Name: isolation and differentiation of stromal vascular cells to beige/brite cells
Uploaded: 2013-03-28T12:00:00
Description: Watch this Scientific Journal Video about Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells at JoVE.com

Vi takker Haruya Ohno, Kosaku Shinoda, Louis Sharp, Emi Tomoda, og Lauren Ruiz for diskussion, teknisk hjælp og redaktionel bistand på manuskriptet. Dette arbejde blev støttet af tilskud fra NIH (DK087853) fra Program for Breakthrough Biomedical Research og fra Asubio Pharm Inc. til SKULA blev støttet af en aktie ph.d.-stipendier fra Københavns Universitet og EU FP7 projekt Diabat (HEALTH-F2 -2.011-278.373) til Lise Madsen og Karsten Kristiansen. Vi anerkender også den DERC center tilskud (NIH P30 DK063720).

1. Forbered Fordøjelse Medium Gøre 5 ml per 5 mus pr væv (ca. 1 ml / 1 g fedtvæv). <ol><li> Afvejes i fordøjelsesenzymer: - Collaginase D: 1,5 u / ml (1108874103, 1 g, Roche, 70.334.223) - Dispase II: 2,4 u / ml (04942078001, 0.980 mg / Lyø, Roche, 11.466.200) </li><li> Tilføj 25 ml PBS og bland godt for at opløse </li><li> Tilføj CaCl2 lige før fordøjelse af vævet ved en slutkoncentration på 10 mM </li></ol> 2. Dissekere fedtvæv...

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Endocrinology and Metabolism. 298, E1244-E1253 (2010).</li><li>Petrovic, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+peroxisome+proliferator-activated+receptor+gamma+(PPARgamma)+activation+of+epididymally+derived+white+adipocyte+cultures+reveals+a+population+of+thermogenically+competent,+UCP1-containing+adipocytes+molecularly+distinct+from+classic+brown+adipocytes.">Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes.</a> The Journal of Biological Chemistry. 285, 7153-7164 (2009).</li><li>Rong, J. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+mitochondrial+biogenesis+is+suppressed+in+db/db+and+high-fat+diet-fed+mice+and+improved+by+rosiglitazone.">Adipose mitochondrial biogenesis is suppressed in db/db and high-fat diet-fed mice and improved by rosiglitazone.</a> Diabetes. 56, 1751-1760 (2007).</li><li>Wilson-Fritch, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+remodeling+in+adipose+tissue+associated+with+obesity+and+treatment+with+rosiglitazone.">Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone.</a> The Journal of Clinical Investigation. 114, 1281-1289 (2004).</li><li>Sell, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome+proliferator-activated+receptor+gamma+agonism+increases+the+capacity+for+sympathetically+mediated+thermogenesis+in+lean+and+ob/ob+mice.">Peroxisome proliferator-activated receptor gamma agonism increases the capacity for sympathetically mediated thermogenesis in lean and ob/ob mice.</a> Endocrinology. 145, 3925-3934 (2004).</li><li>Fukui, Y., Masui, S., Osada, S., Umesono, K., Motojima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+thiazolidinedione,+NC-2100,+which+is+a+weak+PPAR-gamma+activator,+exhibits+potent+antidiabetic+effects+and+induces+uncoupling+protein+1+in+white+adipose+tissue+of+KKAy+obese+mice.">A new thiazolidinedione, NC-2100, which is a weak PPAR-gamma activator, exhibits potent antidiabetic effects and induces uncoupling protein 1 in white adipose tissue of KKAy obese mice.</a> Diabetes. 49, 759-767 (2000).</li><li>Vernochet, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C/EBPalpha+and+the+corepressors+CtBP1+and+CtBP2+regulate+repression+of+select+visceral+white+adipose+genes+during+induction+of+the+brown+phenotype+in+white+adipocytes+by+peroxisome+proliferator-activated+receptor+gamma+agonists.">C/EBPalpha and the corepressors CtBP1 and CtBP2 regulate repression of select visceral white adipose genes during induction of the brown phenotype in white adipocytes by peroxisome proliferator-activated receptor gamma agonists.</a> Molecular and Cellular Biology. 29, 4714-4728 (2009).</li><li>Tai, T. 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Her præsenterer vi et pålideligt cellulært system til at studere udviklingen af ​​beige / Brite celler i primære dyrkede adipocytter i mus. Sammenlignet med adskillige tilgængelige udødeliggjorte cellelinier, dette system vil sandsynligvis give bedre relevans for brunfarvning af hvidt fedt in vivo. Selv om studiet af disse primære adipocytter giver nogle fordele, der også findes visse begrænsninger og bekymringer, der er vigtige at overveje. Dels dette system er meget a...

<html> Fedme er dramatisk stigende på verdensplan, og er nu betragtes som en af de mest alvorlige bekymringer for folkesundheden 1. Denne tilstand er forbundet med en misbalance i energiindtag i forhold til udgifterne og resulterer i overskydende energi lagres som lipid i hvidt adipøst væv (WAT). Forstørret WAT er forbundet med øget masse og vægt, mens brunt fedtvæv har evnen til at sprede overskydende energi til at producere varme. Derfor BAT kan fungere som beskyttelse mod både kulde og fedme 2,3. Dette opnås ved afkobling af elektrontransport i mitokondrierne ved frakobling protein 1 (UCP1). Dette protein betragtes som kendetegnende for nonshivering termogenese i BAT 3. Flere undersøgelser i de senere år har vist, at voksne mennesker har funktionel BAT 4-8 og dermed kan manipulation af BAT i mennesker være en potentiel terapeutisk intervention i kampen mod fedme og dens sygdomme. jove_content &quot;&gt; Aktuel viden indikerer, at to typer af brune adipocyter findes hos gnavere,&quot; klassisk &quot;eller&quot; præ-eksisterende &quot;brunt fedt udvikles under prænatal fase og danner dedikerede brune adipose depoter i interscapular regionen og andre perifere væv På den anden side. En &quot;inducerbar&quot; form af brunt fedt (såkaldte brite eller beige celler) udvikles under postnatale fase og vises indflettet i hvide fedtvæv. De to typer af brune adipocyter er også adskilt af forskellige udviklingsstadier oprindelse. Mens den allerede eksisterende brune adipocyter skyldes myoblastsic-lignende Myf5 forstadier, de inducerbare Brite / beige celler indflettet i WAT opstå fra en ikke-Myf5 linjen 9,10. Derudover er regulatoriske veje af denne celletype kan være forskellig fra den Myf5-afledte brown adipocytter 11. Udviklingen af beige celler (dvs. &quot;brunfarvning&quot; af hvidt fedt) kan aktiveres som reaktion på kronisk kulde ogβ3-adrenoceptor-agonister eller PPARy-agonister hos voksne 12-14. De beige / Brite celler sandsynligvis er en lovende terapeutisk mål for manipulation af samlede energibalance og kan potentielt blive en del af fedme behandling, og derfor er det vigtigt at forstå præcise molekylære mekanismer og signaleringsveje, hvorved miljømæssige signaler styrer udviklingen af ​​beige celler. For at forstå den molekylære kontrol af brunfarvning af hvidt fedt, er in vitro-eksperimenter bedst egnet som differentieringen af præadipocytter foregår temmelig asynkront, og det er vanskeligt at detektere af cellerne in situ 15. Selv om undersøgelser af adipocyt udvikling således er hidtil blevet udført primært på cellelinier, såsom 3T3-L1, 3T3-F442A eller HIB1, disse cellelinjer synes at mangle den molekylære signatur af beige celler. På den anden side, er primære adipocytter isoleret fra subkutan WAT mest sandsynligt, at gentage de forarbejds i brunfarvning af hvidt fedt i en celle selvstændig måde. Her giver vi en protokol for effektiv isolering af det stromale-kar fraktion fra fedtvæv og til at fremkalde den brunfarvning af hvide fedt som reaktion på PPARy-agonister. Rosiglitazon er blevet vist at være et særligt effektivt mediator af brunfarvning i disse celler. Som tidligere antydet 16, kan denne cellulære system anvendes til at tjene et pålideligt cellulært system til at studere udviklingen af beige / Brite celler.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Reagens </td></tr><tr><td> Collaginase D </td><td> Roche </td><td> 11088874103 </td><td></td></tr><tr><td> Dispase II </td><td> Roche </td><td> 04942078001 </td><td></td></tr><tr><td> CaCl2 </td><td></td><td></td><td></td></tr><tr><td> DMEM-medium </td><td> Fisher </td><td> 10.017-CV </td><td> Med 2,5 g / l glucose og L-glutamin, uden natriumpyruvat </td></tr><tr><td> Insulin </td><td></td><td></td><td></td></tr><tr><td> T3 (3,3 &#39;,5-triiod-L-thyronin) </td><td> Sigma </td><td> T-2877 </td><td></td></tr><tr><td> Indomethacin </td><td> Sigma </td><td> I-7378 </td><td></td></tr><tr><td> Dexamethason </td><td> Sigma </td><td> D-1756 </td><td></td></tr><tr><td> IBMX </td><td> Sigma </td><td> I-5879 </td><td></td></tr><tr><td> Rosiglitazon </td><td> Sigma </td><td> R-2408 </td><td></td></tr><tr><td></td><td></td><td></td><td> Udstyr </td></tr><tr><td> Collagen-coatede skåle </td><td> BD </td><td> 354.450 </td><td> 10 cm plader </td></tr><tr><td> 70 um filter </td><td> BD Falcon </td><td> 352.350 </td><td> Cell si, 70 um nylon 1/ea </td></tr></tbody></table>

isolation and differentiation of stromal vascular cells to beige/brite cells

Brune adipocyter har evnen til at afkoble den respiratoriske kæde i mitochondrierne og sprede kemiske energi som varme. Udvikling af UCP1-positive brune adipocyter i hvide fedtvæv (såkaldte beige eller Brite celler) er stærkt induceres af en lang række miljømæssige signaler, såsom kronisk kulde eller PPARy-agonister derfor denne celletype har potentiale som et terapeutisk mål for fedme behandling. Selv om de fleste immortaliserede adipocyt linier ikke kan gentage processen med &quot;brunfarvning&quot; af hvidt fedt i kultur, primære adipocytter isoleret fra stromal vaskulær fraktion i subkutant white adipose tissue (WAT) tilvejebringe et pålideligt cellulært system til at undersøge den molekylære kontrol af beige / Brite celleudvikling . Her beskriver vi en protokol for effektiv isolering af primære præadipocytter og til fremkaldelse af differentiering til beige / Brite celler i kultur. Den bruning effekt kan vurderes ved ekspression af brunt fedt-selektiv mærkeERS såsom UCP1.

Bruning af primære adipocytter kan tilgås ved at måle mRNA-ekspression af UCP1 og andre brunt fedt-specifikke eller selektive gener ved qRT-PCR. Vist i figur 1 er genekspression data i ingvinale WAT-afledte primære adipocyter. Cellerne blev induceret til at differentiere i nærværelse af to forskellige doser af rosiglitazon ved 50 nM og 500 nM. En undergruppe af celler blev behandlet med forskolin ved 10 uM for 4 timer før høst. Dette vil inducere cyklisk-AMP (cAMP) i cellerne og aktiver...

Watch this Scientific Journal Video about Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells at JoVE.com

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Primært hvide præadipocytter isoleret fra hvide fedtvæv hos mus kan opdeles i beige / Brite celler. Præsenteret her er en pålidelig trådløs model system til at undersøge den molekylære regulering af &quot;bruning&quot; af hvidt fedt.

Isolering og Differentiering af Stromale vaskulære celler til Beige / Brite Cells

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

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