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

Detta projekt finansieras genom av NIH / NCI licensnummer R00 CA127357 och University of Kansas Cancer Center Endowment.

1. Isolering och utvinning av givare kollagen från C57BL/6J möss <ol><li> Offra mogna normala kvinnliga C57BL/6J möss med godkända IACUC metoder. </li><li> Skörda svansar och njuta i 70% etanol i 45 minuter för att sterilisera vävnader. Torka svansar med silkespapper, linda i aluminiumfolie. Förvara svansar i -20 ° C tills det behövs. </li><li> Placera svansar i en steril miljö som ett laminärt flöde huva. Använda sax, dela huden vid svansroten och dra bort från svansen. Ta bort från 0,5 till 1 cm frå...

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Den funktionella bidrag fibroblaster i tumörprogression har visats genom transplantation modeller, där cancer förknippas fibroblaster samarbete transplanteras med godartade bröst epitelceller leder till ökad tumörtillväxt och invasiv 5. Konventionella transplantation metoder har inblandade användning av SCID eller nakna möss att samarbeta transplantation stromaceller och epitelceller från olika genetiska mus bakgrunder eller olika arter. Nedsatt immunförsvar möss saknar fungerande T-celler, som sp...

<table border="1"><tbody><tr><th> Reagensets namn </th><th> Företag </th><th> Katalognummer </th><th> Kommentarer (frivilligt) </th></tr><tr><td> C57BL/6N möss </td><td> Harlan </td><td> N / A </td><td></td></tr><tr><td> MMTV-PyVmT transgena möss </td><td> Jackson laboratorier </td><td> 002.374 </td><td></td></tr><tr><td> Fetalt bovinserum </td><td> Fisher </td><td> SH3039603PR </td><td></td></tr><tr><td> DMEM </td><td> VWR </td><td> 10000113873 </td><td></td></tr><tr><td> Penicillin / streptomycin </td><td> Fisher </td><td> MT-30 till 001 </td><td></td></tr><tr><td> amfotericin </td><td> fisher </td><td> BP2645-20 </td><td></td></tr><tr><td> Amicon filtrering kolumner ultracel 50k </td><td> Millipore </td><td> UFC905008 </td><td></td></tr><tr><td> Rör för Beckman TI rotor </td><td> Beckman </td><td> 355.618 </td><td></td></tr><tr><td> Rat svans kollagen </td><td> Fisher </td><td> CB 40.236 </td><td></td></tr><tr><td> 10x EBSS </td><td> Sigma-Aldrich </td><td> E7510-100 ml </td><td></td></tr><tr><td> Trypsin 1X, 0,25% i HBSS w / o Kalcium och Magnesium </td><td> Fisher </td><td> MT-25-050-CI </td><td></td></tr><tr><td> Isättika </td><td> Fisher </td><td> A491-212 </td><td></td></tr><tr><td> Coomasie blå </td><td> Fisher </td><td> BP101 25 </td><td></td></tr><tr><td> Trypsin </td><td> Sigma-Aldrich </td><td> T3924-100ml </td><td></td></tr><tr><td> Kollagenas A </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> DNas </td><td> Sigma-Aldrich </td><td> D5025 </td><td></td></tr><tr><td> Kalejdoskop Protein standard </td><td> Biorad </td><td> 1610375 </td><td></td></tr><tr><td> Glas diabilder </td><td> Fisher </td><td> 12.545-78 </td><td></td></tr><tr><td> Glas täckglas </td><td> VWR </td><td> 101400-042 </td><td></td></tr><tr><td> Vimentin antikropp S-20 </td><td> Santa Cruz Biotechnology </td><td> SC-7558 </td><td></td></tr><tr><td> α-smooth muscle aktin antikropp </td><td> Abcam </td><td> ab5694 </td><td></td></tr><tr><td> CK14 antikropp </td><td> Santa Cruz Biotechnology </td><td> sc-53.253 </td><td></td></tr><tr><td> CK18 antikropp </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-mus biotinylerad </td><td> Vector laboratorier </td><td> BA9200 </td><td> Distribueras via Fisher </td></tr><tr><td> Anti-mus-Alexa-568 </td><td> Invitrogen </td><td> A10037 </td><td></td></tr><tr><td> Anti-mus-Alexa-488 </td><td> Invitrogen </td><td> A11001 </td><td></td></tr><tr><td> Streptavidin-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> Förläng antifade </td><td> Invitrogen </td><td> P-36.930 </td><td></td></tr><tr><td> Kirurgiska saxar </td><td> Fine Science Tools </td><td> 91.400-12 </td><td></td></tr><tr><td> Fin våren sax </td><td> Fine Science Tools </td><td> 15.000-02 </td><td></td></tr><tr><td> Blunt pincett </td><td> Fine Science Tools </td><td> 11.002-12 </td><td></td></tr><tr><td> # 5 fin pincett </td><td> Fine Science Tools </td><td> 11.251-10 </td><td></td></tr><tr><td> Gut kromsyra sutur </td><td> Fisher </td><td> NC9326254 </td><td></td></tr><tr><td> Glas Pasteurpipett </td><td> Fisher </td><td> 22-042-815 </td><td></td></tr><tr><td> Etanol </td><td> Fisher </td><td> A406P 4 </td><td></td></tr><tr><td> Betadine </td><td> fisher </td><td> NC9386574 </td><td></td></tr><tr><td> Sår klipp </td><td> Fisher </td><td> 12.032-07 </td><td></td></tr><tr><td> Sår krampa </td><td> Fisher </td><td> 12.031-07 </td><td></td></tr></tbody></table>

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

Inverkan av stromaceller, bland andra fibroblaster på bröst tumörprogression är väl dokumenterade med hjälp av musmodeller, särskilt genom transplantation av stromaceller och epitelceller i mjölkkörtlar hos möss. Aktuella transplantation modeller innebär ofta användning av nedsatt immunförsvar möss på grund av olika genetiska bakgrunder stromaceller och epitelceller. Extracellulär matriser används ofta för att bädda in två olika celltyper för konsekvent cell-cell interaktioner, men omfattar användning av Matrigel eller råtta svans kollagen, som är immunogena substrat. Avsaknaden av fungerande T-celler från immunsupprimerade möss förhindrar korrekt bedömning av stromaceller om juvertumör progression in vivo, med viktiga konsekvenser för läkemedelsutveckling och effekt. Dessutom nedsatt immunförsvar möss är dyra, svåra att föda upp och kräver särskild omsorg villkor. För att övervinna dessa hinder, har vi utvecklat ett förhållningssätt till orthotopically transplantation stromaceller celler och epitelceller i möss med samma genetiska bakgrunden för att framkalla konsekvent tumörbildning. Detta system innebär avverkning normalt, cancer förknippas fibroblaster, PyVmT bröst celler cancer och kollagen från givare C57BL/6J möss. Cellerna är sedan inbäddade i kollagen och transplanteras i inguinal mjölkkörtlar hos kvinnliga C57BL/6J möss. Transplantation av PyVmT celler ensam bilda palpabla tumörer 30-40 dagar efter transplantation. Endpoint analys på 60 dagar visar att co-transplantation med fibroblaster ökar juvertumör tillväxt jämfört med PyVmT celler transplanterade ensam. Medan celler och matris från C57BL/6J möss användes i dessa studier, kan isolering av celler och matrix och transplantation tillvägagångssätt användas mot möss med olika genetisk bakgrund visar mångsidighet. Sammanfattningsvis kan detta system användas för att undersöka molekylära interaktioner mellan stromaceller och epitelceller, och övervinner kritiska begränsningar hos immunsupprimerade musmodeller.

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

I den här rapporten visar vi ett system för att isolera och odla celler givare från mus juvret och orthotopically transplantera dessa celler i mottagarländerna möss att analysera stromaceller: epiteliala interaktioner under juvertumör utveckling.

Bröst Transplantation av stromaceller och celler carcinoma in C57BL/6J Möss

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

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