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

Todos os espectros de RMN utilizadas como exemplos ilustrativos são derivados de um estudo publicado anteriormente 7, que foi apoiada financeiramente pela Nestlé.

1. Colonização do germe-free animais e coleta de amostras <ol><li> Remover livre de germes animais de isoladores e abrigá-los em uma sala de criação em gaiolas convencionais equipados com filtro na frente dos animais convencionais, que servirão como controles (Figura 1). </li><li> Meia mistura do lixo (3 dias) retirado da gaiola de controle convencional, com a ninhada dos animais germ-free. Mantenha sempre 1 / 3 do lixo sujos convencionais cada vez que é necessário renová-lo, a fim de manter um nível de bact...

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Neste protocolo, descrevemos um procedimento colonização progressiva em um ambiente aberto para investigar o impacto da microbiota intestinal no metabolismo hepático avaliado por 1 H RMN HR MAS perfil da biópsia intacta. Vários métodos de colonização têm sido descritos na literatura. Os métodos mais comuns para colonizar animais com microbiota definida são gavagem oral ou água contaminada 19,20. Inoculação de fezes também pode ser usado como descrito anteriormente 21. O m?...

Tabela de reagentes e equipamentos específicos: <table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Microtubo 2,5 milímetros </td><td> Nova Era </td><td> NE-H5/2.5-V-Br </td><td></td></tr><tr><td> 1,7 milímetros tubo capilar </td><td> Sigma-Aldrich </td><td> NORS175001 </td><td></td></tr><tr><td> Adaptador capilar </td><td> Nova Era </td><td> NE-325-5/1.7 </td><td></td></tr><tr><td> Extração da haste </td><td> Nova Era </td><td> NE-341-5 </td><td></td></tr><tr><td> HR-MAS BL4 rotor com 50 mL kit espaçador esférica Teflon </td><td> Bruker </td><td> HZ07213 </td><td></td></tr><tr><td> Kit de ferramentas para 50 inserções mL </td><td> Bruker </td><td> B2950 </td><td></td></tr><tr><td> Antecedência III 600MHz NMR </td><td> Bruker </td><td></td><td></td></tr><tr><td> 1 H RMN HR MAS sonda sólida </td><td> Bruker </td><td></td><td></td></tr><tr><td> Óxido de deutério 99,9% </td><td> Sigma-Aldrich </td><td> 530867-1L </td><td></td></tr><tr><td> 3 - (trimetilsilil) propiônico ácido-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

É bem conhecido que as bactérias do intestino contribuir significativamente para a homeostase host, fornecendo uma gama de benefícios como a proteção imunológica e síntese de vitaminas. Eles também fornecem o host com uma quantidade considerável de nutrientes, fazendo com que este ecossistema um órgão metabólicas essenciais. No contexto da crescente evidência da relação entre a flora intestinal e síndrome metabólica, a compreensão da interação metabólica entre o anfitrião e sua microbiota intestinal está se tornando um desafio importante da biologia moderna. 1-4 Colonização (também referida como processo de normalização) designa o estabelecimento de micro-organismos em um animal livre de germes ex. Embora seja um processo natural que ocorre no nascimento, é também utilizado em adulto livre de germes animais para controlar o ecossistema intestinal floral e ainda determinar o seu impacto sobre o metabolismo hospedeiro. Um procedimento comum para controlar o processo de colonização é a utilização do método de gavagem com um single ou uma mistura de micro-organismos. Este método resulta em uma colonização muito rápido e apresenta a desvantagem de ser extremamente estressante 5. Portanto, é útil para minimizar o estresse e obter um processo mais lento de colonização para observar gradualmente o impacto da criação de bactérias sobre o metabolismo hospedeiro. Neste manuscrito, nós descrevemos um procedimento para avaliar a modificação do metabolismo hepático durante um processo de colonização gradual utilizando uma técnica não destrutiva de perfil metabólico. Propomos para monitorar a colonização microbiana do intestino através da avaliação da atividade metabólica microbiana do intestino refletida pela excreção urinária de microbiana co-metabólitos por um perfil H RMN baseado metabólica. Isto permite uma apreciação da estabilidade da atividade microbiana do intestino, além do estabelecimento estável do ecossistema microbiano do intestino geralmente avaliado pelo monitoramento bactérias fecais por DGGE (eletroforese em gel de gradiente de desnaturação). 6 Acolonização ocorre em um ambiente convencional aberta e é iniciada por uma maca, suja de animais convencionais, que servirá como controle. Roedores são animais coprófagos, isso garante uma colonização homogênea como descrito anteriormente. 7 Perfil metabólico hepático é medido diretamente a partir de uma biópsia do fígado intacto utilizando 1 H Angle Magia Alta Resolução Spinning espectroscopia de RMN. Esta técnica semi-quantitativa oferece uma forma rápida de avaliar, sem danificar a estrutura da célula, os principais metabolitos, tais como triglicérides, glicose e glicogênio, a fim de estimar mais a complexa interação entre o processo de colonização e do metabolismo hepático 7-10. Este método também pode ser aplicado a qualquer tecido biópsia 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

Um procedimento de colonização progressiva é descrito para melhor avaliar seu impacto sobre o metabolismo hepático host. Colonização é monitorado não invasiva através da avaliação da excreção urinária de microbiana co-metabólitos por RMN baseado em perfil metabólico, enquanto o metabolismo hepático é avaliado pela Alta Resolução Magia Angle Spinning (MAS HR) NMR perfil da biópsia intacta.

Avaliar as alterações metabólicas hepáticas Durante a colonização progressiva de Germ-free Mouse 1 H NMR Spectroscopy

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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

Deteção de colonização Enterohemorrhagic Escherichia Coli murino Host pelo sistema não-invasivo na Vivo bioluminescência

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