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首页> 外文期刊>PLoS One >Establishment of a novel probe-based RT-qPCR approach for detection and quantification of tight junctions reveals age-related changes in the gut barriers of broiler chickens
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Establishment of a novel probe-based RT-qPCR approach for detection and quantification of tight junctions reveals age-related changes in the gut barriers of broiler chickens

机译:建立一种基于探针的RT-QPCR方法,用于检测和定量紧密交界处揭示了肉鸡肠道血管屏障中的年龄相关变化

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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.
机译:紧密交界处(TJS)在肠道屏障形成中起主要作用,因此,解决任何动物物种中的TJ结构是至关重要的,但在快速生长的肉鸡中具有重要意义。它们由细胞内蛋白质和细胞骨架进行分子组成,超微结构和功能。 TJ蛋白根据其功能分类为障碍,脚手架和成孔类型,其可抵扣渗透性后果。尽管他们对肠系健康的重要性及其完整性有限的研究已经调查了鸡中的TJ,包括TJS分子组成和鸡肠道功能的综合评价。在实际研究序列特异性探针以靶向不同的TJ基因(Claudin 1,3,5,7,10,19,Zonula occludens 1(ZO1),occludin(OCLN)和三胰岛素(MD2))是基于探针的RT-QPCR是新开发的。 Claudin(CLDN)1,5,ZO1和CLDN 3,7,MD2在多重RT-QPCR中吞噬,最小化单独反应的数量并使许多样品的鲁棒测试能够进行稳健的测试。所有RT-QPCR都标准化为鸡JEUNUM和CAECUM样品,其使得能够进行特异性检测和定量基因表达。此外,新建立的方案用于研究同一器官样品中1-35天的肉鸡TJ的年龄发育变化。结果表明,在济肠中所有测试的TJS的14至2天的mRNA表达的MRNA表达显着增加。然而,在盲肠中,在1天后,一些TJ的mRNA表达减少,而一些TJS mRNA持续到35天。通过RT-QPCR确定TJ-蛋白表达的细胞特异性变化提供了更深入地了解,对具有各种病因的肉鸡鸡肠道病理生理变化的分子机制提供了更深的了解。

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