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

Dit werk werd ondersteund door subsidie ​​DK078732 van de NIH (naar PTF).

1. Adenovirale transductie en kweken van Rat eilandjes <ol><li> Bereid een 6-wells weefselkweekplaten niet beklede plaat door toevoeging van 2 ml medium (RPMI 1640 medium met 8 mM glucose, 10% foetaal runderserum, 50 eenheden / ml penicilline en 50 ng / ml streptomycine) de gewenste aantal putten. Bijvoorbeeld kan een typisch experiment nodig drie bronnen - een voor elke no-viruscontrole een viruscontrole (bijvoorbeeld GFP-expressie adenovirus) en de experimentele groep. </li><li> Verwarm de plaat 37 ° C door het in ...

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Vaststelling van trajecten waarmee die kunnen worden gegoten om de replicatie te stimuleren en de functie van beta-cellen te verbeteren van belang zijn voor zowel grote vormen van diabetes. Omdat de functionele beta cel massa is afhankelijk van het bestaan ​​en de functie van insuline-afscheidende cellen, de beoordeling van deze determinanten tegelijkertijd heeft zo zijn voordelen. Dit protocol beschrijft een gestroomlijnde protocol om te bepalen of overexpressie of onderdrukking van een eiwit leidt tot veranderinge...

<table border="1"><tbody><tr><td> Naam van het serum </td><td> Vennootschap </td><td> Catalogusnummer </td><td> Reacties </td></tr><tr><td> RPMI 1640 media </td><td> Gibco </td><td> 11879 </td><td></td></tr><tr><td> Penicilline / streptomycine </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> 6-well plaat </td><td> BD-Falcon </td><td> 35-1146 </td><td> Niet-behandelde TC </td></tr><tr><td> [Methyl-3H]-thymidine </td><td> Perkin Elmer </td><td> NET027Z001MC </td><td> 1 mCi / ml </td></tr><tr><td> Micro-centrifugebuizen </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> MgSÜ4 </td><td> Acros </td><td> 41348 </td><td></td></tr><tr><td> CaCl2 </td><td> Acros </td><td> 34961 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H0887 </td><td> 1 M oplossing </td></tr><tr><td> 35% BSA </td><td> Sigma </td><td> A7979 </td><td></td></tr><tr><td> NaHCÛ3 </td><td> Acros </td><td> 42427 </td><td></td></tr><tr><td> d-glucose </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> Scintillatietelling buis </td><td> Sarstedt </td><td> 58.536 </td><td> 7 ml, PP </td&gt; </tr><tr><td> Scintillatietelling dopje </td><td> Sarstedt </td><td> 65.816 </td><td></td></tr><tr><td> Econo-Safe tellen cocktail </td><td> RPI </td><td> 111175 </td><td></td></tr><tr><td> Insuline RIA </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> Uitrusting </td></tr><tr><td> Centrifugeer </td><td> Eppendorf </td><td> 5415R </td><td></td></tr><tr><td> Scintillatietelling buizenrek </td><td> Sarstedt </td><td> 93.1431.001 </td><td></td></tr><tr><td> Vloeistofscintillatieteller </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

Glucose-homeostase wordt voornamelijk bepaald door de endocriene hormonen insuline en glucagon, afgescheiden van de pancreatische beta-en alfa-cellen, respectievelijk. Functionele betacelmassa wordt bepaald door de anatomische betacelmassa alsmede het vermogen van de beta cellen reageren op een nutriënten. Een verlies van functionele beta cel massa staat centraal in zowel grote vormen van diabetes 1-3. Overwegende dat de dalende functionele beta cel massa het gevolg is van een auto-immuunziekte bij type 1 diabetes, bij type 2 diabetes, deze verlagen ontwikkelt zowel vanuit een onvermogen van bèta-cellen op de juiste insuline afscheiden en de vernietiging van de bètacellen van een kader van mechanismen. Zo, de inspanningen om functionele beta cel massa te herstellen zijn van cruciaal belang voor de betere behandeling van en de mogelijke behandelingen voor diabetes. Er wordt gewerkt aan moleculaire pathways die kan worden benut om de replicatie te stimuleren en verbeteren van de functie van beta-cellen te identificeren.Idealiter zou therapeutische doelen verbeteren, zowel beta cel groei en functie. Misschien nog belangrijker echter is vast te stellen of een strategie die beta celgroei stimuleert ten koste gaat van de verslechterende beta cel functie (zoals bij sommige oncogenen) en vice versa. Door systematisch te onderdrukken of overexpressie van de expressie van genen in geïsoleerde eilandjes rat kan men potentiële therapeutische doelen voor het verhogen van functionele bèta-celmassa 4-6. Adenovirale vectoren kan worden gebruikt om efficiënt overexpressie of knockdown eiwitten in geïsoleerde eilandjes rat 4,7-15. Hier presenteren we een methode om genexpressie gebruik te maken van adenovirale transductie te manipuleren en te beoordelen eilandje replicatie-en bèta-cel functie in geïsoleerde eilandjes rat (figuur 1). Deze methode is eerder gebruikt om nieuwe targets beta cel-replicatie of functie 5,6,8,9,16,17 moduleren te identificeren.

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

Dit protocol maakt het mogelijk factoren te identificeren die de functionele beta cel massa moduleren om potentiële therapeutische doelen voor de behandeling van diabetes te vinden. Het protocol bestaat uit een gestroomlijnde methode om eilandje replicatie-en bèta-cel functie te beoordelen in geïsoleerde eilandjes rat na manipulatie van genexpressie met adenovirussen.

Het beoordelen van replicatie en Beta cel functie in Adenovirally-getransduceerde Geïsoleerd Rodent Eilandjes

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

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