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

Dit project werd gefinancierd door middel van door NIH / NCI verlenen nummer R00 CA127357 en de Universiteit van Kansas Cancer Center Endowment.

1. Isolatie en extractie van donor collageen uit C57BL/6J muizen <ol><li> Offer volwassen normale vrouwelijke C57BL/6J muizen met behulp van erkende IACUC methoden. </li><li> Oogst de staarten en genieten in 70% ethanol gedurende 45 minuten aan weefsels steriliseren. Droog de staarten met vloeipapier, wikkel in aluminiumfolie. Bewaar de staarten in de -20 ° C totdat het nodig is. </li><li> Leg de staarten in een steriele omgeving, zoals een laminaire stroming kap. Met een schaar, splitsing van de huid op de staart wo...

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	<li>Hayward, S., Haughney, P. C., Rosen, M. A., Greulich, K. M., Weier, H. U., Dahiya, R., Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+adult+human+prostatic+epithelium+and+rat+urogenital+sinus+mesenchyme+in+a+tissue+recombination+model.">Interactions between adult human prostatic epithelium and rat urogenital sinus mesenchyme in a tissue recombination model.</a> Differentiation. 63, 131-131 (1998).</li><li>Cunha, G., Hom, Y. K., Young, P., Brody, J., Asch, B. B., Ip, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Mammary Gland Biology. , (2000).</li><li>Ethier, S. P., Ammerman, C. A., Dziubinski, M. L., Asch, B. B., Ip, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Mammary Gland Biology. , (2000).</li><li>Medina, D., Kittrell, F., Asch, B. B., Ip, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Mammary Gland Biology. , (2000).</li><li>Kalluri, R., Zeisberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+in+cancer.">Fibroblasts in cancer.</a> Nat Rev Cancer. 6, 392-401 (2006).</li><li>DeNardo, D. G., Johansson, M., Coussens, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cells+as+mediators+of+solid+tumor+metastasis.">Immune cells as mediators of solid tumor metastasis.</a> Cancer Metastasis. 27, 11-18 (2008).</li><li>Naito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+differentiation+and+function+in+health+and+disease.">Macrophage differentiation and function in health and disease.</a> Pathol Int. 58, 143-155 (2008).</li><li>Firestein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+T+cell+cometh:+interplay+between+adaptive+immunity+and+cytokine+networks+in+rheumatoid+arthritis.">The T cell cometh: interplay between adaptive immunity and cytokine networks in rheumatoid arthritis.</a> J Clin Invest. 114, 471-474 (2004).</li><li>Cheng, N., Chytil, A., Shyr, Y., Joly, A., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Hepatocyte+Growth+Factor+Signaling+by+Type+II+Transforming+Growth+Factor-{beta}+Receptor+Knockout+Fibroblasts+Promotes+Mammary+Tumorigenesis.">Enhanced Hepatocyte Growth Factor Signaling by Type II Transforming Growth Factor-{beta} Receptor Knockout Fibroblasts Promotes Mammary Tumorigenesis.</a> Cancer Res. 67, 4869-4877 (2007).</li><li>Qiu, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+expression+profiling+identifies+signatures+of+tumor+virulence+in+MMTV-PyMT-transgenic+mice:+correlation+to+human+disease.">Global expression profiling identifies signatures of tumor virulence in MMTV-PyMT-transgenic mice: correlation to human disease.</a> Cancer Res. 64, 5973-5981 (2004).</li><li>Schaffhausen, B. S., Roberts, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+polyoma+middle+T+antigen+on+signaling+and+transformation:+A+DNA+tumor+virus+contribution+to+the+war+on+cancer.">Lessons from polyoma middle T antigen on signaling and transformation: A DNA tumor virus contribution to the war on cancer.</a> Virology. 384, 304-316 (2009).</li><li>Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+neural+cells+via+retrovirus-mediated+oncogene+transduction.">Immortalization of neural cells via retrovirus-mediated oncogene transduction.</a> Annu Rev Neurosci. 12, 47-65 (1989).</li><li>O'Hare, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+freshly+isolated+human+mammary+fibroblasts+and+endothelial+cells.">Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.</a> Proc Natl Acad Sci U S A. 98, 646-651 (2001).</li><li>Shay, J. W., Wright, W. E., Werbin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+molecular+mechanisms+of+human+cell+immortalization.">Defining the molecular mechanisms of human cell immortalization.</a> Biochim Biophys Acta. 1072, 1-7 (1991).</li><li>Gudjonsson, T., Villadsen, R., Ronnov-Jessen, L., Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+protocols+used+in+cell+culture+models+of+human+breast+morphogenesis.">Immortalization protocols used in cell culture models of human breast morphogenesis.</a> Cell Mol Life Sci. 61, 2523-2534 (2004).</li><li>Raschke, W. C., Baird, S., Ralph, P., Nakoinz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+macrophage+cell+lines+transformed+by+Abelson+leukemia+virus.">Functional macrophage cell lines transformed by Abelson leukemia virus.</a> Cell. 15, 261-267 (1978).</li><li>Shen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+endothelial+cells+differentiated+from+mouse+embryonic+stem+cells.">Immortalization of endothelial cells differentiated from mouse embryonic stem cells.</a> Shi Yan Sheng Wu Xue Bao. 35, 218-228 (2002).</li><li>Wang, S. J., Greer, P., Auerbach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+propagation+of+yolk-sac-derived+endothelial+cells+from+a+hypervascular+transgenic+mouse+expressing+a+gain-of-function+fps/fes+proto-oncogene.">Isolation and propagation of yolk-sac-derived endothelial cells from a hypervascular transgenic mouse expressing a gain-of-function fps/fes proto-oncogene.</a> In Vitro Cell Dev Biol Anim. 32, 292-299 (1996).</li><li>Tiede, B. J., Owens, L. A., Li, F., DeCoste, C., Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mouse+model+for+non-invasive+single+marker+tracking+of+mammary+stem+cells+in+vivo+reveals+stem+cell+dynamics+throughout+pregnancy.">A novel mouse model for non-invasive single marker tracking of mammary stem cells in vivo reveals stem cell dynamics throughout pregnancy.</a> PLoS One. 4, e8035-e8035 (2009).</li><li>Guzman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracarotid+injection+of+fluorescence+activated+cell-sorted+CD49d-positive+neural+stem+cells+improves+targeted+cell+delivery+and+behavior+after+stroke+in+a+mouse+stroke+model.">Intracarotid injection of fluorescence activated cell-sorted CD49d-positive neural stem cells improves targeted cell delivery and behavior after stroke in a mouse stroke model.</a> Stroke. 39, 1300-1306 (2008).</li><li>Duda, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+transplantability+of+tumor-associated+stromal+cells.">Differential transplantability of tumor-associated stromal cells.</a> Cancer Res. 64, 5920-5924 (2004).</li></ol>

De functionele bijdrage van fibroblasten in tumorprogressie is aangetoond door middel van transplantatie-modellen, waarbij carcinoom geassocieerd fibroblasten co-getransplanteerd met goedaardige mamma-epitheliale cellen resulteert in een verhoogde groei van de tumor en invasiviteit 5. Conventionele transplantatie benaderingen hebben betrokken van het gebruik van SCID muizen of naakt aan co-transplantatie stromale en epitheelcellen van verschillende genetische muis achtergronden of verschillende soorten. Immuu...

<table border="1"><tbody><tr><th> Naam van het serum </th><th> Vennootschap </th><th> Catalogus nummer </th><th> Commentaar (optioneel) </th></tr><tr><td> C57BL/6N muizen </td><td> Harlan </td><td> N / A </td><td></td></tr><tr><td> MMTV-PyVmT transgene muizen </td><td> Jackson laboratoria </td><td> 002374 </td><td></td></tr><tr><td> Foetaal runderserum </td><td> Visser </td><td> SH3039603PR </td><td></td></tr><tr><td> DMEM </td><td> VWR </td><td> 10000113873 </td><td></td></tr><tr><td> Penicilline / streptomycine </td><td> Visser </td><td> MT-30-001 </td><td></td></tr><tr><td> amfotericine </td><td> visser </td><td> BP2645-20 </td><td></td></tr><tr><td> Amicon filtratie columns ultracel 50k </td><td> Millipore </td><td> UFC905008 </td><td></td></tr><tr><td> Buizen voor Beckman TI rotor </td><td> Beckman </td><td> 355618 </td><td></td></tr><tr><td> Rattenstaart collageen </td><td> Visser </td><td> CB 40236 </td><td></td></tr><tr><td> 10x EBSS </td><td> Sigma Aldrich </td><td> E7510-100ML </td><td></td></tr><tr><td> Trypsine 1X, 0,25% in HBSS w / o Calcium en Magnesium </td><td> Visser </td><td> MT-25-050-CI </td><td></td></tr><tr><td> Ijsazijn </td><td> Visser </td><td> A491-212 </td><td></td></tr><tr><td> Coomasie blauw </td><td> Visser </td><td> BP101 25 </td><td></td></tr><tr><td> Trypsine </td><td> Sigma Aldrich </td><td> T3924-100ml </td><td></td></tr><tr><td> Collagenase 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> DNase </td><td> Sigma Aldrich </td><td> D5025 </td><td></td></tr><tr><td> Kaleidoscope Protein standaard </td><td> Biorad </td><td> 1610375 </td><td></td></tr><tr><td> Objectglaasjes </td><td> Visser </td><td> 12545-78 </td><td></td></tr><tr><td> Dekglaasjes </td><td> VWR </td><td> 101400-042 </td><td></td></tr><tr><td> Vimentin antilichaam S-20 </td><td> Santa Cruz Biotechnology </td><td> SC-7558 </td><td></td></tr><tr><td> α-gladde spieren actine antilichaam </td><td> Abcam </td><td> ab5694 </td><td></td></tr><tr><td> CK14 antilichaam </td><td> Santa Cruz Biotechnology </td><td> sc-53253 </td><td></td></tr><tr><td> CK18 antilichaam </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-muis gebiotinyleerde </td><td> Vector laboratoria </td><td> BA9200 </td><td> Gedistribueerd via Fisher </td></tr><tr><td> Anti-muis-alexa-568 </td><td> Invitrogen </td><td> A10037 </td><td></td></tr><tr><td> Anti-muis-alexa-488 </td><td> Invitrogen </td><td> A11001 </td><td></td></tr><tr><td> Streptavidine-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> Verleng antifade </td><td> Invitrogen </td><td> P-36930 </td><td></td></tr><tr><td> Chirurgische schaar </td><td> Fijn Science Tools </td><td> 91400-12 </td><td></td></tr><tr><td> Mooie voorjaarsdag schaar </td><td> Fijn Science Tools </td><td> 15000-02 </td><td></td></tr><tr><td> Blunt pincet </td><td> Fijn Science Tools </td><td> 11002-12 </td><td></td></tr><tr><td> # 5 fijn pincet </td><td> Fijn Science Tools </td><td> 11251-10 </td><td></td></tr><tr><td> Gut chroomzuur hechtdraad </td><td> Visser </td><td> NC9326254 </td><td></td></tr><tr><td> Glas Pasteur pipet </td><td> Visser </td><td> 22-042-815 </td><td></td></tr><tr><td> Ethanol </td><td> Visser </td><td> A406P 4 </td><td></td></tr><tr><td> Betadine </td><td> visser </td><td> NC9386574 </td><td></td></tr><tr><td> Wond clips </td><td> Visser </td><td> 12032-07 </td><td></td></tr><tr><td> Wond nietje </td><td> Visser </td><td> 12031-07 </td><td></td></tr></tbody></table>

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

De invloed van stromale cellen, waaronder fibroblasten op borstkanker progressie is goed gedocumenteerd door het gebruik van muismodellen, in het bijzonder door middel van transplantatie van stromale cellen en epitheelcellen in de uier van de muizen. Huidige transplantatie modellen vaak gepaard met het gebruik van muizen immuun te wijten aan de verschillende genetische achtergronden van stromale cellen en epitheelcellen. Extracellulaire matrices worden vaak gebruikt om de twee verschillende soorten cellen insluiten voor een consistente cel-cel interacties, maar het gebruik van Matrigel of rat staart collageen, die zijn immunogeen substraten te betrekken. Het gebrek aan functionele T-cellen van muizen immuun voorkomt accurate beoordeling van de stromale cellen op borstkanker progressie in vivo, met belangrijke gevolgen voor de ontwikkeling van geneesmiddelen en de werkzaamheid. Bovendien immuungecompromitteerde muizen zijn duur, moeilijk te kweken en vereisen speciale zorg voorwaarden. Om deze obstakels, hebben we een aanpak ontwikkeld om stromale cel en epitheelcellen orthotopically transplantatie in muizen uit hetzelfde genetische achtergrond voor een consistente tumorvorming veroorzaken. Dit systeem gaat oogsten normaal, carcinoom geassocieerd fibroblasten, PyVmT mammacarcinoom cellen en collageen van donor C57BL/6J muizen. De cellen worden vervolgens ingebed in collageen en getransplanteerd in de lies borstklieren van vrouwelijke C57BL/6J muizen. Transplantatie van PyVmT cellen alleen vorm voelbare tumoren 30-40 dagen na transplantatie. Endpoint analyse op 60 dagen geeft aan dat co-transplantatie met fibroblasten verbetert mammatumor groei ten opzichte van PyVmT cellen alleen getransplanteerd. Terwijl de cellen en matrix van C57BL/6J muizen werden gebruikt in deze studies, kan de isolatie van cellen en matrix en transplantatie aanpak worden toegepast op muizen met verschillende genetische achtergronden tonen veelzijdigheid. Samenvattend kan dit systeem worden gebruikt om de moleculaire interacties tussen stromale cellen en epitheliale cellen te onderzoeken, en overwint kritische beperkingen bij immuungecompromitteerde muismodellen.

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

In dit rapport tonen we een systeem te isoleren en cultuur donor cellen van de muis borstklier, en orthotopically deze cellen transplantatie in ontvangende muizen om de stromale analyseren: epitheel interacties tijdens mammatumor ontwikkeling.

Borstklieren transplantatie van stromale cellen en carcinoom cellen in C57BL/6J Muizen

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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