Toll样受体在早期离巢乳鸽肠上皮屏障发育中的作用及调控机制

Although artificial feeding early nidifugous pigeon squabs is the key to achieve the transition from traditional pigeon industry to modern one, it will cause severe stress reactions in squabs, such as nutrition, maternal separation, and environment. It is well known that stress can result in injury and reestablishment to intestinal epithelial barrier. However, there have been no reports regarding the effects of stress on gut barrier of altricial birds such as pigeons. Our preliminary study showed that intestinal immune-related differentially expressed genes were mainly enriched in toll-like receptors (TLRs) pathway during the development of early nidifugous squabs, and it has been demonstrated elsewhere that TLRs played an important role in the intestinal epithelial barrier function. Thus, the present study is designed to clarify the specific process of injury and reestablishment in intestinal epithelial barrier by analyzing its morphological and functional changes during the development of early nidifugous squabs. To find out which TLRs are involved in the aforementioned process, the intestinal TLRs distribution and expression pattern and its correlation with intestinal epithelial barrier development will be studied. On the basis, the molecular mechanism and regulatory targets of the selected TLRs in the regulation of intestinal epithelial barrier development will be further revealed by controlling TLRs expression and blocking some specific signaling pathway molecules in early nidifugous squabs, as well as in an established in vitro model of intestinal epithelial cell barrier. The results may enrich the connotation of intestinal immunology-based theory in altricial birds, and provide scientific reference for the nutritional manipulation of gut healthy in early nidifugous pigeon squabs.

乳鸽早期离巢人工哺育是实现鸽业由传统向现代转型的关键，但给乳鸽带来营养、母幼分离和环境等方面的应激。应激会导致肠上皮屏障的损伤与重建。然而，目前关于应激对鸽等晚成鸟肠屏障影响的研究未见报道。申请人前期研究发现早期离巢乳鸽小肠发育过程中免疫相关差异表达基因主要富集在Toll样受体（TLRs）通路，而TLRs与肠上皮屏障功能密切相关。因此，本课题拟在研究早期离巢乳鸽肠上皮屏障形态和功能发育的基础上，探明早期离巢乳鸽肠上皮屏障损伤与重建的具体过程，通过分析早期离巢乳鸽小肠TLRs分布和表达规律及其与肠上皮屏障发育的关系，筛选出在乳鸽早期离巢过程中参与调控肠上皮屏障损伤与重建的TLRs，并通过调控相应TLRs表达，结合肠上皮屏障体外培养及特异信号分子阻断等方法，揭示TLRs调控早期离巢乳鸽肠上皮屏障发育的分子机制和调节靶点，丰富晚成鸟肠道免疫学理论，为营养调控早期离巢乳鸽肠道健康提供科学依据。

与哺乳动物类似，乳鸽生长发育前期需亲鸽哺乳，早期离巢（早期断奶）可减轻种鸽负担，缩短繁殖周期，是实现鸽业由传统向现代转型的关键。乳鸽早期断奶经历着从亲鸽哺育转变为人工哺育、从母幼共居的依赖环境转变为独立生活的变化过程，对乳鸽是一种严重应激。应激会导致肠上皮屏障功能的损伤。肠上皮屏障的完整性对确保幼龄动物健康至关重要。然而，目前关于应激对鸽等晚成鸟类肠屏障功能影响的研究未见报道。本项目在研究早期断奶乳鸽肠上皮屏障形态和功能发育的基础上，结合早期断奶乳鸽小肠Toll样受体（TLRs）表达的发育性变化，并通过调控相应TLRs的表达，筛选出了在乳鸽早期断奶过程中参与调控肠上皮屏障形态和功能发育的2个关键TLRs，即TLR2t2和TLR4。接着，本项目利用Transwell体外细胞培养技术，通过TLR2/TLR4配体刺激模拟早期断奶应激，成功构建了乳鸽体外肠上皮屏障损伤模型。利用上述模型研究发现，在早期断奶乳鸽肠上皮屏障功能发育过程中，激活表达的TLR2t2/TLR4通过MyD88-ERK/JNK信号通路促进促炎细胞因子IL-6和TNF-α的释放，诱导小肠绒毛萎缩，调控细胞旁“孔”通道紧密连接蛋白（即CLDN1、CLDN2和CLDN3）的表达，破坏肠上皮屏障完整性，增加肠上皮屏障通透性，最终损伤肠上皮屏障功能。本项目为国内外首次探讨TLRs在晚成鸟类早期发育过程中对肠上皮屏障发育的调控作用，揭示了TLRs调控早期断奶乳鸽肠上皮屏障功能损伤的分子机制和调节靶点，研究结果可丰富晚成鸟类肠道免疫学基础理论，为营养调控早期断奶乳鸽肠道健康提供科学依据。