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

我们感谢大野治哉，筱田耕作，路易·夏普，友田惠美，劳伦·鲁伊斯的手稿上的讨论，技术帮助和编辑协助。这项工作是支持的股份从哥本哈根与欧盟FP7：项目DIABAT（HEALTH-F2大学的博士研究生奖学金补助金从美国国立卫生研究院（DK087853），从程序突破性的生物医学研究和从Asubio医药公司SKULA的支持-2011-278373）莉莎·马德森和Karsten克里斯蒂安森。我们也承认DERC的中心授予（NIH P30 DK063720）。

1。准备消化液 5毫升，每5只小鼠组织（大约1毫升/ 1克脂肪组织）。 <ol><li>在消化酶称： - Collaginase D：1.5单位/毫升（1108874103，1克，罗氏，70334223） - 中性蛋白酶II：2.4单位/毫升（0980毫克/ LYO，罗氏，04942078001，11466200） </li><li>加入25毫升PBS，拌匀溶解</li><li>的最终浓度为10mM，加入CaCl 2的只是事先消化组织</li></ol> 2。解剖小鼠脂肪组织<...

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+identification+of+bipotential+adipocyte+progenitors+recruited+by+beta3-adrenoceptor+activation+and+high-fat+feeding.">In vivo identification of bipotential adipocyte progenitors recruited by beta3-adrenoceptor activation and high-fat feeding.</a> Cell Metabolism. 15, 480-491 (2012).</li><li>Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transdifferentiation+properties+of+adipocytes+in+the+adipose+organ.">Transdifferentiation properties of adipocytes in the adipose organ.</a> American Journal of Physiology. Endocrinology and Metabolism. 297, E977-E986 (2009).</li><li>Barbatelli, G., 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=The+emergence+of+cold-induced+brown+adipocytes+in+mouse+white+fat+depots+is+determined+predominantly+by+white+to+brown+adipocyte+transdifferentiation.">The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.</a> American Journal of Physiology. 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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在这里，我们提出了一个可靠的蜂窝系统研究的发展，米色/ BRITE细胞的原代培养的脂肪细胞在小鼠体内。几个可用的永生化细胞系相比，这个系统是有可能提供增强相关的的白色脂肪在体内的褐变。 即使这些原代脂肪细胞的研究提供了一定的优势，也存在着一定的局限性和关切问题，是重要的考虑因素。首先，这个系统是高度依赖的细胞的分化能力。如UCP1和<em...

<html>肥胖是显着世界范围内不断增加，现在被认为对公众健康1的最严重的问题之一。这种情况涉及到的能量摄入相对misbalance支出和结果，多余的能量储存在白色脂肪组织（WAT）的脂质。扩大WAT与增加身体的质量和重量，而棕色脂肪组织的能力，以消耗多余的能量以产生热量。因此，BAT可作为寒冷和保护，防止肥胖2,3。这是通过解偶联线粒体解偶联蛋白1（UCP1）的​​电子传输。这种蛋白质被认为是一个标志性非颤抖性产热作用BAT 3。在近几年的几项研究显示，成年人有功能BAT 4-8，因此，在人类的BAT操作可能是一个潜在的干预治疗，对肥胖及其相关疾病的战斗中。 jove_content“目前的证据表明，棕色脂肪细胞中存在两种类型的啮齿动物，”古典“或”以前存在的“棕色脂肪在产前阶段的发展，形成专用的棕色脂肪在肩胛间区的仓库和其他外围组织。另一方面，“诱导”形式的棕色脂肪（所谓BRITE或米色细胞）在产后阶段发展，穿插在白色脂肪组织中出现，这两种类型的棕色脂肪细胞也分离由不同发育起源。虽然预先存在的棕色脂肪细胞从myoblastsic像MYF5前体的产生，诱导BRITE /米色WAT中穿插的细胞从的非MYF5谱系9,10产生，此外，这种细胞类型的调控途径可能是不同的从MYF5派生棕色可以激活慢性暴露于寒冷的环境和发展的米色细胞（ 即 “黑”的白色脂肪）脂肪11。β3-肾上腺素能受体激动剂或PPARγ激动剂在成人中12-14。浅米色/ BRITE细胞有可能是一个有前途的治疗目标操作的整体能源平衡，并有可能成为肥胖治疗的一部分，因此，重要的是要了解确切的分子机制和信号途径，其中环境因素控制的发展米色细胞。 了解的分子控制的白色脂肪的褐变， 在体外实验中是最适合作为发生，而异步的前体脂肪细胞的分化，它是很难检测细胞原位 15。虽然对脂肪细胞发展的研究迄今为止进行主要对3T3-L1，3T3-F442A或HIB1的细胞系，例如，这些细胞系出现米色细胞缺乏的分子签名。另一方面，从皮下WAT中分离的原代脂肪细胞是最有可能重述议事录S褐变的白色脂肪的细胞自主的方式。在这里，我们提供了一个协议，用于从脂肪组织的间质血管部分有效隔离和白色脂肪PPARγ激动剂诱导褐变。罗格列酮已被证明是一种非常有效的调解员在这些细胞中的褐变。正如先前所建议的16，这蜂窝系统可用于为可靠的蜂窝系统研究米色/布里特细胞的发展。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td> Collaginaseð </td><td>罗氏公司</td><td> 11088874103 </td><td></td></tr><tr><td>中性蛋白酶II </td><td>罗氏公司</td><td> 04942078001 </td><td></td></tr><tr><td> 氯化钙 </td><td></td><td></td><td></td></tr><tr><td> DMEM培养基</td><td>费舍尔</td><td> 10017-CV </td><td>与2,5克/升葡萄糖和L-谷氨酰胺，而不丙酮酸钠</td></tr><tr><td>胰岛素</td><td></td><td></td><td></td></tr><tr><td> T3（3,3&#39;，5 - 三碘-L-甲状腺原氨酸） </td><td>西格玛</td><td> T-2877 </td><td></td></tr><tr><td>吲哚美辛</td><td>西格玛</td><td> I-7378 </td><td></td></tr><tr><td>地塞米松</td><td>西格玛</td><td> D-1756 </td><td></td></tr><tr><td> IBMX </td><td>西格玛</td><td> I-5879 </td><td></td></tr><tr><td>罗格列酮</td><td>西格玛</td><td> R-2408 </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>胶原涂层菜</td><td> BD </td><td> 354450 </td><td> 10 CM平板</td></tr><tr><td> 70微米的过滤器</td><td> BD猎鹰</td><td> 352350 </td><td>细胞过滤网，70μm的尼龙1/EA </td></tr></tbody></table>

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

棕色脂肪细胞的能力脱开在线粒体呼吸链和消散化学能量作为热量。 UCP1阳性棕色脂肪细胞在白色脂肪组织（所谓的米色或BRITE细胞）的发展是高度由各种环境线索，如慢性暴露于寒冷的环境或PPARγ激动剂诱导，因此，这种细胞类型具有潜在的治疗靶点肥胖的治疗。虽然大多数永生脂肪细胞线可以不概括的“褐变”的白色脂肪中文化的过程，原代脂肪细胞分离出间质血管部分在皮下白色脂肪组织（WAT）提供一个可靠的蜂窝系统研究的分子控制的米色/ BRITE细胞发展。在这里，我们描述了一个协议，有效隔离的主要前体脂肪细胞和为文化的米色/布里特细胞的诱导分化。褐变效果可评估的棕色脂肪选择性标记的表达雇员再培训计划，如UCP1。

原代脂肪细胞褐变的可访问通过测量UCP1和其他棕色脂肪特异性或选择性由定量RT-PCR的基因的mRNA的表达。示于图1是腹股沟WAT-衍生的原代脂肪细胞中的基因表达数据。将细胞诱导分化，分别在存在两种不同剂量的罗格列酮，在50纳米和500纳米。 4小时收获前10μMforskolin的细胞治疗的一个子集。这将引起循环AMP（cAMP）的细胞和，包括UCP1的表达，激活热基因程序。 <p class="jo...

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

主要从小鼠白色脂肪组织中分离出的白色脂肪前体细胞可以分化成浅米色/ BRITE细胞。这里介绍的是一个可靠的细胞模型系统，研究的分子调控“黑”的白色脂肪。

米色/布里特细胞的间质血管细胞的分离和分化

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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Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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Induction and Analysis of Epithelial to Mesenchymal Transition

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Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

CD146的分离<sup&gt; +</sup&gt;从大鼠肺组织驻地肺间质基质细胞

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

恒河猴脂肪干细胞的分离、增殖和分化

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...