斑马鱼食源性肠炎模型及其免疫细胞成像分析方法的建立

ESTABLISHING THE ZEBRAFISH MODEL OF FOODBORNE-ENTERITIS AND IMAGING THE INVOLVED IMMUNE CELLS’ RESPONSE

  • 摘要: 为了研究豆粕替代鱼粉对鱼类肠黏膜免疫的影响和建立缓解肠炎药物的筛选及功能性饲料添加剂的平台, 研究采用固有免疫或适应性免疫细胞荧光标记的斑马鱼品系, 通过50%的豆粕添加量替代鱼粉作为蛋白源, 共设计了两组饲料, 分别在幼鱼固有免疫或适应性免疫的发育阶段中饲喂荧光标记的斑马鱼, 构建了斑马鱼豆粕诱导的肠炎模型, 对构建的食源性肠炎模型进行了病理、免疫细胞及分子水平的探索。结果表明, 在固有免疫方面, 斑马鱼通过增加中后肠的中性粒细胞和巨噬细胞的招募、诱导巨噬细胞形态发生抗炎/促炎状态的转换(如形成免疫突触、抗炎/促炎细胞因子等), 来应对食源性急性肠炎; 在适应性免疫阶段, 豆粕饲料促进斑马鱼中后肠淋巴细胞的聚集, 包括未成熟的淋巴细胞以及成熟的T淋巴细胞, 并且也可发生抗炎/促炎状态转换。从肠道组织病理来说, 豆粕替代鱼粉蛋白源会造成肠黏膜损伤, 结合切片HE染色显示的肠道物理屏障受到破坏的病理, 目前qPCR结果可推测肠道细胞凋亡增加, 肠上皮层紧密连接变弱并出现代偿性的细胞增生, 同时促炎因子表达增加, 免疫细胞聚集肠道, 暗示免疫屏障也受损。值得一提的是, 斑马鱼幼鱼的适应性免疫发育阶段, 即可发生类似成鱼的肠黏膜免疫调控来应对食源性肠炎, 例如通过提高调节性免疫细胞转录因子(foxp3)及效应因子(il-10、tgf-beta)来提高肠黏膜的免疫耐受。因此, 研究从模式生物的角度, 提供了一种快速、可视化的水产动物的食源性肠炎模型及相关功能饲料添加剂的在体研发平台。

     

    Abstract: Foodborne enteritis caused by plant protein substitution has become one of the major limiting factors in aquaculture. The purpose of this study was to establish a disease model for better understanding the local intestinal immune effect of fish foodborne enteritis. In this study, zebrafish lines of fluorecence labled immune cells (either innate or adaptive populations) were used to construct soybean induced enteritis model, during either the developmental stage of innate or adaptive immunity, respectively. Analysis was carried for both pathology, including both histological and molecular level, and the response of immune cell. Current results showed that in the aspect of innate immunity, zebrafish responded to acute enteritis by recruiting neutrophils and macrophages in the hindgut and inducing the morphological change of macrophages, which indicated the transition between anti-inflammatory/pro-inflammatory states and was also coincidant with expresional changing of cytokines. One the other hand, for the adaptive immunity, soybean meal diet promoted lymphocytes’ aggregation to the hindgut, for both immature lymphocytes and mature T lymphocytes. The mophological change, which suggested the transition of anti-inflammatory/pro-inflammatory states, was observed among the lymphocytes population. Furthermore, at the level of pathology, soybean meal caused damage on intestinal barrier indicated by result of HE staining. In additon to patholocially disrupted barrier, qPCR results molecularly indicated the increased apoptosis of intestinal cells, disturbed tight junction, as well as compensatory hyperplasia for intestinal epithelial cell, paralled with increased expression of proinflammatory factors. It was worth mentioning that during the development stage of adaptive immunity in zebrfish, the intestinal immune regulation, indicated by the increased expression of foxp3, il-10, and tgf-beta, in order to develop the immune tolerance has worked during food borne enteritis. In conclution, this study has provided a effective and visible model of food-borne enteritis in zebrafish and thus set up a platform for the development of related functional feed additives in fish.

     

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