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

Bu çalışma NIH (PTF kadar) hibe DK078732 tarafından desteklenmiştir.

1. Sıçan Adacıklar ve adenoviral İletimi ve Kültürleme <ol><li> Gerekli sayıda ortam, 2 ml (8 mM glikoz,% 10 fetal bovin serumu, 50 ünite / ml penisilin ve 50 ug / ml streptomisin içeren RPMI 1640 ortamı) ilave edilerek bir 6-sıra non-doku kültürü plakası kaplamalı hazırlamak kuyu. No-virüsü kontrolü için tek her biri, bir virüs kontrolü (örn., GFP-eksprese adenovirüs) ve deney grubu - Örneğin, tipik bir deneyde, üç kuyu gerektirebilir. </li><li> En azından 30 dakika için bir doku kült?...

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Çoğaltma teşvik ve beta hücrelerinin işlevini geliştirmek için modüle olabilir kurulması yollarının her iki diyabet büyük formlar alakalı. Işlevsel beta hücre kitlesi bu belirleyicileri değerlendirmek, insülin salgılayan hücrelerin varlığı ve fonksiyonu bağlı olduğundan aynı anda avantajları vardır. Bu protokolün bir proteini aşırı ekspresyonu ya da bastırma sonra in vivo etki için test edilebilir in vitro olarak fonksiyonel beta hücre kütlesi değişikliklere yol a...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> RPMI 1640 ortamı </td><td> Gibco </td><td> 11879 </td><td></td></tr><tr><td> Penisilin / streptomisin </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> 6-iyi plaka </td><td> BD-Falcon </td><td> 35-1146 </td><td> Sigara TC tedavi </td></tr><tr><td> [Metil-3 H] timidin- </td><td> Perkin Elmer </td><td> NET027Z001MC </td><td> 1 mCi / ml </td></tr><tr><td> Mikro-santrifüj tüpü </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> MgSO 4 </td><td> Acros </td><td> 41348 </td><td></td></tr><tr><td> CaCI2 </td><td> Acros </td><td> 34961 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H0887 </td><td> 1 M solüsyon </td></tr><tr><td> % 35 BSA </td><td> Sigma </td><td> A7979 </td><td></td></tr><tr><td> NaHCO 3 </td><td> Acros </td><td> 42427 </td><td></td></tr><tr><td> D-glukoz </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> Sintilasyon tüp sayma </td><td> Sarstedt </td><td> 58.536 </td><td> 7 ml, PP </td&gt; </tr><tr><td> Sintilasyon sayma tüp kap </td><td> Sarstedt </td><td> 65.816 </td><td></td></tr><tr><td> Econo-Güvenli sayma kokteyl </td><td> RPI </td><td> 111175 </td><td></td></tr><tr><td> İnsülin RIA </td><td> Siemens </td><td> TKIN2 </td><td></td></tr><tr><td> BCA Test Kiti </td><td> Thermo Scientific </td><td> 23250 </td><td></td></tr><tr><td></td><td></td><td></td><td> Ekipman </td></tr><tr><td> Santrifüj </td><td> Eppendorf </td><td> 5415R </td><td></td></tr><tr><td> Sintilasyon tüp raf sayma </td><td> Sarstedt </td><td> 93.1431.001 </td><td></td></tr><tr><td> Sıvı sintilasyon sayacı </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

Glikoz homeostazı, öncelikle, sırasıyla, pankreas beta ve alfa hücrelerden salgılanan, endokrin hormonlar insülin ve glukagon tarafından kontrol edilir. Fonksiyonel beta hücre kütlesi anatomik beta hücre kütlesi gibi bir besin yük yanıt beta hücrelerinin yeteneği ile belirlenir. Işlevsel beta hücre kitlesi kaybı diyabet 1-3 iki büyük formlar merkezidir. Tip 1 diyabetin otoimmün bir saldırı azalan işlevsel beta hücre kitlesi sonuçları ise, tip 2 diyabet, bu eksiltme uygun insülin salgılamaya beta hücrelerinin bir yetersizlik ve mekanizmalar bir kadro beta hücrelerinin yok hem de gelişir. Böylece, işlevsel beta hücre kitlesi geri çabaları daha iyi tedavi ve diyabet için potansiyel tedavileri için her şeyden vardır. Çabaları çoğaltma teşvik ve beta hücrelerinin işlevini geliştirmek için kullanılabilir moleküler yolları belirlemek için çalışmalar devam etmektedir.İdeal olarak, tedavi hedefleri beta hücre büyümesi ve fonksiyonu hem de artıracak. Ancak belki de daha önemli beta hücre büyümesini uyarır bir strateji zedeleyen beta hücre fonksiyonu (bazı onkogenler olduğu gibi) ve tam tersi pahasına geliyor olmadığını belirlemektir. Izole sıçan adacıklar hedef genlerin sistematik baskılayarak veya aşırı ifadesi olarak, bir artan işlevsel beta hücre kitlesi 4-6 için potansiyel tedavi hedefleri belirleyebilir. Adenoviral vektörler izole sıçan adacıklar 4,7-15 verimli olarak artmış veya demonte proteinlerine istihdam edilebilir. Burada, adenoviral transdüksiyon kullanarak gen ekspresyon işlemek ve izole sıçan adacıklar (Şekil 1) adacık çoğaltma ve beta hücre fonksiyonu değerlendirmek için bir yöntem sunuyoruz. Bu yöntem, beta hücre çoğaltma veya fonksiyon 5,6,8,9,16,17 modüle yeni hedefler belirlemek için daha önce kullanılmıştır.

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

Bu protokol bir diyabet tedavisi için terapötik hedefleri bulmak için işlevsel beta hücre kitlesi modüle faktörleri belirlemek sağlar. Protokolü adenovirüsler ile gen ekspresyon manipülasyonu takiben izole sıçan adacıklarında adacık çoğaltma ve beta hücre fonksiyonu değerlendirmek için modern bir yöntem oluşur.

Çoğaltma ve Beta Hücre Fonksiyonu değerlendirilmesi Adenovirally-transdük İzole Kemirgen Islets

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