IOCAS-IR
中华绒螯蟹造血组织及造血作用机理的研究
贾志浩
学位类型博士
导师张国范
2018-06
学位授予单位中国科学院大学
学位授予地点中国科学院海洋研究所
学位名称理学博士
学位专业海洋生物学
关键词中华绒螯蟹 血淋巴细胞 造血作用 转录组 活性氧
摘要

造血作用是一系列的复杂过程,包括不同类型的血细胞从特定的造血位点发生、发育以及成熟。造血作用中生成的形态各异的血细胞(免疫细胞),构成了机体细胞免疫和体液免疫的核心和基础,因此了解造血作用中血细胞的发生、分化及其相应的免疫功能,是探究机体免疫防御机制的前提和关键切入点。由于无脊椎动物缺乏适应性免疫系统,它们主要依赖固有免疫系统抵御病原入侵,而其中由细胞免疫发挥了至关重要的作用,细胞免疫所需要的血淋巴细胞主要通过造血作用快速补充。中华绒螯蟹是一种主要养殖于亚洲南部的重要经济甲壳动物,成体内存在独立的造血组织。本研究以中华绒螯蟹为研究对象,鉴定了其成体造血组织的特征,利用流式细胞仪分选了不同血淋巴细胞亚群,通过转录组学分析,探究了造血过程,阐述了活性氧(reactive oxygen species, ROS)和甲壳动物细胞因子EsAstakine对造血过程的调控机制。

中华绒螯蟹的造血组织位于其胃的背侧,为一层一系列的卵圆形囊泡构成的薄膜。其中的囊泡内含有大量细胞核相对较大的圆形细胞。这些细胞由结缔组织包裹,含有大量线粒体,并且多数造血组织细胞处于有丝分裂前期和中期。EdU标记实验也证明了造血组织细胞呈现出相当旺盛的DNA复制活动。造血相关转录因子Runx1 特异性表达在中华绒螯蟹造血组织中,可能存在3个不同的蛋白异构体。GATA1在造血组织和血淋巴细胞中均有表达,但可能以不同的蛋白异构体形式存在,参与新生血淋巴细胞的成熟与释放。

通过流式细胞仪分选,中华绒螯蟹的血淋巴细胞被分为P1P2P1型血淋巴细胞形状较小,内含大量着色较深的嗜酸性颗粒,因此可能主要为粒细胞。P2型血淋巴细胞大小不一且颗粒度较小,可能主要由透明细胞和半粒细胞组成。正常条件下,P1P2型血淋巴细胞都具有吞噬能力,并且吞噬率相似。但是LPS刺激之后,P1型血淋巴细胞的吞噬率显著性上升,而P2型血淋巴细胞未出现显著性变化,表明P1型血淋巴细胞是中华绒螯蟹中行使吞噬功能的主要细胞类群。此外,P1型血淋巴细胞中的ROS、溶酶体及钙离子水平也显著高于P2型血淋巴细胞。RT-PCR结果表明,病原识别、吞噬、杀菌、溶酶体以及抗氧化作用相关的基因在P1血淋巴细胞中特异性高表达;而酚氧化物酶原激活系统相关的基因在P2血淋巴细胞中显著性高表达。P1血淋巴细胞为免疫应答中行使吞噬功能的主要细胞类群,但仍有超过80%P1血淋巴细胞不具有吞噬能力,同时造血组织细胞也具有一定的吞噬能力,结果也揭示了中华绒螯蟹血淋巴细胞并没有完全特化,可能由多种细胞共同发挥类似作用。

本研究也构建了造血组织和血淋巴细胞的六个转录文库,共得到51,229,690条单边变长的序列,拼接后共得到31,346个基因作为参考基因组。经过组装之后,共筛选出362个差异表达基因。造血组织中高表达的301个基因富集在与DNARNA和蛋白质合成、细胞分裂以及能量代谢相关功能群。其中与线粒体ROS产生相关的基因mitochondrial complexes I在造血组织中的表达水平是血淋巴细胞中的8.6倍。这些上调基因表明了造血组织中具有旺盛的细胞分裂活动以及氧化还原反应。在61个血淋巴细胞上调的基因中,参与酚氧化酶原(prophenoloxidase, proPO)激活的蛋白酶以及proPO大量富集,表明proPO可能作为成熟血淋巴细胞的标记分子。和血淋巴细胞相比,造血组织中ROS水平相对较高。当造血组织中的ROS被其清除剂NAC清除之后,可以延迟由嗜水气单胞菌刺激引起的血淋巴细胞增多。推测ROS可促进造血组织中细胞的增殖和分化,是中华绒螯蟹造血的重要调节因子。

Astakine是甲壳动物中发现的参与造血的细胞因子。本研究从中华绒螯蟹的EST库中鉴定并克隆得到了一个全长为1163 bpEsAstakine基因,其编码一个128个氨基酸的短肽。EsAstakine二级结构包括一个信号肽结构域和一个前动力蛋白结构域,其中有9个保守的半胱氨酸残基。EsAstakine与淡水龙虾和斑节对虾中的Astakine在序列和进化上更为接近。EsAstakinemRNA主要在血淋巴细胞和肝胰腺组织中表达,并且鳗弧菌和毕赤酵母的刺激可以显著提高血淋巴细胞中EsAstakine的表达量。在嗜水气单胞菌的刺激下,EsAstakine的表达水平和血淋巴细胞的总数变化趋势一致。注射EsAstakine重组蛋白可以显著提高血淋巴细胞的总数和造血组织细胞的ROS水平,也可以引起JNKmTOR信号通路相关基因的表达水平上升。EsAstakineRNA干扰可以显著延迟由嗜水气单胞菌刺激引起的血淋巴细胞增多并且减少造血组织细胞的DNA复制活动。同时,线粒体上的ROS重要产生基因mitochondrial complexes I的干扰,不仅降低造血组织细胞的ROS水平,同时显著抑制由EsAstakine的重组蛋白注射引起的血淋巴细胞数目的增多。上述结果表明在病原刺激条件下,血淋巴细胞产生大量EsAstakineEsAstakine随循环系统作用于造血组织细胞,引起胞内ROS水平上升,可能通过激活JNK通路,促进造血细胞的分化以及血淋巴细胞的再生。

综上所述,中华绒螯蟹存在特化的造血组织和功能上有一定分化的血淋巴细胞亚群。ROS能够促进造血组织细胞的增殖和分化。EsAstakine通过ROS依赖的信号通路促进造血细胞的增殖及血淋巴细胞的再生。

其他摘要

Hematopoiesis is a complex process that different kinds of hemocytes are formed and released from the restricted hematopoietic tissue. Chinese mitten crab Eriocheir sinensis is one the most economically important crustacean species widely cultivated in South-East Asia. As invertebrate, Chinese mitten crab only has the innate immune system to defense against invading pathogens. The cellular immunity mediated by hemocytes is of vital importance in the immune response of crab, which requires the continuously production of new hemocytes by hematopoiesis. In the present study, the hematopoietic tissue of Chinese mitten crab has been characterized, different sub-population of hemocytes has also been characterized by flow cytometry. The transcriptome libraries of HPT and hemocytes have been constructed in order to find the mechanism of hematopoiesis. In addition, the involvement of ROS and crustacean cytokine Astakine has been explored.

The hematopoietic tissue (HPT) from Chinese mitten crab E. sinensis was a thin and non-transparent sheet located at the dorsolateral side of the stomach, which was composed of a series of ovoid lobules. The lobules contained a large amount of spherical cells with big nucleus and were surrounded by connective tissues. In HPT, mangy of the cells were full of mitochondria and granules, and DNA replication was detected in some cells by EdU labeling technique. Cell proliferation was also observed in HPT by transmission electron microscope (TEM). Three homologues of RUNX1 were detected in the HPT while no signal of RUNX1 was observed in hemocytes, and GATA1 was detected in both HPT and some hemocytes. The results suggested that the stem cells in the hematopoietic tissue of Chinese mitten crab E. sinensis were regulated by transcriptional and humoral factors to generate hemocytes.

The hemocytes of Chinese mitten crab E. sinensis can be directly separate into two groups by flow cytometry, P1 hemocytes with deeply stained cytoplasm which were full of round and abundant granules, P2 hemocytes which had a wide range of sizes and less granularity. Both P1 and P2 hemocytes were detected to possess phagocytic ability and the phagocytic rate were similar. After LPS challenge, the phagocytic rate of P1 hemocytes was significantly up-regulated and no difference was detected in P2 hemocytes. Meanwhile, lysosome content and ROS production were mainly concentrated in P1 hemocytes under both normal and immune stimulations. P1 hemocytes possessed higher intracellular Calcium levels than P2 hemoytes. After P1 and P2 hemocytes were sorted by flow cytometry, and the mRNA transcripts of some immune related genes were further tested. P1 hemocytes expressed higher levels of genes participating in antimicrobial activities, phagocytosis, lysosome and antioxidant activities. While genes enriched in proPO activation system were highly expressed in P2 hemocytes. These results collectively suggested although overlapping function were exist between P1 and P2 hemocytes, the P1 hemocytes were the main immunocompetent hemocytes and P2 hemocytes mainly participated in proPO activation system.

The transcriptomes of HPT and hemocytes were sequenced using the Ion Torrent Proton sequencing platform. A total of 51,229,690 single end reads were obtained from six single-end libraries, which were assembled into 31346 unireads as reference genome. After mapping and transcript assembling, totally 362 differently expressed genes were identified and 301 of them were more abundant in HPT. GO annotation revealed that the 301 HPT up-regulted genes were mostly implicated in DNA, RNA and protein synthesis, cell division, mitochondria activities and energy metabolism. The expression level of mitochondrial complexes I (mitochondrial NADH-ubiquinone oxidoreductase) which was the main natural producers of mitochondrial ROS was found to be 8.6-fold (p < 0.01) higher in HPT than that in hemocytes. In hemocytes, the proteinase associated with proPO activation were the main up-regulated genes which might be the potential markers for mature hemocytes. ROS level in HPT cells was relatively higher which was confirmed with the high expression level of mitochondria related genes identified by transcriptome sequencing. These results collectively indicated that the genes in redox system were more active in HPT. After the ROS level was depressed by N-acetyl-L-cysteine (NAC), the production of hemocytes from HPT was inhibited, and the recovery rate of the total hemocytes counts induced by aeromonas hydrophila was delayed. ROS could function as an important modulator in the hematopoiesis of crab and promote the production of hemocytes from HPT

A novel Astakine gene was identified from Chinese mitten crab E. sinensis (designated as EsAst). The full-length cDNA of EsAst was of 1163 bp, consisting of a 5’ untranslated region (UTR) of 120 bp, a 3’ UTR of 656 bp, and an open reading frame (ORF) of 387 bp encoding a polypeptide of 128 amino acids. There were a signal peptide and a prokineticin domain with nine conserved cysteine residues in the deduced amino acid sequence of EsAst. EsAst shared higher similarity with Astakines from Penaeus monodon and Pacifastacus leniusculus, and it was closely clustered with the Astakine from shrimp P. monodon in the phylogenetic tree. The EsAst mRNA transcript was higher expressed in hemocytes and hepatopancreas. The relative expression level of EsAst in hemocytes was continuously increased from 1.5 to 48 h after Vibro anguillarum challenge compared that in the untreated control group. After Pichia pastoris GS115 challenge, the relative expression level of EsAst in hemocytes was up-regulated, which was significantly higher than that in the untreated control group. After rEsAst injection, ROS levels in HPT cells were also increased at 12 and 24 h, and the total hemocyte counts were also significantly increased at 6, 9, 12, and 24 h post rEsAst injection. The interference of EsAst expression with dsRNA injection could delay the recovery of hemocytes production post A. hydrophila stimulation. When mitochondrial complexes I was knocked down by dsRNA, ROS levels were decreased and THCs were also decreased. Recovery of hemocyte production inducing by A. hydrophila stimulation and rEsAst injection were delayed with dsEsbc1 injection. When ROS levels were increased after RNAi of Lon protease, THCs were also increased. The expression levels of five genes (EsJNK, EsSTAT, EsPI3K, EsAKT1, EsP70S6K) involved in SAPK-JNK and mTOR signaling pathways were up-regulated at 12 and 24 h in rEsAst group and EsLon dsRNA group compared with that in EGFP dsRNA group, and were similar to the trend of ROS levels. EsAst should be a novel Astakine to promote the production of hemocytes in a ROS-dependent way in E. sinensis.

In conclusion, E. sinensis has specialized hematopoietic tissue and functionally diversed hemocytes sub-population. Astakine could be released from hemocytes post challenge and promote the proliferation of hematopoietic cells as well as the regeneration of hemocytes in a ROS-dependent way.

学科领域水产学
学科门类理学 ; 理学::海洋科学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/154509
专题中国科学院海洋研究所
第一作者单位中国科学院海洋研究所;  实验海洋生物学重点实验室
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贾志浩. 中华绒螯蟹造血组织及造血作用机理的研究[D]. 中国科学院海洋研究所. 中国科学院大学,2018.
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