Name: assessing replication and beta cell function in adenovirally-transduced isolated rodent islets
Uploaded: 2012-06-25T13:00:00
Description: Watch this Scientific Journal Video about Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets at JoVE.com

Este trabalho foi apoiado pela concessão DK078732 do NIH (a FPT).

1. Transdução adenoviral e cultura de ilhotas de ratos <ol><li> Prepara-se uma placa de 6 poços de cultura de tecido não-revestido por adição de 2 ml de meio (RPMI 1640 contendo 8 mM de glucose, 10% de soro fetal bovino, 50 unidades / ml de penicilina, e 50 ug / ml de estreptomicina) para o número necessário de poços. Por exemplo, uma experiência típica pode exigir três poços - um para cada um controlo não-vírus, um controle de vírus (por exemplo, GFP-expressando adenovírus), eo grupo experimental. </...

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Vias, que estabelece que pode ser modulada para estimular a replicação e melhorar a função das células beta são relevantes para ambas as formas principais de diabetes. Devido massa celular funcional beta é dependente da existência e função de células secretoras de insulina, avaliando estes determinantes simultaneamente tem as suas vantagens. Este protocolo descreve um protocolo simplificado para identificar se a superexpressão ou supressão de uma proteína leva a alterações na massa funcional das células...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> RPMI 1640 meios </td><td> Gibco </td><td> 11879 </td><td></td></tr><tr><td> A penicilina / estreptomicina </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> 6-bem placa </td><td> BD-Falcon </td><td> 35-1146 </td><td> Non-TC tratada </td></tr><tr><td> [Metil-3 H]-timidina </td><td> Perkin Elmer </td><td> NET027Z001MC </td><td> 1 mCi / ml </td></tr><tr><td> Micro-centrífuga tubos </td><td> Denville </td><td> C2170 </td><td> 1,7 ml </td></tr><tr><td> NaCl </td><td> Sigma </td><td> 59888 </td><td></td></tr><tr><td> KCl </td><td> Acros </td><td> 42409 </td><td></td></tr><tr><td> KH 2 &lt;/ Sub&gt; PO 4 </td><td> Acros </td><td> 20592 </td><td></td></tr><tr><td> MgSO4 </td><td> Acros </td><td> 41348 </td><td></td></tr><tr><td> CaCl 2 </td><td> Acros </td><td> 34961 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H0887 </td><td> Solução 1 M </td></tr><tr><td> 35% de BSA </td><td> Sigma </td><td> A7979 </td><td></td></tr><tr><td> De NaHCO3 </td><td> Acros </td><td> 42427 </td><td></td></tr><tr><td> d-glicose </td><td> Sigma </td><td> G8769 </td><td></td></tr><tr><td> TCA </td><td> Fisher Scientific </td><td> SA9410-1 </td><td> 10% w / v </td></tr><tr><td> NaOH </td><td> Acros </td><td> 12426 </td><td></td></tr><tr><td> Contagem de cintilação tubo </td><td> Sarstedt </td><td> 58,536 </td><td> 7 ml, PP </td&gt; </tr><tr><td> Cintilação tampa do tubo de contagem </td><td> Sarstedt </td><td> 65,816 </td><td></td></tr><tr><td> Econo-Safe cocktail contando </td><td> RPI </td><td> 111175 </td><td></td></tr><tr><td> RIA Insulina </td><td> Siemens </td><td> TKIN2 </td><td></td></tr><tr><td> BCA Assay Kit </td><td> Thermo Scientific </td><td> 23250 </td><td></td></tr><tr><td></td><td></td><td></td><td> Equipamento </td></tr><tr><td> Centrifugar </td><td> Eppendorf </td><td> 5415R </td><td></td></tr><tr><td> Contagem de cintilação suporte de tubos </td><td> Sarstedt </td><td> 93.1431.001 </td><td></td></tr><tr><td> Contador de cintilação líquida </td><td> Perkin Elmer </td><td> Tri-Carb 2910TR </td><td></td></tr></tbody></table>

assessing replication and beta cell function in adenovirally-transduced isolated rodent islets

Homeostase da glucose é principalmente controlada pela insulina hormonas endócrinas e glucagon, secretada a partir da beta pancreáticas e células alfa, respectivamente. Massa celular funcional beta é determinado pela massa anatómica beta celular, bem como a capacidade das células beta para responder a uma carga de nutriente. Uma perda de massa funcional das células beta é central para ambas as formas principais de diabetes 1-3. Considerando que as declínio da capacidade funcional das células beta resultados de massa de um ataque auto-imune em pacientes com diabetes tipo 1, diabetes tipo 2, este decréscimo se desenvolve tanto uma incapacidade das células beta a secretar insulina de forma apropriada e destruição das células beta de um quadro de mecanismos. Assim, os esforços para restabelecer a massa de células beta funcional são fundamentais para o melhor tratamento e possíveis curas para o diabetes. Esforços estão em andamento para identificar caminhos moleculares que podem ser exploradas para estimular a replicação e melhorar a função das células beta.Idealmente, alvos terapêuticos iria melhorar tanto o crescimento das células beta e função. Talvez o mais importante é que para identificar se uma estratégia que estimula o crescimento das células beta está no custo da função das células beta prejudicando (tal como com alguns oncogenes) e vice-versa. Sistematicamente suprimindo ou superexpressão de expressão de genes-alvo em ilhéus isolados de ratos, é possível identificar potenciais alvos terapêuticos para o aumento da massa funcional das células beta 4-6. Vectores adenovirais podem ser empregues para proteínas eficientemente superexpressam ou knockdown em ilhéus isolados de ratos 4,7-15. Aqui, nós apresentamos um método para manipular a expressão do gene utilizando transdução adenoviral e avaliar a replicação de ilhotas e função das células beta em ilhéus isolados de ratos (Figura 1). Este método tem sido utilizado anteriormente para identificar novos alvos que modulam a replicação das células beta ou função 5,6,8,9,16,17.

Watch this Scientific Journal Video about Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets at JoVE.com

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Este protocolo permite uma para identificar factores que modulam a massa de células funcional de beta para encontrar potenciais alvos terapêuticos para o tratamento da diabetes. O protocolo é constituído por um método simplificado para avaliar a replicação de ilhotas e função das células beta em ilhéus isolados de ratos após a manipulação da expressão do gene com adenovírus.

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