牙鲆黏膜IgM与多聚免疫球蛋白受体同源分子的应答动态研究

Dynamic Immune Interaction Between Mucosal IgM and Polymeric Immunoglobulin Receptor-Like Protein in Flounder(Paralichthys olivaceus)

本文采用注射和浸泡灭活鳗弧菌(Vibrio anguillarum)免疫牙鲆(Paralichthys olivaceus),研究其黏液(鳃、皮肤和肠)和胆汁中特异性免疫球蛋白M(IgM)及多聚免疫球蛋白受体同源分子(pIgRL)的变化,分析这两种分子在肠组织中的分布及共定位。研究显示,两种免疫方式均可诱导牙鲆黏液及胆汁中pIgRL和IgM水平升高,pIgRL应答时间早于IgM。浸泡免疫后,牙鲆的鳃、皮肤和肠的黏液及胆汁中pIgRL水平分别于第3、5、7和10天达到峰值,而IgM分别于第7、10、21和21天达到峰值。注射免疫后,上述分泌液中pIgRL分别于第5、7、10和10天达到峰值,而IgM在皮肤和鳃黏液中于第14天达到峰值,在肠黏液和胆汁中于第21天达到峰值。多重染色结果表明:免疫前,pIgRL和IgM阳性信号主要位于肠黏膜固有层;浸泡免疫后,IgM信号增强,随后IgM大量出现于肠上皮层,第14天时荧光最强,pIgRL信号也明显增强且少量出现在肠上皮层,在肠黏膜中观察到代表pIgRL和IgM共定位的橘黄色荧光。ImageJ定量分析显示,二者共定位的像素占比在免疫前为19%,免疫后第3、7、14、21和28天时分别为25%、41%、35%、28%和28%,表明存在IgM-pIgRL复合物的跨上皮转运。研究结果为深入理解pIgRL在黏膜免疫防御中的作用和揭示鱼类黏膜免疫系统功能提供了基础资料。

In this study,flounder(Paralichthys olivaceus)was immunized by immersion and injection of inactivated Vibrio anguillarum,and the dynamic interaction of specific IgM and polymeric immunoglobulin receptor-like(pIgRL)protein in gill,skin and gut mucus and bile were investigated with their distribution and co-localization in gut-associated lymphoid tissue determined.The results showed that the protein levels of pIgRL and specific IgM in mucus and bile of flounder increased first and then decreased,and the up-regulated expression of pIgRL gene was earlier than that of specific IgM gene.After immersion immunization,the protein level of pIgRL in gill mucus,skin mucus,gut mucus and bile maximized on 3,5,7,and 10 d,respectively;while specific IgM reached the peak on 7 and 10 d in gill and skin mucus,and on 21 d in gut mucus and bile,respectively.Post injection immunization,pIgRL level peaked on 5,7,10 and 10 d in gill,skin and gut mucus and the bile,respectively,while IgM peaked on 14 d in skin and gill mucus,and on 21 d in gut mucus and bile.The co-staining results indicated that the positive signals of IgM and pIgRL mainly appeared in the intestinal lamina propria of flounder before immunization.After immersion immunization,IgM positive signals gradually increased in the lamina propria and then appeared in a large number in the intestinal epithelial cells,and the fluorescence was strongest on 14 d;the positive signal of pIgRL was also significantly enhanced in the lamina propria and appeared a little in the epithelial layer,and some orange fluorescence signals which represented the co-localization of IgM and pIgRL was observed in intestinal mucosa.Co-localization analysis by ImageJ software revealed that the percentage of co-localization between IgM and pIgRL was 19%before immunization,which was 25%on 3 d,41%on 7 d,35%on 14 d,28%on 21 d and 28 d,suggesting that pIgRL transported IgM in the form of pIgRL-IgM complex across the intestinal mucosa.These results provided a better understanding of the role of pIgRL in mucosal immune defense and the mucosal immune system of teleost.