Summary

Vurdering Nedsat metaboliske ændringer Under Progressive Kolonisering af Kim-fri mus ved 1 H-NMR-spektroskopi

Published: December 15, 2011
doi:

Summary

En progressiv kolonisering proceduren er beskrevet yderligere at kunne vurdere dens indvirkning på værten hepatisk metabolisme. Colonization overvåges ikke invasivt ved at evaluere urinudskillelsen af ​​mikrobielle co-metabolitter ved NMR-baseret metabolisk profilering, mens levermetabolisme er vurderet af High Resolution Magic Angle Spinning (HR MAS) NMR profilering af intakte biopsi.

Abstract

Det er velkendt, at tarmbakterier bidrage væsentligt til værten homeostase, hvilket giver en række fordele, såsom immunbeskyttelsen og vitamin syntese. De leverer også værten med en betydelig mængde af næringsstoffer, hvilket gør dette økosystem en væsentlig metabolisk organ. I forbindelse med stigende dokumentation for sammenhængen mellem tarmflora og det metaboliske syndrom, forståelse af metabolisk interaktion mellem vært og dens tarm mikrobiota er ved at blive en stor udfordring for moderne biologi. 1-4

Colonization (også kaldet normalisering processen) betegner etablering af mikroorganismer i en tidligere kim-fri dyr. Selv om det er en naturlig proces, der forekommer ved fødslen, er det også anvendes til voksne kim-fri dyr til at kontrollere tarmen blomsterarter økosystemet og yderligere bestemme dets indvirkning på værten stofskiftet. En fælles procedure til at kontrollere de kolonisering processen er at bruge sonde metoden med en single eller en blanding af mikroorganismer. Denne metode resulterer i en meget hurtig kolonisering og præsenterer den ulempe, at ekstremt stressende 5. Derfor er det nyttigt at minimere stress og for at opnå en langsommere kolonisering proces at observere gradvist virkningen af ​​bakteriel etablering på værten stofskiftet.

I dette manuskript, beskriver vi en procedure for at vurdere ændringen af ​​levermetabolisme under en gradvis kolonisering proces med en ikke-destruktiv metabolisk profilering teknik. Vi foreslår at overvåge tarmen mikrobiel kolonisering ved at vurdere tarmen mikrobielle metaboliske aktivitet afspejles i urinudskillelsen af mikrobielle co-metabolitter ved 1 H NMR-baseret metabolisk profilering. Dette giver en vurdering af stabiliteten i tarmen mikrobielle aktivitet ud over den stabile etableringen af tarmen mikrobielle økosystem vurderes normalt ved at overvåge fecal bakterier ved DGGE (denaturering gradient gelelektroforese). 6 Denkolonisering foregår i en konventionel åbent miljø og er initieret af en beskidt kuld tilsølet af konventionelle dyr, der skal tjene som kontrol. Gnavere er coprophagous dyr, dette sikrer en homogen kolonisering som tidligere beskrevet. 7

Nedsat metabolisk profilering måles direkte fra en intakt leverbiopsi bruger 1 H High Resolution Magic Angle Spinning NMR spektroskopi. Denne semi-kvantitative teknik tilbyder en hurtig måde at vurdere, uden at beskadige cellen struktur, hovedmetabolitter som triglycerider, glukose og glykogen for yderligere at vurdere det komplekse samspil mellem kolonisering processen og den hepatiske metabolisme 7-10. Denne metode kan også anvendes på alle væv biopsi 11,12.

Protocol

1. Kolonisering af kim-fri dyr og prøvetagning Fjern kim-fri dyr fra isolatorer og huse dem i et konventionelt husdyrbrug værelse i bure udstyret med filter foran den konventionelle dyr, som vil tjene som kontrol (Figur 1). Bland halvdelen af ​​kuldet (3 dage gammel) taget fra kontrol konventionelle buret med kuld af kimen-frie dyr. Hold altid 1 / 3 af det beskidte konventionelle kuld, hver gang det er nødvendigt at forny det for at opretholde et niveau af bakterier (holde det i det mindste f…

Discussion

I denne protokol, beskrev vi en progressiv kolonisering procedure i et åbent miljø for yderligere at undersøge virkningen af tarme mikrobiota på levermetabolisme vurderet af 1 H HR MAS NMR profilering af intakte biopsi. Forskellige metoder til kolonisering er blevet beskrevet i litteraturen. De mest almindelige metoder til at kolonisere dyr med en defineret mikrobiota er oral sonde eller forurenet drikkevand 19,20. Fecal podningen kan også bruges som tidligere beskrevet 21. Kolonise…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Alle NMR spektre bruges som illustrative eksempler stammer fra en tidligere offentliggjort undersøgelse 7, som blev økonomisk støttet af Nestlé.

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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Cite This Article
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).

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