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长牡蛎免疫适应性(免疫致敏)机制的初步研究
王伟林
学位类型博士
导师王玲玲
2017-05-10
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词长牡蛎 免疫适应性 颗粒细胞 吞噬作用 无氧糖酵解
摘要 适应性是生物的基本特征,是指机体能根据内外环境变化及时调整内部机能、维持自身稳态以更好地生存下来的特性。生物能在形态结构、生理功能上表现出对内外环境变化的适应,其中免疫适应是其生理适应的一个重要方面。研究表明,动物中广泛存在免疫适应性现象,表现为对过往感染经历的记忆,再次遇到相似病原入侵时免疫抗性增强,个体存活机率提高,典型的例子如脊椎动物的“适应性免疫”。由于缺乏经典“适应性免疫”的细胞(B/T淋巴细胞)和分子(Ig分子)基础,无脊椎动物中广泛报道的免疫适应性现象(又称免疫致敏)的具体机制仍不清楚,并严重阻碍了重要水产养殖无脊椎动物病害的免疫防治。本论文以软体动物长牡蛎为研究对象,从细胞和分子两个层面,探究灿烂弧菌连续两次刺激引起免疫适应性现象的具体机制,主要取得以下结果:
一、发现了长牡蛎颗粒细胞发挥主要免疫功能,是免疫适应中的核心细胞类群
形态上,长牡蛎循环血细胞主要可以分为三种类群:无粒细胞、半粒细胞和颗粒细胞。其中,颗粒细胞中吞噬活性、包囊化活性及杀菌活性物质水平(溶酶体活性、ROS含量和NO含量)最强,免疫相关基因(CgTLR、CgClathrin、CgATPeV、CgLysozyme、CgDefensin和CgIL-17)表达量最高,因而颗粒细胞发挥主要免疫功能。此外,灿烂弧菌连续两次刺激后,与首次刺激相比,长牡蛎循环血细胞总数增加,颗粒细胞比例显著提高,且血细胞的抗凋亡能力、吞噬活性及杀菌活性显著增强,表现出免疫适应性特征。因此,颗粒细胞是长牡蛎免疫适应中的核心细胞类群。
二、探明了血细胞吞噬和抗凋亡过程在长牡蛎免疫适应中发挥关键作用
通过灿烂弧菌连续两次刺激,从长牡蛎血细胞转录组中筛选出1800个差异表达基因能同时响应两次免疫刺激,其中有1037个基因在首次和再次免疫刺激后的0 h差异表达,可能与再次免疫刺激时机体更快的免疫响应有关,有996个基因在首次和再次刺激后的6 h差异表达,可能与再次免疫刺激时机体更强的免疫响应有关。这些基因中与免疫识别、细胞形变、内吞过程、吞噬溶酶体成熟和抗凋亡等过程相关基因均显著高表达,因此吞噬过程和抗凋亡过程在长牡蛎免疫适应中发挥关键作用。
三、发现了mTOR调节无氧糖酵解是长牡蛎免疫适应中的关键能量代谢通路
灿烂弧菌连续两次刺激后,血细胞中葡萄糖转运体表达量显著升高,胞内葡萄糖含量增加,糖酵解过程关键限速酶如己糖激酶、葡萄糖激酶、磷酸葡萄糖变位酶等表达显著升高,糖酵解过程加强,葡萄糖消耗增加,次级代谢产物NAD+/NADH水平及乳酸含量显著升高,无氧呼吸加强。同时,mTOR信号通路关键分子如mTOR、HIFα表达显著升高,且mTOR磷酸化(p-mTOR)被抑制后,血细胞中葡萄糖消耗降低,无氧糖酵解降低,血细胞吞噬活性及ROS水平均降低,免疫适应现象消失。因此,mTOR调节无氧糖酵解是长牡蛎免疫适应中的关键能量代谢通路。
四、初步揭示了组蛋白修饰在长牡蛎免疫适应中可能发挥调节作用
长牡蛎组蛋白甲基转移酶CgHMT646基因和乙酰基转移酶CgHAT009基因主要在长牡蛎血细胞中表达,将其分别转染至HEK293细胞中能增强细胞内组蛋白甲基化和乙酰化修饰水平。灿烂弧菌连续两次刺激后,长牡蛎血细胞中CgHMT646基因的表达量显著上调,组蛋白H3K4甲基化修饰水平升高,CgHAT009基因的表达量显著下调,组蛋白乙酰化修饰水平降低,提示组蛋白修饰在长牡蛎免疫适应中可能发挥调节作用。
综上所述,长牡蛎三种类型循环血细胞中,颗粒细胞发挥主要免疫功能,灿烂弧菌连续两次刺激引起的长牡蛎免疫适应性现象,主要依赖于粒细胞的免疫功能,其中吞噬和抗凋亡过程在免疫适应中发挥关键作用,mTOR调节无氧糖酵解是其关键的能量代谢通路,而且提示组蛋白修饰在免疫适应中可能发挥调节作用。这些研究结果在细胞和分子水平进一步深化当前对长牡蛎免疫适应性机制的认识,为无脊椎动物免疫适应性机制的研究奠定了扎实基础,同时为重要水产养殖无脊椎动物的病害防治提供了思路。
其他摘要Adaptability is the basic characteristic of living organisms, which means that the organism can adjust internal function and maintain homeostasis to better survive according to the change of internal and external environment. The organisms can show adaptation to the environmental change in the morphological structure or physiological function, of which immune adaptation is an important aspect. Studies have shown that there is a widespread immune adaptation in animals, which can memory their past infection experiences and increase their immune resistance and survival opportunity when meet the similar pathogen again, the "adaptive immunity" in vertebrates as a good example. Owing to the lack of cellular (B/T lymphocytes) and molecular (Ig antibody) bases of classical "adaptive immunity", the specific mechanisms of immune adaptation in invertebrate (also known as immune priming) are still unclear, which severely prevented the immune prevention and cure of the diseases of important aquaculture invertebrates. This dissertation explores the detail mechanism of the immune adaptation in Pacific oyster caused by continuous stimulation of Vibrio splendidus at the cellular and molecular levels, with the main conclusion obtained as below:
Firstly, the oyster granulocytes are the main immunocompetent hemocytes and the core hemocytes population in the immune adaptation.
Three types of circulating hemocytes were morphologically identified and separated in oyster as agranulocytes, semi-granulocytes and granulocytes. Among of those hemocytes, granulocytes possessed the strongest activities of phagocytosis, encapsulating and bactericidal factors (the lysosome activity, the productions of ROS and NO). Besides, compared to agranulocytes and semi-granulocytes, the RNA transcripts of immune related genes (CgTLR, CgClathrin, CgATPeV, CgLysozyme, CgDefensin and CgIL-17) were the highest expressed in granulocytes. Therefore, the granulocytes were characterized as the main immunocompetent hemocytes in oysters. In addition, after two consecutive stimulations of Vibrio splendidus, the total hemocytes counts and the proportion of granulocytes both increased significantly, compared with that after first stimulation. Meanwhile, the anti-apoptosis capacity, phagocytic activity and bactericidal activity of hemocytes were also enhanced remarkably, displaying a characteristic of immune adaptation. These results collectively suggested that the granulocytes were also the core cell population in immune adaptation of oysters.
Secondly, the processes of hemocytes phagocytosis and anti-apoptosis play a key role in the immune adaptation of oyster.
After two consecutive stimuli of Vibrio splendidus, a total of 1800 differentially expressed genes were screened from hemocytes transcriptom, which were simultaneously responding to the two times immune stimulation. Among of those genes, 1037 differentially expressed were obtained at 0 h after challenging primarily and secondarily, which indicated those genes might be related with faster immune response in the secondary challenge. And 996 genes were differentially expressed at 6 h after challenging primarily and secondary, which suggested that those genes might be involved in the stronger immune response in the secondary challenge. Besides, among all of those differentially expressed genes, the genes in the process such as recognition, cell deformation, endocytosis, phagolysosome maturation and anti-apoptosis were all up-regulated significantly, which suggesting that hemocytes phagocytosis and anti-apoptosis might play indispensable role in the immune adaptation of oysters.
Thirdly, it was found that the mTOR regulating anaerobic glycolysis was the key energy metabolism pathway in the immune adaptation of Pacific oyster.
After two consecutive stimulations, the expression of glucose transporter in hemocytes increased significantly, the key speed limiting enzyme in the process of glycolysis, such as hexokinase, phosphoglucomutase and glucokinase were also up-regulated significantly. Meanwhile, the continued immune challenge could also induce an increase of glucose consumption, the enhancement of anaerobic respiration and glycolysis, and the up-regulation of secondary metabolite NAD+/NADH and lactic acid level. Besides, the expression of key factors in the mTOR pathway such as mTOR and HIFα also increased significantly. As expected, when the phosphorylation of mTOR (p-mTOR) was inhibited, the glucose consumption, the anaerobic glycolysis, the phagocytosis and ROS level in oyster hemocytes were all decreased significantly, blocking the resonse of immune adaptation in oyster. Therefore, the results suggested that anaerobic glycolysis mediated by mTOR is the key energy metabolism pathway in the immune adaptation of oysters.
Forthly, it is preliminary revealed that histone modifications may play a regulatory role in the immune adaptation of Pacific oyster
In oyster, the histone methyl-transferase CgHMT646 gene and histone acetyl-transferase CgHAT009 gene were expressed mainly in the hemocytes, which can enhance the intracellular histone methylation and acetylation modification levels when transferred to HEK293 cells, respectively. After two consecutive stimuli by Vibrio splendidus, the expression of CgHMT646 genes in hemocytes increased markedly, along with elevated histone H3K4methylation level of hemocytes, and the expression of CgHAT009 gene decreased markedly, accompanied by reduced histone acetylation level of hemocytes. All the results suggested that histone modifications might play a regulatory role in the immune adaptation of Pacific oyster.
To sum up, among the three kinds of circulating hemocytes of Pacific oysters, granulocytes are the main immunocompetent hemocytes, which play fundamental role in immune adaptation induced by the two consecutive stimulations of Vibrio splendidus. Moreover, phagocytosis and anti-apoptotic processed play a key role in immune adaptation, mTOR regulates anaerobic glycolysis is the key energy metabolic pathway, and histone modifications may play a regulatory role in immune adaptation. The results will further establish the basis of the immune adaptation mechanism in Pacific oyster at both the cellular and molecular level, promote our understanding of immune adaptation mechanism in invertebrate, and provide some new ideas for the prevention and cure of diseases of important aquaculture invertebrates.
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136637
专题实验海洋生物学重点实验室
作者单位1.中国科学院大学
2.中国科学院海洋研究所
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王伟林. 长牡蛎免疫适应性(免疫致敏)机制的初步研究[D]. 北京. 中国科学院大学,2017.
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