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

Alla NMR-spektra används som belysande exempel är hämtade från en tidigare publicerad studie 7 som ekonomiskt stöddes av Nestlé.

1. Kolonisering av bakterie-fria djur och provtagning <ol><li> Ta bort bakteriefri djur från isolatorer och hus dem i en konventionell djurhållning rum i burar utrustade med filter framför den konventionella djur som kommer att fungera som kontroller (Figur 1). </li><li> Blanda hälften av kullen (3 dagar gamla) som tagits från kontroll konventionella bur med strö av grodden-fria djur. Håll alltid 1 / 3 av det smutsiga konventionella kullen varje gång det är nödvändigt att förnya för att upprätthålla en ...

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I detta protokoll, beskrev vi en progressiv kolonisering förfarande i en öppen miljö för att ytterligare undersöka effekten av tarmfloran om levermetabolism bedömas av en H HR-MAS NMR profilering av intakt biopsi. Olika metoder för kolonisering har beskrivits i litteraturen. De vanligaste metoderna för att kolonisera djur med ett definierat mikrobiota är oral sondmatning eller förorenat dricksvatten 19,20. Fekal inokulationen kan också användas som tidigare beskrivits 21. Kol...

Tabell över specifika reagenser och utrustning: <table border="1"><tbody><tr><th> Reagensets namn </th><th> Företag </th><th> Katalognummer </th><th> Kommentarer </th></tr><tr><td> 2,5 mm mikrorör </td><td> New Era </td><td> NE-H5/2.5-V-Br </td><td></td></tr><tr><td> 1,7 mm kapillärrör </td><td> Sigma-Aldrich </td><td> NORS175001 </td><td></td></tr><tr><td> Kapillär-adapter </td><td> New Era </td><td> NE-325-5/1.7 </td><td></td></tr><tr><td> Extraktion stav </td><td> New Era </td><td> NE-341-5 </td><td></td></tr><tr><td> HR-MAS rotor BL4 med 50 mikroliter sfäriska Teflon Spacersats </td><td> Bruker </td><td> HZ07213 </td><td></td></tr><tr><td> Verktygssats för 50 mikroliter insatser </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 sond </td><td> Bruker </td><td></td><td></td></tr><tr><td> Deuteriumoxid 99,9% </td><td> Sigma-Aldrich </td><td> 530.867-1L </td><td></td></tr><tr><td> 3 - (trimetylsilyl) propionsyra syra-d 4 (TSP) </td><td> Sigma-Aldrich </td><td> 269.913 </td><td></td></tr></tbody></table>

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

Det är väl känt att tarmbakterier bidra avsevärt till värden homeostas, vilket ger en rad fördelar såsom immun skydd och vitamin syntes. De levererar också värd en ansenlig mängd näringsämnen, vilket gör detta ekosystem en viktig metabolisk orgel. I samband med allt fler bevis om kopplingen mellan tarmfloran och det metabola syndromet, förstå den metaboliska interaktionen mellan värden och dess tarmfloran blir en viktig utmaning i modern biologi. 1-4 Colonization (även kallad normaliseringsprocess) betecknar inrättandet av mikroorganismer i en tidigare bakteriefri djur. Även om det är en naturlig process som uppstår vid födseln, är det också ges till vuxna bakteriefri djur för att kontrollera tarmen blommor ekosystemet och ytterligare bestämma dess påverkan på värden ämnesomsättningen. Ett gemensamt förfarande för att kontrollera kolonisationen processen är att använda sondmatning metod med en single eller en blandning av mikroorganismer. Metoden ger en mycket snabb kolonisering och presenterar nackdelen med att vara extremt stressande 5. Det är därför lämpligt att minimera stress och att få en långsammare kolonisering process för att följa successivt effekterna av bakteriell etablering på värden ämnesomsättningen. I detta manuskript, beskriver vi ett förfarande för bedömning ändring av levermetabolismen under en successiv kolonisering process med hjälp av ett icke-förstörande tekniken metabolisk profilering. Vi föreslår att övervaka kolonisering tarmen mikrobiella genom att bedöma i tarmen aktivitet mikrobiell metabolism återspeglas i urinutsöndringen av mikrobiell samarbete metaboliter med 1 H NMR-baserade metabolisk profilering. Detta möjliggör en uppskattning av stabiliteten i tarmen mikrobiell aktivitet utanför stallet inrättandet av tarmen mikrobiella ekosystem vanligtvis kontrolleras genom avföringen bakterier genom DGGE (denaturering elektrofores gradient gel). 6kolonisering sker i en konventionell öppen miljö och inleds med en smutsig kull som förorenats med konventionella djur, som fungerar som kontroller. Gnagare är coprophagous djur, säkerställer detta en homogen kolonisering som tidigare beskrivits. 7 Nedsatt metabolisk profil mäts direkt från en intakt leverbiopsi med ett H Hög Vinkel upplösning Magiska Spinning NMR-spektroskopi. Detta semikvantitativ tekniken erbjuder ett snabbt sätt att bedöma, utan att skada cellstruktur, huvudmetaboliterna som triglycerider, glukos och glykogen för att ytterligare uppskatta det komplexa samspelet mellan kolonisation processen och levermetabolismen 7-10. Denna metod kan också tillämpas på alla vävnad 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 förfarande beskrivs att ytterligare utvärdera dess effekter på värden levermetabolism. Colonization övervakas icke invasivt genom att utvärdera urinutsöndringen av mikrobiell samarbete metaboliter med hjälp av NMR-baserade metabolisk profilering medan levermetabolismen bedöms av tröjan Magiska Angle Spinning (HR MAS) NMR profilering av intakt biopsi.

Bedömning av förändringar Nedsatt Metabola Under Progressiv koloniseringen av bakteriefri mus med 1 H NMR-spektroskopi

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

Molekylär diffusion i plasmamembran primära lymfocyter Mätt genom 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

Påvisande av entrohemoragiska Escherichia Coli koloniseringen i murina mottagande av icke-invasiva In Vivo Mareld System

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