Department for Farm Animals and Veterinary Public Health, Clinic for Poultry and Fish Medicine, University of Veterinary Medicine, Vienna, Austria.
Department of Veterinary Medicine, Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany.
PLoS One. 2021 Mar 5;16(3):e0248165. doi: 10.1371/journal.pone.0248165. eCollection 2021.
Tight junctions (TJs) play a dominant role in gut barrier formation, therefore, resolving the structures of TJs in any animal species is crucial but of major importance in fast growing broilers. They are regulated in molecular composition, ultrastructure and function by intracellular proteins and the cytoskeleton. TJ proteins are classified according to their function into barrier-forming, scaffolding and pore-forming types with deductible consequences for permeability. In spite of their importance for gut health and its integrity limited studies have investigated the TJs in chickens, including the comprehensive evaluation of TJs molecular composition and function in the chicken gut. In the actual study sequence-specific probes to target different TJ genes (claudin 1, 3, 5, 7, 10, 19, zonula occludens 1 (ZO1), occludin (OCLN) and tricellulin (MD2)) were designed and probe-based RT-qPCRs were newly developed. Claudin (CLDN) 1, 5, ZO1 and CLDN 3, 7, MD2 were engulfed in multiplex RT-qPCRs, minimizing the number of separate reactions and enabling robust testing of many samples. All RT-qPCRs were standardized for chicken jejunum and caecum samples, which enabled specific detection and quantification of the gene expression. Furthermore, the newly established protocols were used to investigate the age developmental changes in the TJs of broiler chickens from 1-35 days of age in the same organ samples. Results revealed a significant increase in mRNA expression between 14 and 21days of age of all tested TJs in jejunum. However, in caecum, mRNA expression of some TJs decreased after 1 day of age whereas some TJs mRNA remained constant till 35 days of age. Taken together, determining the segment-specific changes in the expression of TJ- proteins by RT-qPCR provides a deeper understanding of the molecular mechanisms underpinning pathophysiological changes in the gut of broiler chickens with various etiologies.
紧密连接（TJs）在肠道屏障形成中起主导作用，因此，解析任何动物物种的 TJs 结构都至关重要，但在快速生长的肉鸡中尤为重要。它们的分子组成、超微结构和功能受到细胞内蛋白质和细胞骨架的调节。TJ 蛋白根据其功能分为形成屏障、支架和形成孔的类型，对通透性有可推断的影响。尽管它们对肠道健康及其完整性很重要，但对鸡的 TJ 进行的研究有限，包括对鸡肠道 TJ 分子组成和功能的综合评估。在实际研究中，设计了针对不同 TJ 基因（claudin 1、3、5、7、10、19、zonula occludens 1（ZO1）、occludin（OCLN）和 tricellulin（MD2）的序列特异性探针，并新开发了基于探针的 RT-qPCR。Claudin（CLDN）1、5、ZO1 和 CLDN 3、7、MD2 被纳入多重 RT-qPCR 中，最大限度地减少了单独反应的数量，并能够对许多样本进行稳健的测试。所有 RT-qPCR 均针对鸡空肠和盲肠样本进行了标准化，这使得基因表达的特异性检测和定量成为可能。此外，还使用新建立的方案来研究同一器官样本中 1-35 日龄肉鸡 TJ 的年龄发育变化。结果表明，在空肠中，所有检测到的 TJ 的 mRNA 表达在 14 至 21 日龄之间显著增加。然而，在盲肠中，一些 TJ 的 mRNA 表达在 1 日龄后下降，而一些 TJ 的 mRNA 表达在 35 日龄前保持不变。总之，通过 RT-qPCR 确定 TJ-蛋白表达的节段特异性变化，可以更深入地了解具有不同病因的肉鸡肠道中病理生理变化的分子机制。
