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

Questo progetto è stato finanziato attraverso dal NIH / NCI codice di autorizzazione R00 CA127357 e l&#39;Università del Kansas Cancer Center Endowment.

1. L&#39;isolamento e l&#39;estrazione del collagene donatore di topi C57BL/6J <ol><li> Sacrificio matura normali topi di sesso femminile C57BL/6J utilizzando metodi approvati IACUC. </li><li> Raccolta le code e immergersi in etanolo al 70% per 45 minuti per sterilizzare i tessuti. Asciugare le code con carta velina, avvolgerlo in un foglio di alluminio. Conservare le code a -20 ° C fino al momento dell&#39;uso. </li><li> Mettere le code in un ambiente sterile come una cappa a flusso laminare. Utilizzando le forbici,...

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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>

Il contributo funzionale dei fibroblasti nella progressione tumorale è stata dimostrata attraverso modelli di trapianto, in cui il carcinoma fibroblasti associati co-trapiantati con mammarie benigne risultati cellule epiteliali in una maggiore crescita del tumore e l&#39;invasività 5. Approcci di trapianto convenzionale hanno comportato l&#39;utilizzo di topi SCID o nudi di co-trapianto di cellule stromali ed epiteliali provenienti da diversi background genetico del mouse o specie diverse. Topi immunocompro...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> C57BL/6N topi </td><td> Harlan </td><td> N / A </td><td></td></tr><tr><td> MMTV-PyVmT topi transgenici </td><td> Jackson laboratori </td><td> 002374 </td><td></td></tr><tr><td> Siero bovino fetale </td><td> Pescatore </td><td> SH3039603PR </td><td></td></tr><tr><td> DMEM </td><td> VWR </td><td> 10000113873 </td><td></td></tr><tr><td> Penicillina / streptomicina </td><td> Pescatore </td><td> MT-30-001 </td><td></td></tr><tr><td> amfotericina </td><td> pescatore </td><td> BP2645-20 </td><td></td></tr><tr><td> Amicon filtrazione colonne ultracel 50k </td><td> Millipore </td><td> UFC905008 </td><td></td></tr><tr><td> Tubi per Beckman TI rotore </td><td> Beckman </td><td> 355618 </td><td></td></tr><tr><td> Ratto coda collagene </td><td> Pescatore </td><td> CB 40236 </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% in HBSS w / o Calcio e Magnesio </td><td> Pescatore </td><td> MT-25-050-CI </td><td></td></tr><tr><td> Acido acetico glaciale </td><td> Pescatore </td><td> A491-212 </td><td></td></tr><tr><td> Coomasie blu </td><td> Pescatore </td><td> BP101 25 </td><td></td></tr><tr><td> Tripsina </td><td> Sigma Aldrich </td><td> T3924-100ml </td><td></td></tr><tr><td> Collagenasi 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> DNasi </td><td> Sigma Aldrich </td><td> D5025 </td><td></td></tr><tr><td> Caleidoscopio proteine ​​di serie </td><td> Biorad </td><td> 1610375 </td><td></td></tr><tr><td> Vetrini </td><td> Pescatore </td><td> 12545-78 </td><td></td></tr><tr><td> Vetrini coprioggetto </td><td> VWR </td><td> 101400-042 </td><td></td></tr><tr><td> Vimentin anticorpi S-20 </td><td> Santa Cruz Biotechnology </td><td> SC-7558 </td><td></td></tr><tr><td> α-actina del muscolo liscio anticorpi </td><td> Abcam </td><td> ab5694 </td><td></td></tr><tr><td> CK14 anticorpi </td><td> Santa Cruz Biotechnology </td><td> sc-53253 </td><td></td></tr><tr><td> CK18 anticorpi </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-topo biotinilato </td><td> Vector laboratori </td><td> BA9200 </td><td> Distribuiti attraverso Fisher </td></tr><tr><td> Anti-topo-alexa-568 </td><td> Invitrogen </td><td> A10037 </td><td></td></tr><tr><td> Anti-topo-alexa-488 </td><td> Invitrogen </td><td> A11001 </td><td></td></tr><tr><td> Streptavidina-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> Prolungare Antifade </td><td> Invitrogen </td><td> P-36930 </td><td></td></tr><tr><td> Forbici chirurgiche </td><td> Strumenti Scienza multa </td><td> 91400-12 </td><td></td></tr><tr><td> Forbici fine primavera </td><td> Strumenti Scienza multa </td><td> 15000-02 </td><td></td></tr><tr><td> Blunt forcipe </td><td> Strumenti Scienza multa </td><td> 11002-12 </td><td></td></tr><tr><td> # 5 pinza sottile </td><td> Strumenti Scienza multa </td><td> 11251-10 </td><td></td></tr><tr><td> Gut cromico sutura </td><td> Pescatore </td><td> NC9326254 </td><td></td></tr><tr><td> Pipetta Pasteur in vetro </td><td> Pescatore </td><td> 22-042-815 </td><td></td></tr><tr><td> Etanolo </td><td> Pescatore </td><td> A406P 4 </td><td></td></tr><tr><td> Betadine </td><td> pescatore </td><td> NC9386574 </td><td></td></tr><tr><td> Ferita clip </td><td> Pescatore </td><td> 12032-07 </td><td></td></tr><tr><td> Ferita di base </td><td> Pescatore </td><td> 12031-07 </td><td></td></tr></tbody></table>

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

L&#39;influenza delle cellule stromali, inclusi i fibroblasti sulla progressione del tumore mammario è stato ben documentato attraverso l&#39;utilizzo di modelli murini, in particolare attraverso il trapianto di cellule stromali e le cellule epiteliali della ghiandola mammaria di topo. Modelli trapianto attuali che prevedono l&#39;utilizzo di topi immunocompromessi a causa della diversa provenienza genetica delle cellule stromali e cellule epiteliali. Matrici extracellulari sono spesso utilizzati per incorporare i due tipi di cellule diverse per coerenza interazioni cellula-cellula, ma implicano l&#39;uso di Matrigel o collagene coda di ratto, che sono substrati immunogenico. La mancanza di funzionalità delle cellule T di topi immunocompromessi impedisce una valutazione accurata delle cellule stromali sulla progressione del tumore della mammella in vivo, con importanti implicazioni sullo sviluppo di farmaci e l&#39;efficacia. Inoltre, i topi immunocompromessi sono costosi, difficili da allevare e richiedono condizioni di particolare attenzione. Per superare questi ostacoli, abbiamo sviluppato un approccio alla ortotopicamente trapianto di cellule stromali e le cellule epiteliali in topi dallo sfondo stesso genetico di indurre la formazione costante del tumore. Questo sistema prevede la raccolta normale, carcinoma fibroblasti associati, PyVmT cellule di carcinoma mammario e del collagene da donatore topi C57BL/6J. Le cellule sono poi integrati nel collagene e trapiantati nelle ghiandole inguinali mammaria di topo femmina C57BL/6J. Il trapianto di cellule PyVmT solo formare tumori palpabile 30-40 giorni dopo il trapianto. Analisi degli endpoint a 60 giorni indicano che la co-trapianto con fibroblasti favorisce la crescita del tumore mammario rispetto alle cellule trapiantate PyVmT solo. Mentre le cellule e la matrice di topi C57BL/6J sono stati utilizzati in questi studi, l&#39;isolamento delle cellule e la matrice e l&#39;approccio trapianto può essere applicato nei confronti topi provenienti da diversi background genetico dimostrando versatilità. In sintesi, questo sistema può essere utilizzato per studiare le interazioni molecolari tra cellule stromali e le cellule epiteliali, e supera i limiti critici in modelli di topi immunocompromessi.

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 questo rapporto, si dimostra un sistema per isolare la cultura e le cellule del donatore dalla ghiandola mammaria del mouse, e ortotopicamente trapianto di queste cellule in topi riceventi per analizzare stromali: interazioni epiteliali durante lo sviluppo del tumore mammario.

Trapianto mammaria di cellule stromali e cellule di carcinoma in topi C57BL/6J

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

Research

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Medicine

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Generazione sperimentale di carcinoma-Associated fibroblasti (CAF) da fibroblasti umani mammari

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

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Co-coltura di sistema con un Organotipica cervello Slice e 3D Spheroid delle cellule di carcinoma

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Modellazione di Carcinoma renale metastatico spontanea (MRCC) in topi dopo nefrectomia

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Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Iniezione ortotopico di cellule di carcinoma mammario nel mammaria Fat Pad di Mice per studiare la crescita del tumore.

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La valutazione delle proprietà sulle cellule staminali in cellule di carcinoma ovarico umano Utilizzo multi e single cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

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Eterotopico cervicale trapianto cardiaco nei topi

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...