रोगाणु मुक्त माउस के प्रगतिशील द्वारा औपनिवेशीकरण के दौरान यकृत मेटाबोलिक परिवर्तन का आकलन 1 एच एनएमआर स्पेक्ट्रोस्कोपी
Summary
एक प्रगतिशील उपनिवेशन प्रक्रिया आगे मेजबान यकृत चयापचय पर इसके प्रभाव का आकलन करने के लिए वर्णित है. औपनिवेशीकरण गैर invasively एनएमआर आधारित चयापचय रूपरेखा द्वारा माइक्रोबियल सह चयापचयों के मूत्र उत्सर्जन का मूल्यांकन द्वारा नजर रखी है, जबकि यकृत चयापचय उच्च संकल्प जादू कोण स्पिनिंग (मानव संसाधन MAS) बरकरार बायोप्सी के एनएमआर रूपरेखा द्वारा मूल्यांकन है.
Abstract
It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.
Protocol
Discussion
इस प्रोटोकॉल में, हम एक खुले वातावरण में एक प्रगतिशील उपनिवेशन प्रक्रिया का वर्णन करने के लिए आगे यकृत चयापचय पर पेट microbiota के प्रभाव को एच 1 बरकरार बायोप्सी के एचआर MAS एनएमआर रूपरेखा द्वारा मूल्यांकन …
Disclosures
The authors have nothing to disclose.
Acknowledgements
सभी एनएमआर उदाहराणदर्शक उदाहरण के रूप में इस्तेमाल किया स्पेक्ट्रा पहले प्रकाशित 7 अध्ययन है जो आर्थिक रूप से नेस्ले द्वारा समर्थित किया गया था से व्युत्पन्न हैं.
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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