Koiwai Keiichiro, Alenton Rod Russel R, Shiomi Reina, Nozaki Reiko, Kondo Hidehiro, Hirono Ikuo
Laboratory of Genome Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan.
Laboratory of Genome Science, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo 108-8477, Japan.
Mol Immunol. 2017 May;85:1-8. doi: 10.1016/j.molimm.2017.01.024. Epub 2017 Feb 4.
Hemocytes in the circulating hemolymph play important roles for immune responses in shrimp. Previous studies on immune responses by hemocytes in penaeid shrimp were based on gene expression analyses of the entire population of hemocytes and thus may have missed different immune responses of different hemocyte sub-populations. In this study, we separated hemocytes into two sub-populations by Percoll gradient centrifugation, morphological characteristics of each population were then analyzed by May-Giemsa staining, flow cytometry, and FACSCalibur. Results showed hemocytes were divided into an upper layer basophilic, and lower layer of eosinophilic hemocytes. Basophilic hemocytes were larger in size compared to eosinophilic hemocytes, which were more granulated than the basophilic hemocytes. Transcriptome analysis was then conducted through RNA-seq analysis by Miseq, which revealed 16 differentially-expressed transcripts between the two sub-populations. In the upper-layer, the highly expressed transcripts that were homologous to immune-related genes that suggest hemocytes from this layer may play as the regulator of immune system and control the action of other cells to eliminate pathogen. On the other hand, transcripts that were highly expressed in the lower-layer were homologous to the antimicrobial peptide (AMP) crustin, which supports that hemocytes on this layer have granules as crustins are normally secreted from hemocyte granules. The high expression of crustin in the lower-layer also provides insight on the mechanism of the anti-microbial function, where hemocytes produce and store AMPs in its granules. These differentially expressed genes are potential hemocyte molecular markers, and among them we identified one of the highly expressed genes in the hemocytes from the upper-layer (c11736_g1) to be a promising candidate molecular marker predicted to be a surface molecule, which is a common characteristic for molecular markers.
循环血淋巴中的血细胞对虾的免疫反应起着重要作用。先前对对虾血细胞免疫反应的研究基于整个血细胞群体的基因表达分析，因此可能忽略了不同血细胞亚群的不同免疫反应。在本研究中，我们通过Percoll梯度离心将血细胞分离为两个亚群，然后通过May-Giemsa染色、流式细胞术和FACSCalibur分析每个群体的形态特征。结果显示血细胞分为上层嗜碱性细胞和下层嗜酸性细胞。嗜碱性血细胞比嗜酸性血细胞尺寸更大，嗜酸性血细胞比嗜碱性血细胞颗粒更多。然后通过Miseq进行RNA-seq分析进行转录组分析，结果显示两个亚群之间有16个差异表达的转录本。在上层，与免疫相关基因同源的高表达转录本表明该层的血细胞可能作为免疫系统的调节因子，控制其他细胞消除病原体的作用。另一方面，在下层高表达的转录本与抗菌肽（AMP）crustin同源，这支持该层的血细胞含有颗粒，因为crustin通常从血细胞颗粒中分泌。下层crustin的高表达也为抗菌功能机制提供了见解，即血细胞在其颗粒中产生和储存AMPs。这些差异表达的基因是潜在的血细胞分子标记，其中我们鉴定出上层血细胞中高表达的一个基因（c11736_g1）是一个有前景的候选分子标记，预计是一种表面分子，这是分子标记的一个共同特征。
