Name: assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1h nmr spectroscopy
Uploaded: 2011-12-15T13:00:00
Description: Watch this Scientific Journal Video about Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy at JoVE.com

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

1. Kolonisering af kim-fri dyr og prøvetagning <ol><li> 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). </li><li> 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...

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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...

Tabel over specifikke reagenser og udstyr: <table border="1"><tbody><tr><th> Navn på reagenset </th><th> Firma </th><th> Katalog nummer </th><th> Kommentarer </th></tr><tr><td> 2,5 mm mikrorør </td><td> New Era </td><td> NE-H5/2.5-V-Br </td><td></td></tr><tr><td> 1,7 mm kapillarrør </td><td> Sigma-Aldrich </td><td> NORS175001 </td><td></td></tr><tr><td> Kapillær adapter </td><td> New Era </td><td> NE-325-5/1.7 </td><td></td></tr><tr><td> Ekstraktion Rod </td><td> New Era </td><td> NE-341 til 5 </td><td></td></tr><tr><td> HR-MAS rotor BL4 med 50 μL sfæriske Teflon spacer kit </td><td> Bruker </td><td> HZ07213 </td><td></td></tr><tr><td> Værktøjssæt til 50 μL indstik </td><td> Bruker </td><td> B2950 </td><td></td></tr><tr><td> Advance III 600MHz NMR </td><td> Bruker </td><td></td><td></td></tr><tr><td> 1 H HR MAS NMR fast sonde </td><td> Bruker </td><td></td><td></td></tr><tr><td> Deuteriumoxid 99,9% </td><td> Sigma-Aldrich </td><td> 530867-1L </td><td></td></tr><tr><td> 3 - (trimethylsilyl) propionsyre syre-d 4 (TSP) </td><td> Sigma-Aldrich </td><td> 269913 </td><td></td></tr></tbody></table>

assessing hepatic metabolic changes during progressive colonization of germ-free mouse by 1h nmr spectroscopy

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.

Watch this Scientific Journal Video about Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy at JoVE.com

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

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.

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Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Molekylær diffusion i plasma Membraner af primær lymfocytter målt ved fluorescenskorrelationsspektroskopi

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Påvisning af Enterohemorrhagic Escherichia Coli kolonisering i Murine vært ved Non-invasiv In Vivo bioluminescens System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...