Name: mammary transplantation of stromal cells and carcinoma cells in c57bl/6j mice
Uploaded: 2011-08-12T12:00:00
Description: Watch this Scientific Journal Video about Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice at JoVE.com

Este projecto foi financiado através de NIH / NCI número de concessão R00 CA127357 e da Universidade de Kansas Cancer Center Endowment.

1. Isolamento e extração de colágeno de doadores de camundongos C57BL/6J <ol><li> Sacrifício madura fêmeas normais C57BL/6J usando métodos aprovados IACUC. </li><li> Colher as caudas e mergulhar em etanol 70% por 45 minutos para esterilizar tecidos. Seque o rabo com papel de seda, embrulhe em papel alumínio. Armazenar os rabos em -20 ° C até ser necessário. </li><li> Coloque o rabo em um ambiente estéril, como uma capela de fluxo laminar. Com uma tesoura, divida a pele na raiz da cauda e se desfazer da caud...

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A contribuição funcional de fibroblastos na progressão tumoral tem sido demonstrado através de modelos de transplantes, em que os fibroblastos carcinoma associados co-transplantados com resultados benignos mamários células epiteliais no crescimento do tumor aumentou e invasão 5. Abordagens transplante convencional envolveram o uso de camundongos SCID ou nuas para co-transplante de células do estroma e do epitélio de diferentes origens genéticas do rato ou de espécies diferentes. Ratinhos imunocompr...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Camundongos C57BL/6N </td><td> Harlan </td><td> N / A </td><td></td></tr><tr><td> MMTV-PyVmT camundongos transgênicos </td><td> Jackson laboratórios </td><td> 002374 </td><td></td></tr><tr><td> Soro fetal bovino </td><td> Pescador </td><td> SH3039603PR </td><td></td></tr><tr><td> DMEM </td><td> VWR </td><td> 10000113873 </td><td></td></tr><tr><td> Penicilina / estreptomicina </td><td> Pescador </td><td> MT-30-001 </td><td></td></tr><tr><td> anfotericina </td><td> pescador </td><td> BP2645-20 </td><td></td></tr><tr><td> Filtração Amicon colunas ultracel 50k </td><td> Millipore </td><td> UFC905008 </td><td></td></tr><tr><td> Tubos para rotor Beckman TI </td><td> Beckman </td><td> 355618 </td><td></td></tr><tr><td> Colágeno da cauda de rato </td><td> Pescador </td><td> CB 40.236 </td><td></td></tr><tr><td> EBSS 10x </td><td> Sigma Aldrich </td><td> E7510-100ML </td><td></td></tr><tr><td> Tripsina 1X, 0,25% em HBSS cálcio w / o e Magnésio </td><td> Pescador </td><td> MT-25-050-CI </td><td></td></tr><tr><td> Ácido acético glacial </td><td> Pescador </td><td> A491-212 </td><td></td></tr><tr><td> Coomasie azul </td><td> Pescador </td><td> BP101 25 </td><td></td></tr><tr><td> Tripsina </td><td> Sigma Aldrich </td><td> T3924-100 ml </td><td></td></tr><tr><td> A colagenase </td><td> Sigma AldrichC0130-50 </td><td></td><td></td></tr><tr><td> hyalronidase </td><td> Sigma Aldrich </td><td> H3884 </td><td></td></tr><tr><td> DNase </td><td> Sigma Aldrich </td><td> D5025 </td><td></td></tr><tr><td> Padrão de proteínas caleidoscópio </td><td> Biorad </td><td> 1610375 </td><td></td></tr><tr><td> Lâminas de vidro </td><td> Pescador </td><td> 12545-78 </td><td></td></tr><tr><td> Lamínulas de vidro </td><td> VWR </td><td> 101400-042 </td><td></td></tr><tr><td> Vimentina anticorpo S-20 </td><td> Santa Cruz Biotechnology </td><td> SC-7558 </td><td></td></tr><tr><td> α-actina de músculo liso de anticorpos </td><td> Abcam </td><td> ab5694 </td><td></td></tr><tr><td> Anticorpo CK14 </td><td> Santa Cruz Biotechnology </td><td> sc-53253 </td><td></td></tr><tr><td> Anticorpo CK18 </td><td> Abcam </td><td> ab668 </td><td></td></tr><tr><td> DAPI </td><td> Sigma Aldrich </td><td> D9542 </td><td></td></tr><tr><td> Anti-rato biotinilado </td><td> Vector laboratórios </td><td> BA9200 </td><td> Distribuídos através de Fisher </td></tr><tr><td> Anti-mouse-alexa-568 </td><td> Invitrogen </td><td> A10037 </td><td></td></tr><tr><td> Anti-mouse-alexa-488 </td><td> Invitrogen </td><td> A11001 </td><td></td></tr><tr><td> Estreptavidina-alexa-488 </td><td> Invitrogen </td><td> S11226 </td><td></td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D21490 </td><td></td></tr><tr><td> Prolongar Antifade </td><td> Invitrogen </td><td> P-36930 </td><td></td></tr><tr><td> Tesoura cirúrgica </td><td> Multa Ferramentas Ciência </td><td> 91400-12 </td><td></td></tr><tr><td> Multa tesoura primavera </td><td> Multa Ferramentas Ciência </td><td> 15000-02 </td><td></td></tr><tr><td> Blunt forceps </td><td> Multa Ferramentas Ciência </td><td> 11002-12 </td><td></td></tr><tr><td> # 5 pinça fina </td><td> Multa Ferramentas Ciência </td><td> 11251-10 </td><td></td></tr><tr><td> Sutura crômico Gut </td><td> Pescador </td><td> NC9326254 </td><td></td></tr><tr><td> Pipeta Pasteur de vidro </td><td> Pescador </td><td> 22-042-815 </td><td></td></tr><tr><td> Etanol </td><td> Pescador </td><td> A406P 4 </td><td></td></tr><tr><td> betadine </td><td> pescador </td><td> NC9386574 </td><td></td></tr><tr><td> Grampos de sutura </td><td> Pescador </td><td> 12032-07 </td><td></td></tr><tr><td> Grampo ferida </td><td> Pescador </td><td> 12031-07 </td><td></td></tr></tbody></table>

mammary transplantation of stromal cells and carcinoma cells in c57bl/6j mice

A influência das células do estroma, incluindo fibroblastos na progressão do tumor mamário tem sido bem documentada por meio do uso de modelos de mouse, em particular através de transplante de células do estroma e células epiteliais na glândula mamária de camundongos. Modelos atuais de transplante freqüentemente envolvem o uso de camundongos imunocomprometidos devido à diferentes origens genéticas das células do estroma e células epiteliais. Matrizes extracelulares são muitas vezes utilizados para incorporar os dois tipos de células diferentes para consistentes interações célula-célula, mas envolvem o uso de Matrigel ou colágeno da cauda de rato, que são substratos imunogênica. A falta de funcionais de células T de camundongos imunocomprometidos impede uma avaliação precisa das células estromais sobre a progressão do tumor mamário in vivo, com importantes implicações no desenvolvimento de medicamentos e eficácia. Além disso, os camundongos imunocomprometidos são caras, difíceis de produzir e requerem condições de cuidados especiais. Para superar estes obstáculos, temos desenvolvido uma abordagem para transplante de células do estroma ortotopicamente e células epiteliais em camundongos da mesma origem genética para induzir a formação de tumor consistente. Este sistema envolve a coleta normal, os fibroblastos carcinoma associados, PyVmT células de carcinoma de mama e de colágeno de camundongos C57BL/6J doador. As células são então incorporados em colágeno e transplantadas nas glândulas mamárias inguinais de camundongos fêmeas C57BL/6J. Transplante de células PyVmT sozinho formar tumores palpáveis ​​pós-transplante 30-40 dias. Análise Endpoint menos 60 dias indica que co-transplante com fibroblastos aumenta o crescimento do tumor mamário em comparação com as células transplantadas PyVmT sozinho. Enquanto as células e matriz de camundongos C57BL/6J foram utilizadas nesses estudos, o isolamento de células e matriz e abordagem transplante pode ser aplicado para ratos de diferentes origens genéticas demonstrando versatilidade. Em resumo, este sistema pode ser usado para investigar as interações moleculares entre as células do estroma e células epiteliais, e supera as limitações críticos em modelos de ratos imunocomprometidos.

Watch this Scientific Journal Video about Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice at JoVE.com

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

Neste relatório, nós demonstramos um sistema para isolar e cultura de células de doadores da glândula mamária do mouse, e ortotopicamente transplante dessas células em ratos para analisar estroma destinatário: interações epiteliais durante o desenvolvimento do tumor mamário.

Transplante mamária de células estromais e células de carcinoma em camundongos C57BL/6J

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