Name: dual effects of melanoma cell-derived factors on bone marrow adipocytes differentiation
Uploaded: 2018-08-23T12:30:00.000+00:00
Duration: 7 min
Description: Watch this Scientific Journal Video about Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation at JoVE.com

我们感谢 Dov Zipori (魏茨曼科学研究所, 雷霍沃特, 以色列) 亲切地为我们提供小鼠骨髓基质细胞线14F1.1。这项研究得到了中国国家自然科学基金 (81771729 号) 和重庆医科大学永川医院的资助。YJQN201330;YJZQN201527)。

注: 本协议中使用的所有细胞在解冻后, 应至少生长三代。
1. 收获黑色素瘤细胞衍生因子
<ol><li>
准备
	<ol>
<li>获得 B16F10 细胞和小鼠黑色素瘤细胞系。 注: 本协议中, 从中国科学院干细胞库获得了一条小鼠黑色素瘤细胞系。</li>
		<li>制作一个完整的培养基为 B16F10 细胞培养 (100 毫升)。使用 Dulbecco 的改良鹰的培养基 (DMEM) 补充10% 胎牛血清 (血清), 50 毫升青霉素和50µg/毫升链霉素。</li>
		<li>为 B16F10 细胞 (50 毫升) 的饥饿做无血清培养基。使用 DMEM 培养基, 不加血清, 50 毫升青霉素和50µg/毫升链霉素。</li>
	</ol>
</li>
	<li>
制备黑色素瘤条件培养基 (厘米)
	<ol>
<li>种子 2.0 x 105 B16F10 细胞在100毫米盘与10毫升10% 血清 DMEM。然后在 5% CO2 24 小时的大气层中孵化出37摄氏度孵化器中的细胞。</li>
		<li>当细胞达到80% 的汇合, 移除培养基, 用5毫升磷酸盐缓冲盐水 (PBS) (1x, pH 7.4) 清洗细胞, 并用10毫升无血清培养基替代培养基, 以使饥饿。</li>
		<li>将细胞归还给 18–24 h 的孵化器。</li>
		<li>饥饿后, 收集条件培养基 (CM) 与吸管和转移到一个新鲜的50毫升离心管。 注: 条件培养基为 B16F10 细胞培养的上清液。确保 B16F10 细胞在收集 CM 之前状态良好。</li>
		<li>离心机 CM 为5分钟在 1000 x g在4°c。</li>
		<li>将上清液吸管移到新鲜的无菌管或瓶中。 注意: 避免吹打在管子底部的颗粒。</li>
		<li>要去除细胞碎片, 过滤 CM 通过通过注射器连接0.45 µm 过滤器在无菌瓶或管子之上。 注: 0.22 µm 过滤器是最佳的过滤 CM。</li>
		<li>将过滤后的上清液贮存在摄氏-20 摄氏度, 直至使用。 注: 最好不要将上清液贮存在摄氏-20 摄氏度以上的一个月以上。或者, 将 CM 保存在-80 摄氏度以上, 以延长脂肪细胞的分化时间。</li>
	</ol>
</li>
</ol>2. 黑色素瘤细胞衍生因子诱导骨髓脂肪组织的研究
<ol><li>
准备
	<ol>
<li>使100毫升的脂肪感应介质28。使用完整的 DMEM 补充5µg/毫升胰岛素, 0.5 毫米3异丁基 1-甲基黄嘌呤和1µM 地塞米松。存储在4摄氏度的两个星期。</li>
		<li>准备100毫升的脂肪维护培养基。使用完整的 DMEM 或混合50% 黑色素瘤 CM 补充5µg/毫升胰岛素。</li>
		<li>做油红色 O 库存解决方案。将30毫克的油红 O 粉溶于10毫升异丙醇 (≥ 99.5%), 并将其与涡流混合均匀。 注: 储存1年在4摄氏度。</li>
		<li>做一个油红色 O 工作解决方案。在油烟机中, 稀释油红色 O 型股票溶液 (从步骤 2.1.3) 与去离子水的比例为3:2。在室温下孵化混合物15分钟, 并在使用前通过0.22 µm 过滤器进行过滤。 注: 仅在2小时内使用新的工作解决方案。</li>
	</ol>
</li>
	<li>
脂肪细胞分化
	<ol>
<li>培养一个基质细胞线14F1.1 从小鼠骨髓29在60毫米菜与一个完全 DMEM 在80% 融合在37°c 孵化器在 5% CO2的大气中。 注: 或者, 用原发骨髓间充质细胞取代 14F1.130。</li>
		<li>胰蛋白酶处理细胞与1.5 毫升的0.25% 胰蛋白酶溶液在一个孵化器中的最小。</li>
		<li>在细胞中加入5毫升10% 的血清, 用玻璃 hemocytometer 计数细胞数;然后调整细胞密度约 105/毫升。</li>
		<li>种子 5 x 104的14F1.1 细胞每井在24井板与0.5 毫升的脂肪诱导培养基。经过两天的孵化, 细胞达到90% 的汇合。 注意: 这一步非常关键。骨髓基质细胞在没有脂肪诱导培养基的情况下不能诱导为脂肪组织。</li>
		<li>每次取出感应介质, 用1毫升的 PBS 清洗两次细胞。</li>
		<li>改变到1毫升的脂肪维持培养基, 并继续培养细胞直到脂肪成熟。 注: 在显微镜下, 脂肪细胞看起来像小或大肥皂泡, 很容易辨认。通常, 成熟的脂肪细胞存在于 6日或 8天。</li>
		<li>用油红色 O 染色成熟脂肪细胞或收集脂肪细胞进行定量聚合酶链反应 (qPCR) 分析。</li>
	</ol>
</li>
	<li>
脂肪细胞染色
	<ol>
<li>用 PBS 清洗分化的14F1.1 细胞 2x, 并将其固定在3.7% 甲醛中1小时。</li>
		<li>冲洗60% 异丙醇的细胞, 使其完全干燥。</li>
		<li>在油井中加入0.2 毫升油红色 O 工作溶液, 在室温下孵化0.5 至2小时。</li>
		<li>用清水冲洗细胞, 除去未粘合的染料。</li>
		<li>在光显微镜下拍摄染色脂肪细胞。</li>
	</ol>
</li>
</ol>3. 骨髓脂肪细胞与黑色素瘤共培养的研究
<ol><li>
准备
	<ol>
<li>制备成熟的骨髓脂肪细胞: 用脂肪诱导培养基在24井板中诱导14F1.1 干细胞成成熟的 BM 脂肪, 2 天, 然后在脂肪维持培养基中维持6–8天。</li>
		<li>准备 B16F10 细胞: 培养5毫升 DMEM 培养基的60毫米菜肴中的 B16F10 细胞。</li>
		<li>滋润膜插入物 (如transwell): 添加150µL 的无血清 DMEM 培养基到0.4 µm 孔大小膜插入, 并浸泡在24井板的水井, 填充500µL 的无血清 DMEM 培养基。</li>
	</ol>
</li>
	<li>
共培养设置
	<ol>
<li>慢慢地把每个插入物的培养基吸走。 注意: 去除介质时不要破坏膜。</li>
		<li>种子的 B16F10 细胞 (0/103/105/106) 在0.4 µm 孔插入从步骤3.2.1。</li>
		<li>用步骤3.1.1 中的成熟骨髓脂肪细胞取代脂肪的维持培养基。与600µL 完全 DMEM 培养基。</li>
		<li>将填充有 B16F10 细胞的插入物 (从步骤 3.2.2) 转移到与成熟的骨髓脂肪脂 (从步骤 3.2.3) 的水井上共培养实验。</li>
		<li>孵化的 cocultures 在37摄氏度和 5% CO2 2 天。</li>
		<li>48小时的孵化后, 取出共培养的插入物, 然后用500µL 1x PBS 清洗脂肪细胞2x。 注意: 如果需要, 可以选择在插入中收集 B16F10 单元格进行分析。</li>
		<li>用油红 O 工作溶液染色脂肪细胞, 或收集脂肪细胞进行 qPCR 分析。 注: 请参阅步骤2.3 中的染色过程。</li>
		<li>在光显微镜下拍摄染色脂肪细胞。</li>
	</ol>
</li>
</ol>4. 脂肪细胞特异基因特征的 qPCR 分析
<ol><li>在成熟脂肪细胞准备检测的24井板中, 每井添加0.5 毫升的可随时使用的 RNA 隔离试剂。</li>
	<li>遵循一个标准协议的 RNA 提取和 cDNA 合成31。</li>
	<li>使用18S 和 GAPDH 的底漆作为对照, CEBPα/β, PPARγ, FABP4, 瘦素, Pref-1, 或脂联素为脂肪细胞特异基因签名 (表 1)。</li>
	<li>在热循环仪中运行以下反应:95 °c 二十年代, 其次40周期95°c 为 1 s 和60°c 为二十年代。</li>
	<li>使用 2-ΔΔCt方法计算褶皱变化32。</li>
</ol>

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A., Nguyen, V. T., Levi, B., James, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+methods+of+adipogenic+differentiation+of+mesenchymal+stem+cells.">Current methods of adipogenic differentiation of mesenchymal stem cells.</a> Stem Cells and Development. 20 (10), 1793-1804 (2011).</li><li>Zipori, D., Toledo, J., von der Mark, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+among+stromal+cell+lines+from+mouse+bone+marrow+disclosed+in+their+extracellular+matrix+composition+and+interactions+with+normal+and+leukemic+cells.">Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells.</a> Blood. 66 (2), 447-455 (1985).</li><li>Maridas, D. E., Rendina-Ruedy, E., Le, P. T., Rosen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Culture,+and+Differentiation+of+Bone+Marrow+Stromal+Cells+and+Osteoclast+Progenitors+from+Mice.">Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.</a> Journal of Visualized Experiments. (131), e56750 (2018).</li><li>Iguchi, T., Niino, N., Tamai, S., Sakurai, K., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+Quantification+of+Plasma+MicroRNA+Levels+in+Cynomolgus+Monkeys,+Using+Quantitative+Real-time+Reverse+Transcription+PCR.">Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR.</a> Journal of Visualized Experiments. (132), e56850 (2018).</li><li>Bozec, A., Hannemann, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Regulation+of+Adipocyte+Numbers+in+Adult+Organisms+Through+Differentiation+and+Apoptosis+Homeostasis.">Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis.</a> Journal of Visualized Experiments. (112), e53822 (2016).</li><li>Shafat, M. S., 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=Leukemic+blasts+program+bone+marrow+adipocytes+to+generate+a+protumoral+microenvironment.">Leukemic blasts program bone marrow adipocytes to generate a protumoral microenvironment.</a> Blood. 129 (10), 1320-1332 (2017).</li><li>Gazi, E., 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=Direct+evidence+of+lipid+translocation+between+adipocytes+and+prostate+cancer+cells+with+imaging+FTIR+microspectroscopy.">Direct evidence of lipid translocation between adipocytes and prostate cancer cells with imaging FTIR microspectroscopy.</a> The Journal of Lipid Research. 48 (8), 1846-1856 (2007).</li><li>Brown, M. D., 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=Influence+of+omega-6+PUFA+arachidonic+acid+and+bone+marrow+adipocytes+on+metastatic+spread+from+prostate+cancer.">Influence of omega-6 PUFA arachidonic acid and bone marrow adipocytes on metastatic spread from prostate cancer.</a> British Journal of Cancer. 102 (2), 403-413 (2010).</li><li>Hardaway, A. L., Herroon, M. K., Rajagurubandara, E., Podgorski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marrow+adipocyte-derived+CXCL1+and+CXCL2+contribute+to+osteolysis+in+metastatic+prostate+cancer.">Marrow adipocyte-derived CXCL1 and CXCL2 contribute to osteolysis in metastatic prostate cancer.</a> Clinical &amp; Experimental Metastasis. 32 (4), 353-368 (2015).</li><li>Aebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+metastasis+in+the+elderly.">Spinal metastasis in the elderly.</a> European Spine Journal. 12, 202-213 (2003).</li><li>Wagner, M., Bjerkvig, R., Wiig, H., Dudley, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+adipocyte+specification+and+necrosis+augment+tumor-associated+inflammation.">Loss of adipocyte specification and necrosis augment tumor-associated inflammation.</a> Adipocyte. 2 (3), 176-183 (2013).</li><li>Bochet, 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=Cancer-associated+adipocytes+promotes+breast+tumor+radioresistance.">Cancer-associated adipocytes promotes breast tumor radioresistance.</a> Biochemical and Biophysical Research Communications. 411 (1), 102-106 (2011).</li><li>Hirano, T., 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=Enhancement+of+adipogenesis+induction+by+conditioned+media+obtained+from+cancer+cells.">Enhancement of adipogenesis induction by conditioned media obtained from cancer cells.</a> Cancer Letters. 268 (2), 286-294 (2008).</li><li>Gordeev, A. A., Chetverina, H. V., Chetverin, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+arrangement+of+eukaryotic+cells+in+merged+hydrogels+combines+the+advantages+of+3-D+and+2-D+cultures.">Planar arrangement of eukaryotic cells in merged hydrogels combines the advantages of 3-D and 2-D cultures.</a> Biotechniques. 52 (5), 325-331 (2012).</li></ol>

作者没有什么要申报的。

Cocultures 与插入物已被广泛用于研究细胞间的相互作用。2D 共培养系统是观察两个部位在体外进行串扰的有效方法, 我们在这里显示了两种不同的癌细胞驱动对骨髓脂肪细胞的影响。许多实验室利用这种方法来研究脂肪细胞与癌细胞6、12、27、39之间的串扰。
因此, 一般而言, 2D 共培养是细胞间串扰研究中最直接的方法。然而, 在为进一步研究准备一个未分化的细胞系时, 有一些独特的挑战。在这里描述的协议中, 骨髓基质细胞的健康和功能状态对脂肪的分化是至关重要的。该协议可以修改, 以取代14F1.1 细胞与原发骨髓间充质干细胞。此外, 14F1.1 细胞在脂肪诱导培养基中的预孵化数天是另一个关键步骤。没有这种预孵化, 骨髓基质细胞将无法分化成脂肪, 这已经被发现在我们和其他的研究40。这些现象也解释了为什么一些类似的协议可能导致不同的或甚至相反的效果, 并强调了这样一个简单的, 可重复的, 详细的协议的重要性, 对肿瘤细胞和骨髓细胞的串扰研究。
虽然 2D 共培养可能会丢失一些关键信息与 3D 或前体文化相比 , 它仍然很容易被标准化 , 监测 , 并最终纵进一步使用20,21,41。技术的未来应用可能决定成骨细胞在与癌细胞共培养时如何改变。

骨转移在晚期癌症患者中普遍存在, 但治疗仍不可用。除了专门储存能量作为脂肪, 脂肪细胞可以支持肿瘤生长和转移骨髓和其他器官1,2,3,4,5,6。此外, 脂肪细胞在调节癌细胞生物学7、8、9、10和新陈代谢方面起着至关重要的作用,4、11、12 ,13,14,15,16, 以及在骨转移1,4,12。在骨髓小生境中, 脂肪细胞还会影响癌细胞4、6、17的生物学行为。骨髓脂肪细胞与癌细胞 osteotropism 的相互作用对了解骨转移有重要意义。然而, 鲜为人知。
根据目前的研究, 各种方法适用于脂肪细胞, 包括两个或三维 (2/3 d) 和体外培养17,18,19,20,21。最近, Herroon等人设计了一种新的 3 d-培养方法来研究骨髓脂肪细胞与癌细胞的相互作用22。虽然3D 共培养是最佳的模仿脂肪细胞和癌细胞在体内的生理相互作用, 它的重现性差22,23。与2D 共培养系统相比, 3D 共培养系统可以提供不同的细胞表型, 如细胞形态学21、22、24、25、26。此外, 分离的松质骨组织片段的体外培养可以导致骨髓细胞培养出17的生长脂肪体。
然而, 与以前的模型相比, 2D 细胞培养模型仍然是一种经典的、可靠的、易于快速扫描候选分子的技术, 在体外1的脂肪细胞或癌细胞中的表型改变, 4,6,12,15,27.为更好地了解骨髓脂肪细胞与黑色素瘤的相互串扰, 我们提供了一个详细的协议, 为2D 共培养系统的骨髓脂肪细胞与黑色素瘤。

<table><tbody><tr><td>DMEM</td><td>Invitrogen Inc.</td><td>11965092</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Invitrogen Inc.</td><td>16000&amp;ndash;044</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Invitrogen Inc.</td><td>14190-144</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>91077C</td><td></td></tr><tr><td>3-isobutyl-1-methyl-xanthine</td><td>Sigma-Aldrich</td><td>I5879</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>Oil Red o</td><td>Sigma-Aldrich</td><td>O0625</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>CLS3527</td><td></td></tr><tr><td>Transwell insert</td><td>Millipore</td><td>MCHT24H48</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>isopropanol</td><td>Sigma-Aldrich</td><td>I9516</td><td></td></tr><tr><td>0.25% trypsin</td><td>Thermo Scientific</td><td>25200056</td><td></td></tr><tr><td>hemocytometer</td><td>Bio-Rad</td><td>1450016</td><td></td></tr><tr><td>Culture incubator</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>50 mL falcon</td><td>Corning</td><td>CLS430828</td><td></td></tr><tr><td>Clean Bench</td><td>Thermo Scientific</td><td>-</td><td></td></tr><tr><td>Microscopy</td><td>Olympus</td><td>-</td><td></td></tr><tr><td>200 &amp;mu;L pipet tips</td><td>BeyoGold</td><td>FTIP620</td><td></td></tr><tr><td>1,000 mL pipet tips</td><td>BeyoGold</td><td>FTIP628</td><td></td></tr></tbody></table>

dual effects of melanoma cell-derived factors on bone marrow adipocytes differentiation

骨髓脂肪细胞与癌细胞的串扰在骨转移过程中起着至关重要的作用。有多种方法可用于研究显著的串扰;然而, 共培养的二维 transwell 系统仍然是这种串扰研究的经典、可靠、简便的方法。在这里, 我们提出了一个详细的协议, 显示了骨髓脂肪细胞和黑色素瘤的共培养。然而, 这种共培养系统不仅有助于研究骨髓脂肪细胞诱导的癌细胞信号 transductions, 而且可以为今后骨转移的机械学研究提供新的治疗靶点。转移。

在骨髓, 脂肪细胞可以出现在肿瘤微环境1,13,33,34,35在早期阶段支持肿瘤进展通过可溶性因素或激活 osteoclastogenesis6,12,36, 特别是在肥胖6,12和/或衰老37的情况下。我们以前的研究1已经指出, 骨髓脂肪细胞的数量在黑色素瘤转移到骨1的早期发生了显著变化。事实上, 脂肪细胞在肿瘤区积聚, 并支持当小鼠以高脂肪饮食喂养6时骨髓中的癌细胞生长, 表明骨髓脂肪细胞可能在转移骨中起重要作用。骨髓利基37。然而, 肿瘤细胞和骨髓脂肪瘤的动态串扰仍不清楚。因此, 我们致力于解决这一问题的2D 共培养系统的骨髓脂肪细胞和黑色素瘤。
肿瘤衍生因子促进骨髓脂肪细胞分化: 图 1一个典型的工作流诱导骨髓脂肪细胞与肿瘤条件培养基 (中医)。有代表性的实验表明, 14F1.1 细胞在第一2天脂肪诱导介质存在时, 在密度为 5 x 104细胞的单个细胞中被镀成单细胞, 然后在脂肪维护介质中保持 (有或没有中医) 6–8天。成熟的脂肪细胞含有脂滴;因此, 区分脂肪细胞可以很容易地识别出油红色 O 染色 (图 1b) 和量化的 qPCR 与脂肪细胞特异基因签名 (图 1c)。
黑色素瘤细胞超载诱导骨髓脂肪组织的脱分化: 肿瘤细胞可以改变环境生存, 特别是在缺氧条件下1,4,6。为了模拟骨髓脂肪细胞的肿瘤负担次生效应, 我们执行了2D 共培养系统, 在上部插入物中继续增加黑色素瘤单元数 (图 2a)。图 2b显示脂肪细胞的脂质贮存在黑色素瘤 (106细胞/井) 的最高密度范围内被剥夺, 与黑色素瘤细胞最低频率的剥夺相比 (103细胞/好吧)。图 2c给出了共培养脂肪细胞中这些脂肪细胞的基因分析。与黑色素瘤细胞密度最低的共培养相比, pref-1 水平明显增加, 瘦素、脂联素、FABP4、CEBPβ和 PPARγ的表达减少, 这表明肿瘤负担次生效应可归因于脂肪细胞的脱分化过程。此外, 肿瘤细胞超载可诱发脂肪瘤38的坏死。
总之, 我们观察到一个相反的黑色素瘤细胞驱动的影响骨髓脂肪组的可溶性因素。对骨髓脂肪细胞的相反作用是很高的兴趣, 这表明骨髓脂肪瘤是癌细胞转移骨髓的动态调节因子。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57329/57329fig1.jpg"> 图 1:肿瘤衍生因子促进脂肪细胞分化.(a) 本小组展示了使用14F1.1 细胞系和黑色素瘤衍生条件培养基的实验设置的示意图。(b) 本小组显示分化的骨髓脂肪细胞的影像学。(c) 本小组显示了 qPCR 对脂肪细胞特异基因签名的分析。中医 = 肿瘤条件培养基。qPCR: 定量聚合酶链反应。刻度条 = 50 µm。这一数字已由王等修改。1.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57329/57329fig1large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a> 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57329/57329fig2.jpg"> 图 2:肿瘤负担对脂肪细胞的影响.(a) 本小组展示了实验性环境的示意图: 共培养14F1.1 细胞系和黑色素瘤细胞。(b) 本小组显示共培养系统中骨髓脂肪细胞的影像学。(c) 本小组显示了 qPCR 对脂肪细胞的基因分析。qPCR: 定量聚合酶链反应。刻度条 = 50 µm。这一数字已由王等修改。1.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57329/57329fig2large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a> 
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td colspan="3">引</td>
		</tr>
<tr>
<td>基因名称</td>
			<td>向前序列 (5 ' 到 3 ')</td>
			<td>反向序列 (5 ' 到 3 ')</td>
		</tr>
<tr>
<td>PPARγ</td>
			<td>CTGATGCACTGCCTATGAGC</td>
			<td>GGGTCAGCTCTTGTGAATGG</td>
		</tr>
<tr>
<td>CEBPα</td>
			<td>AAG AGC CGC GAC AAG GC</td>
			<td>agc</td>
		</tr>
<tr>
<td>CEBPβ</td>
			<td>CAACCTGGAGACGCAGCACAAG</td>
			<td>GCTTGAACAAGTTCCGCAGGGT</td>
		</tr>
<tr>
<td>FABP4</td>
			<td>TGAAATCACCGCAGACGACAGG</td>
			<td>GCTTGTCACCATCTCGTTTTCTC</td>
		</tr>
<tr>
<td>瘦 素</td>
			<td>AAG 的 TCA gc</td>
			<td>GGT 民航局 GC</td>
		</tr>
<tr>
<td>Pref-1</td>
			<td>GACACTCGAAGCTCACCTGG</td>
			<td>GGAAGGCTGGGACGGGAAAT</td>
		</tr>
<tr>
<td>脂 联 素</td>
			<td>GCG ata AGC GGC</td>
			<td>GGC GGA 猫 C</td>
		</tr>
</tbody></table>表 1: 用于 qPCR 的引物表。

Watch this Scientific Journal Video about Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation at JoVE.com

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

在这里, 我们提出了一个可靠和直接的二维 (2D) 共培养系统, 研究肿瘤细胞和骨髓细胞之间的相互作用, 这表明黑色素瘤干细胞衍生因子的双重作用对骨髓脂肪的影响分化, 也为骨转移的机械学研究提供了经典的方法。

黑色素瘤细胞衍生因子对骨髓脂肪分化的双重影响

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are ...

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JoVE Journal

Cancer Research

6.1K 次观看.  Shanghai Jiao Tong University. 在这里, 我们提出了一个可靠和直接的二维 (2D) 共培养系统, 研究肿瘤细胞和骨髓细胞之间的相互作用, 这表明黑色素瘤干细胞衍生因子的双重作用对骨髓脂肪的影响分化, 也为骨转移的机械学研究提供了经典的方法。

视频: Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation

Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1&#945; protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes.

Adipose tissue (AT) can influence whole body homeostasis, therefore understanding the molecular mechanisms of adipocyte differentiation and function is of importance. We provide a protocol for gaining new insights into these processes by analyzing adipocyte homeostasis, differentiation and hypoxia exposure as a model for induced adipocyte apoptosis.

成人脂肪细胞生物体的数字调控机制，通过分化与凋亡的动态平衡

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of ...

科研

发育生物学

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

突变黑色素细胞干细胞引发小鼠黑色素瘤的时空控制

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

癌症研究

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the development of hypertension during obesity. The extent of hypertrophy and hyperplasia depends on depot location, sex, and the type of Adipocyte Progenitor Cell (APC) phenotypes present. Techniques used for APC and preadipocytes isolation in the last 10 years have drastically improved the accuracy at which individual cells can be identified based on specific cell surface markers. However, isolation of APC and adipocytes can be a challenge due to the fragility of the cell, especially if the intact cell must be retained for cell culture applications.
Magnetic-activated Cell Sorting (MCS) provides a method of isolating greater number of viable APC per weight unit of adipose tissue. APC harvested by MCS can be used for in vitro protocols to expand preadipocytes and differentiate them into adipocytes through use of growth factor cocktails allowing for analysis of the prolific and adipogenic potential retained by the cells. This experiment focused on the aortic and mesenteric PVAT depots, which play key roles in the development of cardiovascular disease during expansion. These protocols describe methods to isolate, expand, and differentiate a defined population of APC. This MCS protocol allows isolation to be used in any experiment where cell sorting is needed with minimal equipment or training. These techniques can aid further experiments to determine the functionality of specific cell populations based on the presence of cell surface markers.

Here we report a method for isolation of Adipocyte Progenitor Cell (APC) populations from Perivascular Adipose Tissue (PVAT) using Magnetic-activated Cell Sorting (MCS). This method allows for an increased isolation of APC per gram of adipose tissue when compared to Fluorescence-Activated Cell Sorting (FACS).

通过磁性活化细胞分选分离的血管周围脂肪组织的脂肪细胞祖细胞的扩增和脂肪生成诱导

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

表皮黑色素和防止晒斑的人性化小鼠模型的诱导药物

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

医学

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

The protocol allows the generation of a pure adipocyte population from induced pluripotent stem cells (iPSCs). Retinoic acid is used to differentiate iPSCs into mesenchymal stem cells (MSCs) which are used for producing adipocytes. Then, a sorting approach based on Nile red staining is used to obtain pure adipocytes.

将人 iPSC 稳健分化为纯脂肪细胞群以研究脂肪细胞相关疾病

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

免疫与感染

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

生物学

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

带谱系特异标记的间充质细胞脂肪分化电位的可视化与量化

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

新生小鼠肩胛间脂肪组织基质血管部分棕色脂肪细胞的分化和成像

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

小鼠白色脂肪组织的半自动分离

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

作者聚焦：使用组织解离剂从小鼠白色脂肪组织中半自动分离基质血管组分  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...