实验海洋生物学重点实验室知识库

牙鲆（Paralichthys olivaceus）是我国具有重要经济价值的海水养殖品种。迟缓爱德华氏菌（Edwardsiella tarda，E. tarda）是牙鲆的一种重要病原，给养殖业造成了严重经济损失。红细胞作为牙鲆血液中含量最丰富的细胞类型，具有细胞核和细胞器，能够进行转录和翻译，具备免疫应答的能力。本论文主要研究了牙鲆红细胞对不同大小非己颗粒及E. tarda的内吞能力和内吞途径，利用转录组学技术筛选出了E. tarda诱导的内吞相关分子并研究了其免疫功能。同时，本研究借助蛋白质组技术对E. tarda感染牙鲆不同时期的血浆蛋白进行了鉴定，揭示了牙鲆血细胞应对E. tarda感染的免疫应答。主要结果如下：

在牙鲆红细胞对不同大小非己颗粒的内吞能力和内吞途径研究方面，我们首先利用激光共聚焦显微镜、免疫荧光显微镜、透射电镜等观察到了牙鲆红细胞对荧光乳胶微球和E. tarda的内吞活性及其形态特征。随后我们发现：红细胞对0.1-1.0 μm范围内的荧光乳胶微球具有很高的内吞效率，而对2.0 μm荧光乳胶微球的内吞效率急剧降低；红细胞对于0.1-1.0 μm范围的荧光乳胶微球的内吞作用依赖于巨胞饮途径和小窝蛋白介导的内吞途径；红细胞对于活E. tarda的内吞作用依赖于网格蛋白介导的内吞途径；红细胞对灭活E. tarda的内吞作用则主要依赖网格蛋白介导的内吞途径，部分依赖巨胞饮途径和小窝蛋白介导的内吞途径。

我们利用转录组技术，分析了E. tarda体外感染诱导的红细胞基因表达。我们鉴定了95个差异表达基因，其中6个差异表达基因显著富集到了内吞通路（Endocytosis）。我们选定了一个内吞通路的差异表达基因，Cytohesin-1（Cyth1），开展了研究。组织特异性表达研究表明，Cyth1在牙鲆血液当中的表达量最高；基因敲降研究表明，敲降Cyth1显著抑制了E. tarda侵染牙鲆FG-9307细胞，也显著抑制了牙鲆红细胞对E. tarda的内吞能力；利用SecinH3抑制Cyth1的活性后，红细胞对E. tarda的内吞能力被显著抑制。

利用转录组技术，我们进一步研究了E. tarda体内感染诱导的牙鲆红细胞基因表达谱。结果表明，与对照组相比，E. tarda感染诱导了1720个基因差异表达，这些基因显著富集到12条免疫通路。这些免疫通路中的111个基因构成了复杂的基因互作网络。我们从这111个基因中筛选出21个Hub基因进行分析。结果表明，这些Hub基因主要参与了以下过程：NLR受体识别与NF-κB激活、抗原加工与呈递、抗炎反应以及剪接体和剪接。前三个过程中的Hub基因均显著上调，而剪接体和剪接过程中的Hub基因均显著下调。这些结果暗示，E. tarda感染牙鲆红细胞后，被红细胞NLR受体识别并激活了NF-κB活性，促进了红细胞的抗原加工和呈递过程以及抗炎反应。

利用蛋白质组技术，我们研究了E. tarda感染早、中期诱导的牙鲆血浆蛋白谱。与对照组相比，我们发现了17个早期差异蛋白和26个中期差异蛋白。差异蛋白分析结果表明：E. tarda感染早期，血浆中过氧化氢酶、组蛋白H4和CD16浓度上升，可能借此增强机体的抗氧化能力及对E. tarda的直接杀伤及吞噬能力；血浆中C1q补体丰度极大地下调，可能抑制了C1q介导的补体激活。在E. tarda感染中期，血浆中补体C5和C9的丰度显著上调，提示红细胞可能通过激活补体杀灭细菌。其它受E. tarda感染影响的血浆蛋白包括：参与宿主炎症反应的急性期蛋白、高绒毛膜酶1（High choriolytic enzyme 1）、免疫球蛋白轻链以及肽酶等。

综上所述，我们首次确定了牙鲆红细胞对非己颗粒及E. tarda的内吞能力和内吞途径，发现了参与内吞的重要分子及其功能，在转录组和蛋白质组水平揭示了牙鲆血细胞应对E. tarda的免疫响应。本论文研究结果提升了我们对牙鲆血细胞抗感染免疫功能的理解。

Japanese flounder (Paralichthys olivaceus) is a marine aquaculture species with important economic value in China. Edwardsiella tarda (E. tarda) is an important pathogen of flounder, causing serious economic losses to the aquaculture industry. As the most abundant cell type of flounder blood, red blood cells (RBCs) have nuclei and organelles, which can perform transcription and translation, and have the ability for exerting immune responses. In this paper, we studied the ability and pathway of flounder RBCs to ingest different-sized “non-self” particles and E. tarda, and identified E. tarda-induced endocytosis-related molecule via transcriptome analysis and examined its immune function. At the same time, we used proteomic technology to identify the plasma proteins of E. tarda infected flounder at different stages, and revealed the immune response of flounder blood cells to E. tarda infection. The main results are described as follows:

We firstly observed the endocytosis activity and morphological characteristics of flounder RBCs against fluorescent latex microspheres by confocal microscopy, immunofluorescence microscopy and transmission electron microscopy. We found that the endocytosis efficiency of RBCs was high for 0.1-1.0 μm microspheres and low for 2.0 μm microspheres; The endocytosis of 0.1-1.0 μm microspheres depended on macropinocytosis and caveolin-mediated endocytosis; the endocytosis of live E. tarda depended on the clathrin-mediated endocytosis pathway; the endocytosis of inactivated E. tarda by RBCs mainly depended on the clathrin-mediated endocytosis pathway, and partly depended on the macropinocytosis pathway and the caveolin-mediated endocytosis pathway.

Using transcriptomic technology, we analyzed RBCs gene expression induced by E. tarda infection in vitro. We identified 95 differentially expressed genes, 6 of which were significantly enriched in the endocytosis pathway. We selected one gene of the endocytosis pathway, Cytohesin-1 (Cyth1), and examined its function. The expression of Cyth1 was highest in the blood of flounder. Knockdown of Cyth1 significantly inhibited E. tarda infection of flounder FG-9307 cells, and also significantly inhibited the endocytosis of E. tarda by flounder RBCs. Blocking the activity of Cyth1 with SecinH3 significantly inhibited the ability of RBCs to uptake E. tarda.

Using transcriptome technology, we further investigated the gene expression profile of flounder RBCs induced by E. tarda infection in vivo. The results showed that compared with control group, E. tarda infection induced 1720 differential expression genes, which were significantly enriched in 12 immune pathways. There were 111 genes in these immune pathways that constituted a complex network of gene interactions. We selected 21 hub genes from the 111 genes for analysis. The results showed that these hub genes were mainly involved in the following processes: NLR receptor recognition and NF-κB activation, antigen processing and presentation, anti-inflammatory response, and spliceosome and splicing. The hub genes in the first three processes were significantly upregulated, while the hub genes in the spliceosome and splicing process were significantly downregulated. These results suggested that after infection of flounder RBCs, E. tarda was likely recognized by NLR receptors, activated the NF-κB activity, promoted the antigen processing and presentation, and promoted the anti-inflammatory response of RBCs.

Using proteomic techniques, we investigated flounder plasma proteins induced by E. tarda infection in the early and middle stages. Compared with the control group, 17 and 26 proteins were differentially expressed in the early and middle stages, respectively. In the early stage of E. tarda infection, the levels of catalase, histone H4 and CD16 increased, which may enhance the antioxidant capacity and promote the killing and phagocytosis of E. tarda. In contrast, the level of C1q was greatly downregulated, suggesting possible inhibition of C1q-mediated complement activation. The abundances of complement C5 and C9 were significantly upregulated during the middle stage of E. tarda infection, suggesting activation of the complement system. Other plasma proteins affected by E. tarda infection included acute-phase proteins involved in host inflammatory responses, high choriolytic enzyme 1, immunoglobulin light chains, and peptidases.

In conclusion, we determined for the first time the endocytosis ability and endocytosis pathway of flounder RBCs against “non-self” particles and E. tarda, discovered an important endocytosis associated molecule and its function, and revealed the immune response of flounder RBCs to E. tarda infection at the transcriptome and proteomic levels. The results of this paper improved our understanding of the anti-infection immune function of flounder blood cells.