草鱼IgM的表达及B淋巴细胞的抗菌活性

为了更加了解草鱼B淋巴细胞吞噬活性,本研究采用实时荧光定量PCR(qRTPCR)检测了IgM在胚胎发育中的表达量,并且检测了IgM在不同组织中的分布以及细菌刺激下IgM的转录情况。结果显示,IgM在卵裂期到出膜前期的表达量变化不明显,在出膜后开始显著增加;IgM在检测的组织中均有分布,在头肾中表达量最高,并且其表达量在不同细菌的刺激下均显著上调。从草鱼外周血中分离纯化得到B淋巴细胞,并通过吉姆萨染色、qRT-PCR和间接免疫荧光进行了验证。细菌吞噬实验结果显示,B淋巴细胞对嗜水气单胞菌具有一定的吞噬能力,并随孵育时间增加而逐渐增强。细菌刺激B淋巴细胞后活性氧(reactive oxygen species, ROS)和一氧化氮(nitric oxide,NO)释放量显著上升;血清调理实验结果显示,通过血清调理可以显著促进B淋巴细胞ROS的释放,然而对NO的释放水平没有显著影响。研究表明,草鱼B淋巴细胞对细菌具有一定的吞噬能力,并且可以通过呼吸爆发等非特异性免疫的方式直接参与抗菌免疫。

IgM expression and B lymphocyte antibacterial activity of grass carp (Ctenopharyngodon idella)

B lymphocytes are the key cells of adaptive immune response, can secrete a variety of immunoglobulins which play an important role in the immune response. Respiratory burst is an oxygen-dependent killing mechanism of immune cells when pathogenic microorganisms invade the body. Previous studies suggested that fish B lymphocytes can participate in non-specific immune process through phagocytosis. However, the study of the phagocytic activity of B lymphocytes in grass carp is still not well known. To elucidate the immune function of IgM, the expression of IgM in embryonic stage, tissue distribusion of grass carp were determined by real-time quantitative PCR (qRT-PCR). The results showed that during embryonic development, the expression of IgM was not significantly changed from cleavage stage to pre-hatching stage, and significantly increased after hatching stage. The tissue distribution assay showed that the expression of IgM in the head kidney was the highest, and its expression was significantly up-regulated during the infection of bacteria. To further reveal the role of B lymphocytes in anti-bacterial immunity, B lymphocytes were isolated and purified from grass carp blood and then verified by giemsa staining, RT-PCR and indirect immunofluorescence. The results of bacterial phagocytosis test showed that B lymphocytes showed phagocytic ability to Aeromonas hydrophila. Furthermore, we also found that the production of reactive oxygen species (ROS) and nitric oxide (NO) in grass carp B cells could be significantly up-regulated after infected by microbes. Finally, the serum oposonization assay showed that the release of ROS from B lymphocytes could be enhanced after organized by serum, however, there was no influence to NO production. Taken together, the results of this study showed that B lymphocytes of grass carp have the potential of phagocytic ability to bacteria and can directly participate in anti-bacterial immunity by producing non-specific immunity such as respiratory burst.