天津市应用基础与前沿技术研究计划 (19JCYBJC29700，15JCZDJC33800)；天津市水产生态及养殖重点实验室开放基金 (TJAE2015005)；国家自然科学基金 (31472299)
血细胞是机体免疫系统的核心，为了对其进行精细的类群划分和功能分析，了解动物免疫防御的机制，实验以体质量为17~23 g的凡纳滨对虾为对象，分别根据显微观察的胞内可辨颗粒结构和流式测量的细胞侧向散射特征探讨了其血淋巴的细胞类群，并进一步探讨了在副溶血性弧菌和白斑综合征病毒两种病原感染早期，主要响应的血细胞类群及其数量变化等特征。结果显示，两种方法都能从凡纳滨对虾血淋巴中辨识出无颗粒、小颗粒、中颗粒以及大颗粒四种血细胞，其中无颗粒细胞占比都接近70%，另外3种颗粒细胞合计占比均为30%左右，但细分比例统计有差异。脂质染料DID显示胞内颗粒普遍被膜，中颗粒和大颗粒细胞内的颗粒大小较均匀，提示了其内源性的形成机制；大颗粒细胞大小分布区间最窄且充满颗粒，表明其可能处于发育终末阶段，功能与分泌相关。通过测量发现，在两种病原感染早期，承担吞噬功能的无颗粒细胞和小颗粒细胞是主要响应类群，总体趋势均表现为细胞计数先降后升，推测感染后大量血细胞由于被用于清除病原而导致短时间内损失较多。副溶血性弧菌感染24 h后细胞总数降至低点，而白斑综合征病毒感染后的低点出现在36~48 h，相较略有延后并持续较长时间，可能与病毒需先入胞并进行复制等过程有关。研究表明，甲壳动物的血细胞可以用相近的量化标准分群，它们通过不同的方式参与病原刺激的免疫应答。
Blood cells play an important role in animal immune system. Accurate classification and functional analysis will facilitate the understanding of immune response mechanism. In this research, hemocyte populations from Litopenaeus vannamei of 17-23 g in weight were identified through micro-observation and flow cytometry, which referred to distinguishable intracellular partical structures and measurable side scattering characteristics of cells separately. Further analyses of verifying the response hemocyte groups and their quantitative changes in early stage of bacteria or virus infection were also carried out. Four hemocyte populations of non-granular, small-granular, intermediate-granular and large-granular hemocyte could be identified by micro-observation and flow cytometry, accounting for (69.88%±4.71%)/(74.88%±3.97%), (9.04%±5.06%)/(20.75%±4.07%), (11.13%±7.02%)/(2.94%±0.72%) and (9.96%±1.31%)/(1.25%±0.58%) of the total hemocytes respectively. In both methods, non-granular hemocyte accounted for about 70%, but proportions of other 3 granular hemocytes were different depending on the method. Lipophilic tracer DID staining showed intracellular granules were covered by lipid membrane. Size similarity of granules in both intermediate-granular hemocyte and large-granular hemocyte inferred an endogenous mechanism, supporting granules were derived from golgi apparatus. The size distribution range of large-granule hemocytes was narrower and their cytoplasm was full of granules, indicating they might be at a mature stage. Further results from low intensity challenging of bacterial or virus pathogens showed that in the early time of Vibrio parahaemolyticus or WSSV infection, total cell counting reflected a similar trend of “down and up”; non-granular and small-granular hemocytes, the two major phagocytic populations, were main response groups. The low point of cell counting in V. parahaemolyticus infection experiment was observed at 24 h; but for WSSV infection, low counting period was between 36 and 48 h, which should be related to the longer time required by virus for entering cell and replicating. These support that a large number of hemocyte could be lost in a short period of time after infection. On the other hand, full of granules and showing little count fluctuation after infection, both intermediate-granular hemocyte and large-granular hemocyte might participate in immune defense through secreting bioactive molecular other than phagocytosis. Above results indicated that hemocytes populations from different crustacean species can be distinguished with similar quantitative criteria and they respond to pathogen invasion in different ways after infection.
LI Rong, REN Xingchao, ZHANG Ning, WANG Yuan, LIU Yichen, ZHANG Yichen, GENG Xuyun, SUN Jinsheng. Imaging flow cytometry measurement and pathogens infection analysis of Litopenaeus vannamei hemocytes[J]. Journal of Fisheries of China,2022,46(6):1094~1103复制