YIC-IR  > 中科院烟台海岸带研究所知识产出
菲律宾蛤仔血细胞胞外陷阱的产生特征及其免疫活性研究
韩怡静
Subtype博士
Thesis Advisor赵建民
2021-05-15
Training institution中国科学院烟台海岸带研究所
Degree Grantor中国科学院烟台海岸带研究所
Place of Conferral中国科学院烟台海岸带研究所
Degree Name理学博士
Degree Discipline海洋生物学
Keyword胞外陷阱 菲律宾蛤仔 活性氧 抗菌蛋白 抑菌作用 Extracellular traps R. philippinarum Reactive oxygen species Antibacterial proteins Antibacterial effect
Abstract菲律宾蛤仔等贝类通常生活在富含微生物的水环境中,经常面临着多种病原微生物的侵袭。既往贝类免疫学研究,大多集中在以抗菌肽为基础的体液免疫和 以吞噬作用为基础的细胞免疫研究等方面。作为细胞免疫反应的重要组成部分,胞外陷阱(Extracellular traps,ETs)是近些年发现的一种抵抗病原微生物入侵的免疫防御反应。在抵御病原入侵过程中,ETs 以细胞内核酸为骨架,搭载弹性蛋白酶、组织蛋白酶、组蛋白以及防御素等免疫多肽组成纤维网状结构,对病原菌起到包裹、限制及杀伤作用。在此过程中,ETs 可集中多种活性多肽而使局部抗菌肽浓度升高,起到抑制病原菌入侵作用。目前,针对海洋贝类 ETs 的研究较少,对 ETs 的产生特征和免疫功能方面,仍然缺乏系统而深入的研究。在本研究中,选取菲律宾蛤仔血细胞为实验对象,探讨 ETs 的产生特征和免疫活性,分析 ETs的抗菌组分及抑菌机制,并体外构建 ETs 网状结构,研究结果可为深入了解双壳贝类的固有免疫提供理论基础,亦可为海洋药物的开发提供理论借鉴。本论文的研究结果如下: (1)菲律宾蛤仔 ETs 的鉴定与功能研究 利用 ETs 的典型诱导物酵母多糖对菲律宾蛤仔血细胞进行诱导,观察菲律宾蛤仔血细胞ETs形态特征;检测ETs形成过程中线粒体响应和胞内活性氧(ROS) 的变化规律,分析菲律宾蛤仔血细胞 ETs 的发生特征;结合免疫基因表达规律和对病原菌的抑制作用,确定 ETs 的抑菌功能。研究发现,酵母多糖能够诱导菲律宾蛤仔血细胞形成 ETs,且较优的诱导浓度和诱导时间分别为 0.5 μg/mL 和 30 分钟。在该诱导条件下,细胞内 ROS 和髓过氧化物酶含量升高,同时,ROS 相关基因(PI3K、AKT 和 HIF)的表达量显著升高。作为 ROS 主要来源场所,线粒体向细胞表面迁移。为了响应 ROS 的爆发,线粒体膜电位降低,线粒体膜通透性转换孔打开。在添加线粒体 ROS 抑制剂(Mito-TEMPO)后,ETs 的形成受到抑制,上述结果表明线粒体 ROS 是 ETs 形成所必需的。作为 ROS 的主要调控蛋白,NADPH 氧化酶受到抑制后(DPI 作用),ETs 的形成亦受到抑制,证明了 ETs形成与 NADPH 氧化酶活性成正相关。此外,我们发现,酵母多糖诱导的 ETs 对革兰氏阴性菌(鳗弧菌、哈氏弧菌和大肠杆菌)和革兰氏阳性菌(藤黄微球菌)表现出抗菌活性,表明 ETs 可能是菲律宾蛤仔的一种抗菌方式,可能在免疫防御反应中发挥重要作用。 (2)菲律宾蛤仔 ETs 对病原微生物入侵的响应 海洋弧菌是菲律宾蛤仔等双壳贝类的主要病原菌,我们选择鳗弧菌进行 ETs在细胞免疫应答中的作用研究。ETs 参与了鳗弧菌入侵时的免疫反应。在此过程 中,ROS 和髓过氧化物酶产量增多,在添加 NADPH 氧化酶抑制剂(DPI)或髓过氧化物酶抑制剂(ABAH)后,ETs 的形成均受到抑制,证明 ROS 和髓过氧化 物酶参与 ETs 的发生。在能量方面,ETs 发生过程中,糖酵解相关基因的表达量显著升高,糖酵解相关酶(丙酮酸激酶和己糖激酶)活性增强,表明糖酵解参与了鳗弧菌诱导的 ETs 过程,且可能是 ETs 发生的能量来源之一。在杀菌能力方面,ETs 能够截留并杀死入侵的鳗弧菌,对鳗弧菌具有显著的杀伤作用。探究鳗弧菌诱导 ETs 发生的主效成分,我们选取四种细菌细胞壁成分(脂多糖、类脂 A、磷壁酸和乙酰胞壁酸)对 ETs 进行诱导,结果发现脂多糖的诱导效果最为显著。在脂多糖诱导 ETs 的过程中,细胞内 ROS 的产生增多,但 NADPH氧化酶抑制剂(DPI)不能抑制 ETs 的形成,表明脂多糖诱导的 ETs 不依赖于NADPH 氧化酶。此过程中,核膜凸起,与染色质有分离的趋势。此外,线粒体膜电位降低,线粒体膜通透性转换孔打开,释放至胞外的线粒体 ROS 增多。线粒体 ROS 抑制剂(Mito-TEMPO)也显著抑制了 ETs 的形成,表明线粒体 ROS在脂多糖诱导下的 ETs 形成过程中是必需的。上述结果表明,鳗弧菌诱导的 ETs发生可能主要是脂多糖等细胞壁的作用,而且脂多糖诱导的 ETs 的发生不依赖于NADPH 氧化酶,这不同于酵母多糖诱导 ETs 的形成机制,证明菲律宾蛤仔 ETs的发生存在多种诱导机制。 (3)菲律宾蛤仔 ETs 免疫多肽的挖掘与功能研究 ETs 的效应过程中往往伴随着抗菌肽的产生与作用。前期课题组关于菲律宾蛤仔抗菌肽的挖掘已取得一定进展。在此基础上,探讨参与 ETs 效应的抗菌多肽 种类、功能及杀菌机制。研究发现,防御素(Rpdef1α)、Macins(RpMacin-1 和RpMacin-2)和泛素(RpUbi)参与了脂多糖诱导的血细胞 ETs 的免疫反应,初步证明 ETs 的抑菌功能可能主要来源于免疫活性多肽。随后,重组防御素蛋白(rRpdef1α)、Macin 蛋白(rRpMacin-1、rRpMacin-2)和泛素蛋白(rRpUbi)的体外抑菌实验表明,它们对革兰氏阴性菌和革兰氏阳性菌均表现出杀伤作用。其中,rRpdef1α 不仅对弧菌具有广谱抗菌活性,而且抑制了细菌生物被膜的形成。在杀菌机制研究中,rRpdef1α 能够增加细菌膜通透性,导致细菌内容物溢出,造成细菌死亡。这些结果表明,Rpdef1α 可作为一种强力抗菌剂,参与了胞外陷阱对病原菌的抵御作用。对于 Macin 蛋白,1.0 倍最小抑菌浓度的 rRpMacin-1 和rpMacin-2 可分别在 400 和 1000 分钟内完全杀灭应试菌株大肠杆菌。值得注意的是,rRpMacin-2 对海洋弧菌等革兰氏阴性菌的生长及生物被膜的形成具有较好的抑制作用,其抑制效果优于RpMacin-1。此外,rRpMacin-1 和 rRpMacin-2 对细胞膜的具有破坏作用。上述结果表明,rRpMacin-2 的较强抑菌作用可能与其较强的细胞膜破坏作用有关,而且同种不同活性的抗菌肽同时参与了 ETs 的效应过程,协同提高 ETs 的免疫活性。对于泛素蛋白,rRpUbi 仍然表现出较好的抑菌活性,其抑菌活性主要是通过与细菌 DNA 结合从而发挥抑菌作用。上述结果表明,不同杀菌作用方式的抗菌肽参与 ETs 的形成,可同时提高 ETs 的杀菌活性和杀菌效率,维持生物机体的健康和稳定。 (4)菲律宾蛤仔 ETs 的体外构建与活性研究 体外获得 ETs 结构,并确定分离获得的 ETs 抑菌功能;进一步,使用 DNA和固相合成防御素制备仿 ETs 微网结构,评估体外构建 ETs 结构的可行性。结果表明,体外分离的 ETs 结构依然完整,且能够捕获副溶血弧菌并可破坏细菌膜。等质量分数的 DNA(负电荷)和固相合成防御素(正电荷)组成的微网结构带有正电荷,并与体外分离的 ETs 具有相似结构。通过测定构建的 ETs 的抑菌活性,发现微网结构对大肠杆菌和鳗弧菌具有强烈的抑制作用。上述结果表明,ETs可通过体外制备的方式获得,且获得的 ETs 网状结构具有较好的抑菌活性。 综上所述,本论文鉴定了菲律宾蛤仔血细胞 ETs 的产生,探讨了 ETs 对病原微生物的入侵的响应,初步研究了 ETs 的抗菌组分及抗菌机制,并在体外构建了类似 ETs 的微网结构。研究结果较全面的揭示了 ETs 在菲律宾蛤仔细胞免疫中的作用,为贝类细胞免疫学发展和核酸-多肽复合体的应用提供了理论基础。
Other AbstractShellfish such as Ruditapes philippinarum usually live in a water environment rich in microorganisms and are often attacked by a variety of pathogens. Most of the previous studies on shellfish immunology focused on humoral immunity based on antimicrobial peptides and cellular immunity based on phagocytosis. As an important part of cellular immunity, extracellular traps (ETs) are a kind of cellular immune defense response discovered in recent years to resist the invasion of pathogens. In the process of resisting pathogen invasion, ETs take intracellular nucleic acid as the skeleton, and carry immune polypeptides such as elastase, cathepsin, histone and defensins to form a fibrous network structure, which can encapsulate, restrict and kill pathogens. ETs can inhibit the invasion of pathogens by concentrating diverse active polypeptides to increase the concentration of local antimicrobial peptides. At present, few studies about ETs have been identified in marine shellfish, and there is still a lack of systematic and in-depth studies on the production characteristics and immune function of ETs. In this study, we selected the hemocytes of R. philippinarum as the research object, explored the characteristics and immune activity of ETs, analyzed the antibacterial components and antibacterial mechanisms of ETs, and constructed the microweb of ETs in vitro. The results can provide a theoretical basis for in-depth understanding the innate immunity of bivalve mollusks, as well as a theoretical reference for the development of marine drugs. The results of the study are as follows: (1) Identification and function of ETs from R. philippinarum The hemocytes of R. philippinarum were induced by zymosan, which is a typical inducer of ETs, and the morphological characteristics of ETs in hemocytes of R. philippinarum was observed. The changes of mitochondrial parameters and intracellular reactive oxygen species (ROS) were detected to analyze the characteristics of ETs induced by zymosan in hemocytes of R. philippinarum. The bacteriostatic function of ETs was evaluated by the expression of antimicrobial related genes and the inhibitory effect on pathogens. The results showed that zymosan could induce the formation of ETs, and the optimal concentration and induction time were 0.5 μg/mL and 30 minutes. Under the induction condition, the production of intracellular ROS and myeloperoxidase increased and the expression of ROS-related genes (PI3K, AKT and HIF) was up-regulated. As the main source of ROS, mitochondria migrated to the cell surface. In response to the ROS burst, the mitochondrial membrane potential decreased, and the mitochondrial permeability transition pore opened. Mitochondrial ROS inhibitor (Mito-TEMPO) significantly inhibited the formation of ETs, suggesting that mitochondrial ROS were necessary for the formation of ETs. NADPH oxidase is a major regulatory protein of ROS. Inhibition of NADPH oxidase (DPI) also inhibited ET formation, demonstrating a positive correlation between the formation of ETs and the activity of NADPH oxidase. In addition, we found that zymosan-induced ETs exhibited antibacterial activities against gram-negative bacteria (Vibrio anguillarum, Vibrio harveyi and Escherichia coli) and gram-positive bacteria (Micrococcus luteus), suggesting that ETs may be an antimicrobial mode in R. philippinarum and play an important role in immune defense responses. (2) Response of ETs to pathogen invasion in R. philippinarum Marine Vibrio is the main pathogen of bivalve shellfish such as R. philippinarum. We selected V. anguillarum to study the role of ETs in cellular immune response. ETs were involved in the immune response to V. anguillarum invasion. During the process, the production of ROS and myeloperoxidase increased, and the formation of ETs was inhibited by the addition of NADPH oxidase inhibitors (DPI) and myeloperoxidase inhibitors (ABAH), confirming that ROS and myeloperoxidase were involved in ETs.In terms of energy, the expression of glycolysis related genes and the activities of glycolysis related enzymes (pyruvate kinase and hexokinase) were significantly enhanced during the process of ETs, suggesting that glycolysis was involved in the V. anguillarum-induced ETs and may be one of the energy sources of ETs. In terms of bactericidal activity, ETs could entrap and kill the invading V. anguillarum and had a significant killing effect on V. anguillarum. To explore the main components of V. anguillarum that induced ETs, we selected four bacterial cell wall components (lipopolysaccharide, lipid A, teichoic acid and acetylmuramic acid) to induce ETs, and the results showed that lipopolysaccharide induced the most significant effect. The ET formation induced by lipopolysaccharide was accompanied by increased production of intracellular ROS. However, NADPH oxidase inhibitors (DPI) cannot inhibit the formation of ETs, demonstrating that lipopolysaccharide induced ETs was independent of NADPH oxidase. In this process, the nuclear membrane bulges, tended to separate from chromatin. In addition, the mitochondrial membrane potential decreased, the mitochondrial permeability transition pore opened, and the increased mitochondrial ROS released into the extracellular. Mitochondrial ROS inhibitor (Mito-TEMPO) also significantly inhibited the formation of ETs, suggesting that mitochondrial ROS were required in the formation of ETs induced by lipopolysaccharide. These results suggested that ETs induced by V. anguillarum may be mainly caused by cell walls such as lipopolysaccharide. Lipopolysaccharide induced-ETs were independent of NADPH oxidase, which was different from the mechanism of zymosan-induced ETs, suggesting that there were multiple induction mechanisms for ETs in R. philippinarum. (3) Discovery and function of immune polypeptides from ETs in R. philippinarum The effect of ETs is often accompanied by the production and action of antimicrobial peptides. In the early stage, the research group had made some progress in antimicrobial peptides from R. philippinarum. On this basis, the types, functions and bactericidal mechanisms of antimicrobial peptides involved in ETs were explored. Defensin (Rpdef1α), Macins (RpMacin-1 and RpMacin-2) and ubiquitin (RpUbi) were involved in the immune response of hemocyte ETs induced by lipopolysaccharide, which preliminarily indicated that the antibacterial function of ETs may mainly come from the immunoactive peptides. Subsequently, the bacteriostatic experiments in vitro of recombined protein of defensin (rRpdef1α), Macin (rRpmacin-1 and rRpmacin-2) and ubiquitin (rRpUbi) showed that they had killing effect on both gram-negative and gram-positive bacteria. Among them, rRpdef1α not only exhibited broad-spectrum antimicrobial activity against Vibrio species, but also inhibited the formation of bacterial biofilms. In the study of bactericidal mechanism, rRpdef1α could increase the bacterial membrane permeabilization, leading to the overflow of bacterial contents and cause bacterial death. These results suggested that Rpdef1α was a potent antimicrobial agent, participating in the resistance of ETs to pathogen invasion. For Macin proteins, rRpMacin-1 and rRpMacin-2 killed E. coli within 400 and 1000 min at a minimum inhibitory concentration, respectively. It was worth noting that rRpMacin-2 had a stronger inhibition effect on the growth and biofilm formation of gram-negative bacteria such as marine Vibrio than that of rRpMacin-1. In addition, rRpMacin-1 and rRpMacin-2 had a strong damage effect on bacterial membrane. These results suggested that the stronger antimicrobial activity of rRpMacin-2 may be related to the stronger destruction effect of bacterial membrane. Moreover, the same type of antimicrobial peptides with different activities simultaneously participated in the process of ETs, which can synergistically improve the immune activity of ETs. For ubiquitin protein, rRpUbi still showed intense antibacterial activity, which was mainly through binding to bacterial DNA. These results indicated that antimicrobial peptides with different bactericidal modes participated in the formation of ETs, which can improve the bactericidal activity and efficiency of ETs, and maintain the health and stability of organisms. (4) Construction and activity of ETs in vitro from R. philippinarum The structure of ETs was isolated, and the antibacterial function of the separated ETs was evaluated. Furthermore, DNA and solid phase synthetic defensins were mixed to prepare the microweb structure of ETs to evaluate the feasibility of constructing ETs structure in vitro. The results showed that the structure of ETs isolated in vitro was still intact, and it could capture Vibrio parahaemolyticus and destroy the bacterial membrane system. The microweb structure composed of equal mass ratio of DNA (negative charge) and solid phase synthetic defensins (positive charge) was positive charged and had similar structure to the ETs isolated in vitro. The bactericidal activity of the constructed ETs showed that the microweb structure had a strong killing effect on E. coli and V. anguillarum. These results indicated that ETs could be prepared in vitro, and the constructed microweb structure obtained had good antibacterial activity. In summary, this study identified the production of ETs in hemocytes of ETs in R. philippinarum, discussed the response of ETs to invasion of pathogenic microorganisms, preliminarily studied the antibacterial components and antibacterial mechanism of ETs, and constructed microweb structure similar to ETs in vitro. The results comprehensively revealed the role of ETs in cellular immunity of R. philippinarum, and provided a theoretical basis for the development of cell immunity of mollusks and the application of nucleic acid-polypeptide.
Language中文
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/29341
Collection中科院烟台海岸带研究所知识产出
Recommended Citation
GB/T 7714
韩怡静. 菲律宾蛤仔血细胞胞外陷阱的产生特征及其免疫活性研究[D]. 中国科学院烟台海岸带研究所. 中国科学院烟台海岸带研究所,2021.
Files in This Item: Download All
File Name/Size DocType Version Access License
博士学位论文-韩怡静-2021.6.7改(6087KB)学位论文 开放获取CC BY-NC-SAView Download
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Google Scholar
Similar articles in Google Scholar
[韩怡静]'s Articles
Baidu academic
Similar articles in Baidu academic
[韩怡静]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[韩怡静]'s Articles
Terms of Use
No data!
Social Bookmark/Share
File name: 博士学位论文-韩怡静-2021.6.7改.pdf
Format: Adobe PDF
All comments (0)
No comment.
 

Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.