,而网格蛋白介导的内吞抑制剂、小窝蛋白介导的内吞抑制剂和巨胞饮途径抑制剂都可以显著抑制外周血IgM细胞的内吞活性。在脾脏IgM细胞中,网格蛋白介导的内吞抑制剂和巨胞饮途径抑制剂预处理能够显著抑制细胞的内吞活性。以上结果表明,虽然外周血IgM+ B细胞和脾脏IgM+ B细胞表现出不同的内吞活性和颗粒大小依赖的内吞效率,但两者皆主要依赖于巨胞饮途径实现对非己颗粒的内吞。

    在哺乳动物中,CD22B细胞表面的共受体分子,对B细胞的免疫活性起关键的调节作用。然而,鱼类CD22B细胞调控功能未知。本研究中我们首次研究了鱼类两个CD22分子,即牙鲆PoCD22和半滑舌鳎CsCD22PoCD22CsCD22具有CD22家族保守的胞外段免疫球蛋白结构域和胞质区免疫受体抑制性酪氨酸基序。其中,PoCD22蛋白广泛表达于外周血白细胞和脾脏白细胞中,且分布于几乎全部IgM+ B细胞和部分淋巴细胞样IgM细胞的细胞膜表面。抗体封闭PoCD22显著降低外周血IgM+ B细胞对病原细菌的内吞,同时导致胞内ROS活性显著降低;相反,抗体封闭PoCD22显著增强脾脏白细胞中的IgM+ B细胞的内吞活性以及胞内ROS含量。以上结果表明,CD22分子对牙鲆外周血 IgM+ B细胞的免疫激活起正向调控作用,而对脾脏 IgM+ B细胞免疫激活起负向调控作用。抗体封闭CsCD22显著增强半滑舌鳎外周血白细胞的增殖活性、吞噬活性和抗菌活性,说明CsCD22在半滑舌鳎外周血白细胞的免疫激活中起负调控作用。

    综上所述,本研究首次发现了硬骨鱼类的IgM+ B细胞对非己颗粒的内吞效率与颗粒物大小呈现相关性,且不同免疫器官(外周血和脾脏)中的IgM+ B细胞内吞能力存在差异;硬骨鱼类的IgM+ B细胞对非己颗粒的内吞方式主要依赖于巨胞饮途径,且B细胞共受体分子CD22IgM+ B细胞及白细胞的免疫激活中起重要调节作用。这些结果为我们研究B细胞的吞噬和进化起源提供了新的思路,同时为海洋经济鱼类的病害防控提供了理论指导。

其他摘要

    Increasing evidence has demonstrated support for the endocytic capacities of teleost B lymphocytes (B cells for shot). It was first investigated in rainbow trout in 2006 that particles could be ingested by B cells and endocytic B cells have been demonstrated to have phagolysosome formation and serial downstream degradative activities. Subsequently, endocytic B cells were found in a variety of teleosts, as well as in amphibians, reptiles and mammals. However, only phenomena have been discovered for this process and lack of study on endocytosis mechanism. In this study, the endocytosis efficiency and endocytic pathway of IgM+ B cells in teleost fish were examined, the similarities and differences of endocytosis activity and pathway of IgM+ B cells in different immune organs were analysed, the mechanism in immune activation of B cells and peripheral blood leukocytes by B cell co-recepter CD22 was investigated by using multiple approaches including immunology, cell biology and molecular biology.

    Flow cytometry, immunofluorescence and electron microscopy were used to identify endocytic IgM+ B cells in turbot peripheral leukocytes and their morphological characteristics. Moreover, their endocytosis efficiency and endocytic pathway for non-self particles in different sizes were investigated. IgM+ B cells with endocytic capacity have larger nuclears, smaller volumes and less intracellular granularity. IgM+ B cells showed different endocytosis enfficiency for different size fluorescent latex microspheres (in a diameter of 0.1, 0.5, 1 and 2 μm), with highest uptake capacity for 0.5 μm beads, moderate for 1 μm beads, and low for 0.1 μm and 2 μm beads. In the presence of macropinocytosis inhibitors, the endocytic activity of turbot peripheral blood leukocytes IgM+ B cells on 0.5 μm fluorescence latex microspheres was significantly decreased, while the endocytic activity of IgM cells was clathrin- and caveolin- dependent. For the uptake of 1 μm beads, IgM+ B cells relied mainly on macropinocytosis and partially on caveolae-mediated pathway, while IgM cells utilized the routes similar to that of internalizing 0.5 μm beads. Consistently, the internalized microspheres were co-localized with high-molecular-mass dextran in IgM+ endocytic cells. In addition to latex beads, IgM+ B cells could also ingest inactivated bacteria predominately through macropinocytosis and caveolae-mediated endocytosis. These results collectively indicated that macropinocytosis is principally responsible for particle uptake by turbot IgM+ B cells.

    The percentage of IgM+ B cells in flounder spleen leukocytes is higher than that in peripheral blood leukocytes, while the endocytic activity is lower than that in PBL IgM+ B cells. Both peripheral blood leukocytes IgM+ B cells and spleen leukocytes IgM+ B cells exhibited endocytic capacities but different endocytosis efficiency. The uptake capacity of PBL IgM+ B cells was higher for 0.5 mm beads than that for 1 mm beads, while uptake capacity of SL IgM+ B cells showed a contrary result. IgM+ B cells in both peripheral blood leukocytes and spleen leukocytes employed apparently macropinocytosis-dependent endocytic pathway. However, in peripheral blood leukocytes IgM cells, the uptake activity was significantly reduced in the presence of macropinocytosis, clathrin- and caveolin- mediated endocytosis inhibitors; in spleen leukocytes IgM cells, both clathrin-mediated endocytosis inhibitors and macropinocytosis inhibitors could reduceed the uptake activity. These results suggest that although IgM+ B cells in peripheral blood leukocytes and spleen leukocytes showed different endocytic activity and particle size-dependent endocytosis efficiency, both of them mainly depended on macropinocytosis upon uptake of particles.

    In mammals, CD22 is a co-receptor on the surface of B cells and plays a crucial role in regulating the immune activity of B cells. However, the function of fish CD22 is still unclear. In this study, we characterized for the first time two fish CD22 from Japanese flounder and tongue sole, separately. Sequence alignment and domain analysis showed that PoCD22 and CsCD22 have extracellular immunoglobulin domains and cytoplasmic immunoreceptor tyrosine-based inhibition motifs, which are conserved in CD22 family. Majority of Japanese flounder IgM+ B cells in peripheral blood leukocytes and spleen leukocytes are CD22 positive, and partial IgM cells also express CD22 protein. During bacterial infection, the endocytic activity and ROS activity of peripheral blood leukocytes IgM+ B cells was significantly reduced in the presence of rPoCD22 antiserum, while those of spleen leukocytes IgM+ B cells was significantly induced. Results above indicate that CD22 positively regulates the immune activation of peripheral blood leukocytes IgM+ B cells, and negatively regulates that of spleen leukocytes IgM+ B cells. In the presence of rCsCD22 antiserum, the proliferation, phagocytosis, and antibacterial activity of peripheral blood leukocytes were significantly increased. These results indicate that CsCD22 plays an inhibitory role in PBL activation.

    To sum up, it was found for the first time that the endocytosis efficiency of teleost IgM+ B cells on non-self particles was size-dependent, and the endocytic capacity had difference in IgM+ B cells of different immune organs (peripheral blood and spleen). Nonetheless, macropinocytosis is principally responsible for particle uptake by teleost IgM+ B cells. B cell co-receptor CD22 played a crucial role in regulating the immune activity of teleost IgM+ B cells as well as peripheral blood leukocytes. These provide us with new ideas for the evolution origin of B lymphocytes and promote our understanding for the prevention of diseases in commercial fish farming.

学科领域生物学
学科门类理学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/154423
专题中国科学院海洋研究所
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李逸群. 鲆鲽鱼IgM+ B细胞内吞作用及其调控机制研究[D]. 中国科学院大学 中国科学院海洋研究所. 中国科学院大学,2018.
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