在无菌无鼠标进定植评估肝代谢的变化 1 H核磁共振光谱
Summary
一个渐进的殖民化过程进行了介绍，以进一步评估其主机上的肝脏代谢的影响。定植被监视的非侵入性的，而基于核磁共振的代谢分析评估微生物的代谢产物的尿中排泄肝脏代谢高分辨率魔角旋转（人力资源的MAS）核磁共振完好活检的分析评估。
Abstract
这是众所周知的，肠道细菌有助于显著主机的动态平衡，提供一系列的好处，如免疫保护和维生素的合成。他们还提供主机与大量的营养物质，使这个生态系统的一个重要的代谢器官。在肠道菌群和代谢综合征之间的联系的理解之间的主机和其肠道菌群的代谢相互作用，越来越多的证据的范围内正在成为现代生物学的一个重要挑战。1-4
定植（也称为正常化的进程）指定建立在前者的无菌动物的微生物。虽然这是一个自然的过程，在出生时发生，它也用于成人无菌动物来控制肠道花卉的生态系统，并进一步确定其主机上的代谢的影响。控制殖民化进程的一个普通的程序是使用SINGL灌胃方法E或微生物的混合物。这种方法的结果，并提出在一个非常快的殖民统治的极端压力5的缺点。因此，它是有用的，以尽量减少压力和获得殖民化进程较慢，逐步观察细菌建立在主机上的新陈代谢的影响。
在这个手稿中，我们描述了一个过程，在一个渐进的殖民过程中，使用非破坏性的代谢分析技术，以评估肝脏代谢的修改。我们建议监测评估肠道微生物的代谢活性微生物的代谢产物 1 H核磁共振的代谢分析尿中排泄反映肠道微生物定植。这使得肠道微生物的活动超出了通常由粪便细菌DGGE（变性 梯度凝胶电泳）监测评估的肠道微生物生态系统的稳定建立稳定的升值 。6定植在传统的开放式环境，是由传统的动物，这将作为控件沾脏的垃圾发起。正在coprophagous动物的啮齿动物，这确保了同质定植如前所述。
肝代谢分析是直接从一个完整的肝活检使用高分辨率的1 H魔角旋转核磁共振光谱测量。这半定量的技术提供一个快速的方法进行评估，不破坏细胞结构，如甘油三酯，葡萄糖和糖原的主要代谢产物，以进一步估计殖民化进程和肝脏代谢 7-10之间复杂的相互作用。这种方法也可以适用于任何组织活检 11,12 。
Protocol
1。定植无菌动物和样品采集从隔离和房子他们在传统畜牧业室配备带过滤器在传统的动物将作为对照组（图1）面前笼中取出无菌动物。 混合垃圾的一半（3天），从常规笼无菌动物的垃圾。始终保持1 / 3脏传统的枯枝落叶每次它是必要的更新，以保持水平（至少要保持3天）的细菌。 收集在1.5毫升的微管的尿液处理管鼠标，帮助轻轻按摩排便排尿。管理单元立即在液氮冻结。…
Discussion
在这个协议中，我们描述了一个渐进的殖民化过程，在一个开放的环境，进一步探讨肝脏代谢的影响肠道菌群的1 H HR MAS完整活检核磁共振分析评估。殖民化的各种方法在文献中被描述。最常用的方法来定义的菌群与殖民动物灌胃或污染的饮用水19,20。粪便接种也可以使用先前描述21。定植方法，这里介绍的是来自一个“正常化”无菌动物库普曼JP等描述的方法。在1986年…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
来自以前发表的研究报告，这是财政支持雀巢作为说明示例中使用的所有NMR谱。
Materials
Table of specific reagents and equipment:
| Name of the reagent | Company | Catalogue number | Comments |
|---|---|---|---|
| 2.5 mm microtube | New Era | NE-H5/2.5-V-Br | |
| 1.7 mm capillary tube | Sigma-Aldrich | NORS175001 | |
| Capillary adapter | New Era | NE-325-5/1.7 | |
| Extraction rod | New Era | NE-341-5 | |
| HR-MAS rotor BL4 with 50 μL spherical Teflon spacer kit |
Bruker | HZ07213 | |
| Tool kit for 50 μL inserts | Bruker | B2950 | |
| Advance III 600 MHz NMR | Bruker | ||
| 1H HR MAS NMR solid probe | Bruker | ||
| Deuterium oxide 99.9 % | Sigma-Aldrich | 530867-1L | |
| 3-(trimethylsilyl)propionic acid-d4 (TSP) |
Sigma-Aldrich | 269913 |
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Citar este artículo
Heath, P., Claus, S. P. Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy. J. Vis. Exp. (58), e3642, doi:10.3791/3642 (2011).