Zhang Tao, Qiu Limei, Sun Zhibin, Wang Lingling, Zhou Zhi, Liu Rui, Yue Feng, Sun Rui, Song Linsheng
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; University of Chinese Academy of Sciences, Beijing 100049, China.
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
Dev Comp Immunol. 2014 Jul;45(1):141-50. doi: 10.1016/j.dci.2014.02.015. Epub 2014 Mar 6.
The increasing experimental evidences suggest that there are some forms of specific acquired immunity in invertebrates, but the underlying mechanism is not fully understood. In the present study, Pacific oyster (Crassostrea gigas) stimulated primarily by heat-killed Vibrio splendidus displayed stronger immune responses at cellular and molecular levels when they encountered the secondary challenge of live V. splendidus. The total hemocyte counts (THC) increased significantly after the primary stimulation of heat-killed V. splendidus, and it increased even higher (p < 0.01) and reached the peak earlier (at 6 h) after the secondary challenge with live V. splendidus compared with that of the primary stimulation. The number of new generated circulating hemocytes increased dramatically (p < 0.01) at 6 h after the pre-stimulated oysters received the secondary stimulation with live V. splendidus, and the phagocytic rate was also enhanced significantly (p < 0.01) at 12 h after the secondary stimulation. Meanwhile, the enhanced phagocytosis of hemocytes was highly specific for V. splendidus and they could distinguish Vibrio anguillarum, Vibrio coralliilyticus, Yarrowia lipolytica, and Micrococcus luteus efficiently. In addition, the mRNA expression of 12 candidate genes related to phagocytosis and hematopoiesis were also monitored, and the expression levels of CgIntegrin, CgPI3K (phosphatidylinositol 3-kinase), CgRho J, CgMAPKK (mitogen-activated protein kinase kinase), CgRab32, CgNADPH (nicotinamide adenine dinucleotide phosphate) oxidase, CgRunx1 and CgBMP7 (bone morphogenetic protein 7) in the hemocytes of pre-stimulated oysters after the secondary stimulation of V. splendidus were higher (p < 0.01) than that after the primary stimulation, but there was no statistically significant changes for the genes of CgPKC (protein kinase C), CgMyosin, CgActin, and CgGATA 3. These results collectively suggested that the primary stimulation of V. splendidus led to immune priming in oyster with specifically enhanced phagocytosis and rapidly promoted regeneration of circulating hemocytes when the primed oysters encountered the secondary challenge with V. splendidus.
越来越多的实验证据表明，无脊椎动物中存在某些形式的特异性获得性免疫，但其潜在机制尚未完全了解。在本研究中，主要受到热灭活灿烂弧菌刺激的太平洋牡蛎（Crassostrea gigas）在遇到活的灿烂弧菌的二次攻击时，在细胞和分子水平上表现出更强的免疫反应。热灭活灿烂弧菌初次刺激后，总血细胞计数（THC）显著增加，与初次刺激相比，在受到活的灿烂弧菌二次攻击后，总血细胞计数增加得更高（p < 0.01）且更早达到峰值（6小时时）。预刺激的牡蛎在受到活的灿烂弧菌二次刺激后6小时，新产生的循环血细胞数量显著增加（p < 0.01），二次刺激后12小时吞噬率也显著提高（p < 0.01）。同时，血细胞吞噬作用的增强对灿烂弧菌具有高度特异性，它们能够有效区分鳗弧菌、溶珊瑚弧菌、解脂耶氏酵母和藤黄微球菌。此外，还监测了12个与吞噬作用和造血相关的候选基因的mRNA表达，预刺激的牡蛎在受到灿烂弧菌二次刺激后，血细胞中Cg整合素、CgPI3K（磷脂酰肌醇3激酶）、CgRho J、CgMAPKK（丝裂原活化蛋白激酶激酶）、CgRab32、CgNADPH（烟酰胺腺嘌呤二核苷酸磷酸）氧化酶、CgRunx1和CgBMP7（骨形态发生蛋白7）的表达水平高于初次刺激后（p < 0.01），但CgPKC（蛋白激酶C）、Cg肌球蛋白、Cg肌动蛋白和CgGATA 3基因无统计学显著变化。这些结果共同表明，灿烂弧菌的初次刺激导致牡蛎产生免疫致敏，当致敏的牡蛎遇到灿烂弧菌的二次攻击时，吞噬作用特异性增强，循环血细胞再生迅速促进。
