作者希望在森塔拉减肥中心的临床协调员贝基. 马奎斯的帮助下, 她的病人筛查和同意的过程。这项研究得到了 R15HL114062 对 Anca d Dobrian 的支持。

东弗吉尼亚医学院的机构审查委员会批准了研究和收集人体脂肪组织样本用于研究。从病人那里收集了知情的书面同意书。
1. 编制缓冲器、媒体和仪器
<ol><li>准备一个克雷布斯的碳酸氢钠-缓冲溶液 (KRBBS): 135 毫米氯化钠, 5 毫米氯化钾, 1 毫米硫酸镁, 0.4 毫米磷酸钾二元, 5.5 毫米葡萄糖, 1 毫米腺苷, 0.01% 抗生素/防霉混合 (50 µg/毫升青霉素,50µg/毫升链霉素, 30 µg/毫升庆大霉素, 15 ng/毫升两性霉素) 和10毫米 HEPES (pH = 7.4)。这个解决方案是准备新鲜的每一次的组织收集和消化。</li>
	<li>制备新的胶原酶溶液: 1 毫克/毫升的胶原酶, 类型 1, 在 KRBSS;做3毫升/克的脂肪。在组织消化前, 在水浴中预热37摄氏度的胶原酶溶液。</li>
	<li>准备一个 CD34 细胞隔离缓冲: 2% 胎牛血清 (血清) 和1毫米 EDTA 在无菌磷酸盐缓冲生理盐水 (PBS)。</li>
	<li>准备成脂培养基: DMEM/F12, 5% 血清, 50 µg/毫升青霉素/链霉素, 1.25 µL/毫升的人胰岛素 (5 µg/毫升或144亩/毫升), 0.36 µL/毫升2.8 毫米罗格列酮 (1 µM)。</li>
	<li>用0.3% 油红 O (破产) 库存溶液 (重量/容积) 将0.3 克的破产, 溶解为100毫升异丙醇。</li>
</ol>2. 脂肪间质血管分数隔离
注: 该研究包括一组2例病态肥胖型糖尿病 (T2D) 和非糖尿病患者, 年龄18–65岁, 在森塔拉代谢和减肥手术中心 (森塔拉医学小组, 诺福克, VA) 进行减肥手术。排除标准包括一种自身免疫性疾病, 包括1型糖尿病、需要长期免疫抑制剂治疗的条件、抗炎药物、thiazolinendiones、有效烟草使用、慢性或急性感染或历史在过去12月内治疗的恶性肿瘤。T2D 被定义为空腹血糖为126毫克/dl 或更大, 葡萄糖200毫克/dl 或更大后2小时葡萄糖耐受性测试, 或使用抗糖尿病药物。
<ol><li>收集人网膜 (OM) 和皮下 (SC) 脂肪组织 (AT) 从人体进行减肥手术。</li>
	<li>保持 OM 和 SC 在单独的小瓶包含汉克的缓冲盐溶液与50µg/毫升青霉素/链霉素在室温下立即组织提取。</li>
	<li>当样品在组织提取后到达实验室时, 用70% 乙醇擦拭剪刀和镊子仔细清洁和去除组织中的纤维化和烧灼部分。</li>
	<li>对脂肪组织进行称量, 将其分成5克 (或更少) 整除数胶原酶消化。</li>
	<li>用两对剪刀, 在室温闪烁的小瓶中, 在5毫升的胶原酶溶液中, 切碎5克。</li>
	<li>添加额外的10毫升胶原酶溶液的切碎组织为15毫升总。</li>
	<li>在连续晃动的水浴中, 将样品消化1小时, 摄氏37摄氏度。</li>
	<li>切断20毫升注射器的尖端, 使钝端。</li>
	<li>从250µm 网格中剪下一个9厘米 x 9 厘米的正方形, 用钝端注射器将其推到50毫升圆锥管中。</li>
	<li>将样品倒入20毫升钝端注射器中, 通过250µm 尼龙网过滤成50毫升锥形管, 将脂肪细胞和基质血管干细胞从未消化的组织中分离出来。 注: 不要使用超过5克的每50毫升圆锥管, 因为网将被堵塞由于过度纤维化未消化的材料。</li>
	<li>用10毫升的 KRBBS 冲洗出闪烁的瓶子, 然后通过过滤器将其倒入收集残余细胞。</li>
	<li>在室温下孵化过滤样品5分钟。 注意: 这一步是很重要的, 让脂肪细胞漂浮在管的顶部和一些基质血管干细胞, 以解决在管的底部。</li>
	<li>利用20毫升注射器和一个20克 x 6 在吹打针去除基质血管分数层, 并将其转移到一个干净的50毫升圆锥管。</li>
	<li>添加10毫升的 KRBBS, 并允许样品坐在板凳上5分钟, 在室温下。</li>
	<li>重复步骤2.12–2.14 两次。 注: 这些步骤将确保无污染的基质血管细胞与漂浮脂肪。</li>
	<li>将两个仓库的基质血管分数保持在冰上进行进一步处理, 如下面的步骤所述。 注意: 不要把细胞放在冰上超过1小时, 因为这可能会影响细胞的生存能力。脂肪细胞可以在液态氮中被闪光冷冻, 用于长期贮存或用于实验。</li>
</ol>3. 脂肪组织内皮细胞的分离
<ol><li>将基质血管馏分 (SVF) 在 500 x g处旋转5分钟, 在4摄氏度。</li>
	<li>从离心机上取出样品, 仔细地倒上清液;保持含有 SVF 细胞的颗粒。</li>
	<li>在5毫升的 PBS 中轻轻并用重悬细胞颗粒。 注: 如果5克以上的 a 在车厂被处理成多个整除数 (见步骤 2.4), 把细胞颗粒聚集在一起。</li>
	<li>旋转样品在 500 x g 5 分钟, 在4摄氏度。</li>
	<li>移除上清, 并用重悬1毫升的 PBS 细胞, 并计数细胞。 注: 在这里, 一个自动细胞计数器用于细胞计数。细胞的生存能力是通过混合12µL 细胞悬浮与12µL 的吖啶橙/碘碘 (AO/PI) 解决方案 (见材料表)。每个车厂每克的平均单元数为: (a) OM 车厂: 1.69 x 105 @ 4.51 x 104;(b) SC 车厂: 1.24 x 105 @ 6.45 x 104。两个仓库的细胞的生存能力是 > 90%。</li>
	<li>将样品按 500 x g分5分钟, 在4摄氏度。 注: CD34 磁选择协议 (见材料表) 已适应步骤3.7–3.15。</li>
	<li>并用重悬100µL CD34 隔离缓冲器中的颗粒。 注: 总细胞计数要求以下再悬浮容量: (a) < 2 x 107个细胞: 并用重悬颗粒在100µL;(b) 2 x 108–5 x 108细胞: 并用重悬在1毫升的颗粒。在步骤3.9–3.16 中, 所用试剂的体积缩小为 < 2 x 107细胞。</li>
	<li>添加10µL CD34 鸡尾酒, 并在室温下孵化样品15分钟。</li>
	<li>在室温下添加5µL 的磁性珠子, 并孵育样品10分钟。</li>
	<li>在每个样品中加入2.5 毫升的 CD34 隔离缓冲器, 将样品放入磁铁中, 在室温下放置5分钟。</li>
	<li>将磁铁反转5秒, 将隔离缓冲器倒掉。</li>
	<li>从磁铁中取出样品, 重复步骤3.10 和 3.11 4x。</li>
	<li>从磁铁中取出管子, 加入2毫升的 CD34 隔离缓冲器。</li>
	<li>将细胞以 500 x g为5分钟, 在4摄氏度。</li>
	<li>并用重悬细胞颗粒在1毫升的 CD34 隔离缓冲和计数细胞。 注: 在这里, 每克的平均细胞数是: (a) OM 车厂: 4.75 x 104 @ 3.36 x 104;(b) SC 车厂: 1.06 x 104 @ 9.53 x 103。CD31 塑料 immunobead 协议适用于步骤 3.16–3.34 (见材料表)。</li>
	<li>颗粒细胞在 500 x g 5 分钟和并用重悬的颗粒在250µL 的缓冲 B 和250µL 的洗涤缓冲器。</li>
	<li>涡流管彻底并用重悬 CD31 珠。</li>
	<li>添加20µL 的 CD31 珠。 注: 由于总单元数 > 1 x 106, 卷根据制造商的输入被缩小了。</li>
	<li>在室温下用摇摆30分钟孵化样品。</li>
	<li>将过滤器连接到无菌50毫升圆锥管。 注: 过滤器的较大开口必须在顶部。</li>
	<li>在细胞分离之前, 加入1毫升的洗涤缓冲器, 平衡过滤器。将步骤3.16 下生成的混合物应用于过滤器。</li>
	<li>用5毫升的洗涤缓冲器在圆形运动中清洗过滤器, 总容积为20毫升。将接头连接至无菌50毫升圆锥管, 并关闭鲁尔锁。</li>
	<li>将过滤器连接到接头。在滤网壁上添加1毫升洗涤缓冲器。在滤网壁上添加1毫升的活化缓冲 D。轻轻旋转样品, 在室温下孵化10分钟。</li>
	<li>添加1毫升的洗涤缓冲器。将细胞从珠子中分离出来, 吹打10x。 注意: 避免产生气泡。</li>
	<li>打开鲁尔锁, 允许分离的细胞流入50毫升锥形管。每次用1毫升的洗涤缓冲器清洗过滤器10x。</li>
	<li>丢弃接头和过滤器, 离心样品在 300 x g 10 分钟, 在4摄氏度。并用重悬细胞颗粒在2毫升完整的 EGM-2 培养基中进行培养。 注: 此时每克的平均细胞数为: (a) OM 车厂: 5.92 x 103 @ 4.27 x 103;(b) SC 车厂: 4.12 x 103 @ 2.1 x 103。两个仓库的细胞的生存能力超过90%。</li>
</ol>4. 成脂在离体内皮细胞中的诱导
<ol><li>在37摄氏度, 5% CO2孵化器中, 用完整的 EGM-2 培养基在6井组织培养处理的板材中生长细胞。每3–4天更换介质, 直到细胞达到80–90% 汇合。 注: 人口加倍是在2–3天之间。</li>
	<li>在100毫米培养皿上涂上细胞, 然后将细胞分成1:3 次, 然后将其展开。在这个阶段, 细胞在2通道上。</li>
	<li>使用自动单元格计数器计数单元格 (参见材料表)。</li>
	<li>种子大约100–200,000 细胞成6井板确保每个车厂重复井, 并培养他们在完整的 EGM-2 媒体2天。</li>
	<li>吸取任何生长培养基, 并用2毫升的 DMEM/F12 与5% 的血清和50µg/毫升的青霉素/链霉素的控制细胞, 并取代它与2毫升的成脂培养基 (见步骤 1.4) 的治疗细胞。</li>
	<li>在湿润 5% CO2孵化器中孵育37摄氏度的细胞13天, 每3到4天更换各自的培养基。 注: 在治疗4天后, 细胞将开始积聚脂质。</li>
	<li>根据公布的方法15-17进行破产和尼罗河红染色。</li>
	<li>要分离 RNA 提取的细胞, 从6井感应板中取出介质, 用1毫升的无菌 PBS 冲洗。</li>
	<li>添加350µL guanidium 硫氰酸盐-苯酚-氯仿溶液 (见材料表), 并在室温下孵化5–10分钟。</li>
	<li>用细胞刮刀刮掉盘子上的细胞, 将细胞转移到1.5 毫升的离心管上。 注: 样品可存储在-80 °c 冷冻库中, 用于 RNA 提取和以后进一步处理。</li>
	<li>使用一种适应的 RNA 萃取协议从样品中提取 mRNA (见材料表)。</li>
	<li>使用以下探针进行实时聚合酶链反应 (rt-pcr) 来评估基因表达: 脂联素、UCP-1 和 CIDEA (见材料表)。 注: 在这里, RT PCR 程序是采用以下标准协议: 变性 (95 °c; 十五年代), 退火和延长 (60 °c; 1 分钟) 40 循环。用 CT值 > 35 表达基因的样本被认为是无法检测的。</li>
</ol>

<ol>
	<li>Tang, W., 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=White+fat+progenitor+cells+reside+in+the+adipose+vasculature.">White fat progenitor cells reside in the adipose vasculature.</a> Science. 322 (5901), 583-586 (2008).</li><li>Lee, Y. H., Petkova, A. P., Granneman, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+adipogenic+niche+for+adipose+tissue+remodeling+and+restoration.">Identification of an adipogenic niche for adipose tissue remodeling and restoration.</a> Cell Metabolism. 18 (3), 355-367 (2013).</li><li>Min, S. Y., 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=Human+'brite/beige'+adipocytes+develop+from+capillary+networks,+and+their+implantation+improves+metabolic+homeostasis+in+mice.">Human 'brite/beige' adipocytes develop from capillary networks, and their implantation improves metabolic homeostasis in mice.</a> Nature Medicine. 22 (3), 312-318 (2016).</li><li>Gupta, R. K., 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=Zfp423+expression+identifies+committed+preadipocytes+and+localizes+to+adipose+endothelial+and+perivascular+cells.">Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells.</a> Cell Metabolism. 15 (2), 230-239 (2012).</li><li>Tran, K. V., 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+vascular+endothelium+of+the+adipose+tissue+gives+rise+to+both+white+and+brown+fat+cells.">The vascular endothelium of the adipose tissue gives rise to both white and brown fat cells.</a> Cell Metabolism. 15 (2), 222-229 (2012).</li><li>Rodeheffer, M. S., Birsoy, K., Friedman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+white+adipocyte+progenitor+cells+in+vivo.">Identification of white adipocyte progenitor cells in vivo.</a> Cell. 135 (2), 240-249 (2008).</li><li>Scott, M. 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>Macotela, Y., 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=Intrinsic+differences+in+adipocyte+precursor+cells+from+different+white+fat+depots.">Intrinsic differences in adipocyte precursor cells from different white fat depots.</a> Diabetes. 61 (7), 1691-1699 (2012).</li><li>Lee, Y. H., Petkova, A. P., Mottillo, E. P., Granneman, J. 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 (4), 480-491 (2012).</li><li>Xue, Y., Xu, X., Zhang, X. Q., Farokhzad, O. C., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+diet-induced+obesity+in+mice+by+adipose+tissue+transformation+and+angiogenesis+using+targeted+nanoparticles.">Preventing diet-induced obesity in mice by adipose tissue transformation and angiogenesis using targeted nanoparticles.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (20), 5552-5557 (2016).</li><li>Villaret, A., 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+tissue+endothelial+cells+from+obese+human+subjects:+differences+among+depots+in+angiogenic,+metabolic,+and+inflammatory+gene+expression+and+cellular+senescence.">Adipose tissue endothelial cells from obese human subjects: differences among depots in angiogenic, metabolic, and inflammatory gene expression and cellular senescence.</a> Diabetes. 59 (11), 2755-2763 (2010).</li><li>Miranville, A., 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=Improvement+of+postnatal+neovascularization+by+human+adipose+tissue-derived+stem+cells.">Improvement of postnatal neovascularization by human adipose tissue-derived stem cells.</a> Circulation. 110 (3), 349-355 (2004).</li><li>Sengenes, C., Lolmede, K., Zakaroff-Girard, A., Busse, R., Bouloumie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preadipocytes+in+the+human+subcutaneous+adipose+tissue+display+distinct+features+from+the+adult+mesenchymal+and+hematopoietic+stem+cells.">Preadipocytes in the human subcutaneous adipose tissue display distinct features from the adult mesenchymal and hematopoietic stem cells.</a> Journal of Cellular Physiology. 205 (1), 114-122 (2005).</li><li>Moller, D. E., Flier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance--mechanisms,+syndromes,+and+implications.">Insulin resistance--mechanisms, syndromes, and implications.</a> The New England Journal of Medicine. 325 (13), 938-948 (1991).</li><li>Pittenger, M. F., 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=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284 (5411), 143-147 (1999).</li><li>Novikoff, A. B., Novikoff, P. M., Rosen, O. M., Rubin, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+relationships+in+cultured+3T3-L1+preadipocytes.">Organelle relationships in cultured 3T3-L1 preadipocytes.</a> The Journal of Cell Biology. 87 (1), 180-196 (1980).</li><li>Satish, 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=Expression+analysis+of+human+adipose-derived+stem+cells+during+in+vitro+differentiation+to+an+adipocyte+lineage.">Expression analysis of human adipose-derived stem cells during in vitro differentiation to an adipocyte lineage.</a> BMC Medical Genomics. 8 (41), (2015).</li><li>Gimbrone, M. A., Cotran, R. S., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+vascular+endothelial+cells+in+culture.+Growth+and+DNA+synthesis.">Human vascular endothelial cells in culture. Growth and DNA synthesis.</a> The Journal of Cell Biology. 60 (3), 673-684 (1974).</li><li>Burridge, K. A., Friedman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environment+and+vascular+bed+origin+influence+differences+in+endothelial+transcriptional+profiles+of+coronary+and+iliac+arteries.">Environment and vascular bed origin influence differences in endothelial transcriptional profiles of coronary and iliac arteries.</a> American Journal of Physiology-Heart and Circulatory Physiology. 299 (3), H837-H846 (2010).</li><li>Paruchuri, 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=Human+pulmonary+valve+progenitor+cells+exhibit+endothelial/mesenchymal+plasticity+in+response+to+vascular+endothelial+growth+factor-A+and+transforming+growth+factor-beta2.">Human pulmonary valve progenitor cells exhibit endothelial/mesenchymal plasticity in response to vascular endothelial growth factor-A and transforming growth factor-beta2.</a> Circulation Research. 99 (8), 861-869 (2006).</li><li>Hewett, P. W., Murray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+microvessel+endothelial+cells:+isolation,+culture+and+characterization.">Human microvessel endothelial cells: isolation, culture and characterization.</a> In Vitro Cellular &amp; Development Biology. 29 (11), 823-830 (1993).</li><li>Hewett, P. W., Murray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lung+microvessel+endothelial+cells:+isolation,+culture,+and+characterization.">Human lung microvessel endothelial cells: isolation, culture, and characterization.</a> Microvascular Research. 46 (1), 89-102 (1993).</li><li>Hewett, P. W., Murray, J. C., Price, E. A., Watts, M. E., Woodcock, M. <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+characterization+of+microvessel+endothelial+cells+from+human+mammary+adipose+tissue.">Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue.</a> In Vitro Cellular &amp; Development Biology. 29 (4), 325-331 (1993).</li><li>Xiao, L., McCann, J. V., 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=Isolation+and+culture+expansion+of+tumor-specific+endothelial+cells.">Isolation and culture expansion of tumor-specific endothelial cells.</a> Journal of Visualized Experiments. (105), e53072 (2015).</li><li>Berry, R., Rodeheffer, M. S., Rosen, C. J., Horowitz, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+residing+progenitors+(adipocyte+lineage+progenitors+and+adipose+derived+stem+cells+(ADSC).">Adipose tissue residing progenitors (adipocyte lineage progenitors and adipose derived stem cells (ADSC).</a> Current Molecular Biology Reports. 1 (3), 101-109 (2015).</li><li>Zhou, 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=In+vitro+evaluation+of+endothelial+progenitor+cells+from+adipose+tissue+as+potential+angiogenic+cell+sources+for+bladder+angiogenesis.">In vitro evaluation of endothelial progenitor cells from adipose tissue as potential angiogenic cell sources for bladder angiogenesis.</a> PLoS One. 10 (2), e0117644 (2015).</li><li>Curat, C. A., 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=From+blood+monocytes+to+adipose+tissue-resident+macrophages:+induction+of+diapedesis+by+human+mature+adipocytes.">From blood monocytes to adipose tissue-resident macrophages: induction of diapedesis by human mature adipocytes.</a> Diabetes. 53 (5), 1285-1292 (2004).</li><li>Sidney, L. E., Branch, M. J., Dunphy, S. E., Dua, H. S., Hopkinson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+evidence+for+CD34+as+a+common+marker+for+diverse+progenitors.">Concise review: evidence for CD34 as a common marker for diverse progenitors.</a> Stem Cells. 32 (6), 1380-1389 (2014).</li><li>Traktuev, D. O., 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=A+population+of+multipotent+CD34-positive+adipose+stromal+cells+share+pericyte+and+mesenchymal+surface+markers,+reside+in+a+periendothelial+location,+and+stabilize+endothelial+networks.">A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks.</a> Circulation Research. 102 (1), 77-85 (2008).</li><li>Frontini, A., Giordano, A., 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=Endothelial+cells+of+adipose+tissues:+a+niche+of+adipogenesis.">Endothelial cells of adipose tissues: a niche of adipogenesis.</a> Cell Cycle. 11 (15), 2765-2766 (2012).</li><li>Sengenes, C., Miranville, A., Lolmede, K., Curat, C. A., Bouloumie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+endothelial+cells+in+inflamed+adipose+tissue.">The role of endothelial cells in inflamed adipose tissue.</a> Journal of Internal Medicine. 262 (4), 415-421 (2007).</li><li>Ong, W. K., 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=Identification+of+specific+cell-surface+markers+of+adipose-derived+stem+cells+from+subcutaneous+and+visceral+fat+depots.">Identification of specific cell-surface markers of adipose-derived stem cells from subcutaneous and visceral fat depots.</a> Stem Cell Reports. 2 (2), 171-179 (2014).</li><li>Ong, W. K., Sugii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+fatty+potentials+for+therapy.">Adipose-derived stem cells: fatty potentials for therapy.</a> The International Journal of Biochemistry &amp; Cell Biology. 45 (6), 1083-1086 (2013).</li><li>Tchkonia, 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=Abundance+of+two+human+preadipocyte+subtypes+with+distinct+capacities+for+replication,+adipogenesis,+and+apoptosis+varies+among+fat+depots.">Abundance of two human preadipocyte subtypes with distinct capacities for replication, adipogenesis, and apoptosis varies among fat depots.</a> American Journal of Physiology-Endocrinoloy and Metabolism. 288 (1), E267-E277 (2005).</li><li>van de Vyver, M., Andrag, E., Cockburn, I. L., Ferris, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazolidinedione-induced+lipid+droplet+formation+during+osteogenic+differentiation.">Thiazolidinedione-induced lipid droplet formation during osteogenic differentiation.</a> Journal of Endocrinology. 223 (2), 119-132 (2014).</li></ol>

作者没有什么可透露的。

本文的重点是为 CD34+CD31+ 内皮细胞从内脏和皮下的脂肪组织的分离、扩增和脂肪诱导提供一种方法。
已报告的方法, 以隔离从不同的啮齿动物或人类的血管床内皮细胞, 主要涉及使用 CD31 抗体荧光标记或耦合磁性珠18,19,20,21,22,23....

最近的证据显示, 在小鼠和人类中, 在脂肪组织血管中的细胞子集可以被区分成白色或米色/褐色脂肪体1,2,3。这种细胞的表型是一个争议的主题, 有证据支持内皮细胞, 平滑肌/周细胞, 或一光谱的中间表型4,5,6,7。这种方法的发展范围是测试从肥胖人群中分离出不同脂肪库的 CD34+CD31+ 内皮细胞的脂肪电位。文献中的其他研究集中在脂肪电位的总基质血管分数或已知脂肪细胞祖细胞2,8,9。由于目前现有的技术可以专门针对脂肪组织内皮细胞的药物传递10, 了解这种细胞的潜力接受脂肪诱导向白色或米色脂肪体是重要的未来靶向治疗。
不同的组报告, CD31 和 CD34 标记作为代理人, 以隔离内皮细胞从人脂肪组织11,12,13。通常情况下, 隔离是使用两个连续步骤和一个使用磁性珠子的正选择来执行的。在本报告中, 利用 CD34+ 磁珠结合 CD31 塑料珠 immunoseparation。我们发现这种技术优于顺序磁性 immunoseparation 的保存典型的鹅卵石内皮形态学。此外, 我们能够产生足够的细胞, 以扩大和脂肪诱导开始从尽可能少的脂肪 g。小样本皮下脂肪活检足以为下游应用生产所需的细胞数量。这方面是潜在的重要, 特别是如果这一方法将被用于筛选对脂肪诱导的人的对象的反应。
与文献报道的其他系统不同, 这种方法仅利用两种成分脂肪诱导 CD34+CD31+ 细胞: PPARγ激动剂-罗格列酮-和人胰岛素。重要的是, 使用胰岛素的数量在正常/高范围内循环后吸收胰岛素的人14。Akt 磷酸化测定的细胞体外对胰岛素的反应程度与它们对诱导性鸡尾酒反应的能力没有关联。有趣的是, 使用这种诱导鸡尾酒和实验条件, 由白色和米色/褐色细胞的混合, 由细胞内脂质滴的大小和数量和分子标记的表达决定。这种简单和经济高效的诱导协议以及对应答器细胞表型的定量评估 (白色与米色) 允许筛选可能改变差异性米色平衡的药剂。: 白色脂肪细胞。
该方法还为了解人体脂肪组织中血管内皮祖细胞成脂的基本机制提供了一种平移方法。使用这种特殊的隔离/分化技术, 调查人员可以审问各种途径, 负责成脂在血管内皮细胞的子集, 从不同脂肪库的瘦身和肥胖的人。

<table><tbody><tr><td> &lt;p&gt;&lt;strong&gt;Large Equipment&lt;/strong&gt;&lt;/p&gt; </td><td></td><td></td><td></td></tr><tr><td> &lt;p&gt;Biosafety Cabinet&lt;/p&gt; </td><td> &lt;p&gt;Nuaire&lt;/p&gt; </td><td> &lt;p&gt;nu-425-400&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Cell Culture Incubator&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;800 DH&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Water Bath&lt;/p&gt; </td><td> &lt;p&gt;Forma Scientific&lt;/p&gt; </td><td> &lt;p&gt;2568&lt;/p&gt; </td><td> &lt;p&gt;Reciprocal Shaker&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;RT-PCR Machine&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;CFX96-C1000&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Electrophoresis Box&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;Mini PROTEAN 3 Cell&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Transblot Box&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;Mini Trans-Blot Cell&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Electrophoresis Power Supply&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;PowerPac Basic&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;ELISA Reader&lt;/p&gt; </td><td> &lt;p&gt;Molecular Devices&lt;/p&gt; </td><td> &lt;p&gt;SpectraMax M5&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Blot Reader&lt;/p&gt; </td><td> &lt;p&gt;LI-COR&lt;/p&gt; </td><td> &lt;p&gt;Odyssey&lt;/p&gt; </td><td> &lt;p&gt;Near Infrared&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Refrigerated Centrifuge&lt;/p&gt; </td><td> &lt;p&gt;Eppendorf&lt;/p&gt; </td><td> &lt;p&gt;5810 R&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Tabletop Centrifuge&lt;/p&gt; </td><td> &lt;p&gt;Eppendorf&lt;/p&gt; </td><td> &lt;p&gt;MiniSpin Plus&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Fluorescent Microscope&lt;/p&gt; </td><td> &lt;p&gt;Olympus&lt;/p&gt; </td><td> &lt;p&gt;BX50&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Inverted Microscope&lt;/p&gt; </td><td> &lt;p&gt;Nikon&lt;/p&gt; </td><td> &lt;p&gt;TMS&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;&lt;strong&gt;KRBSS Buffer&lt;/strong&gt;&lt;/p&gt; </td><td></td><td></td><td></td></tr><tr><td> &lt;p&gt;HEPES&lt;/p&gt; </td><td> &lt;p&gt;Research Products International&lt;/p&gt; </td><td> &lt;p&gt;H75030&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Sodium bicarbonate&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;792519&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Calcium chloride dihydrate&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;C7902&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Potassium phosphate monobasic&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;P5655&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Magnesium sulfate&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;M2643&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Sodium chloride&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;746398&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Sodium phosphate monobasic monohydrate&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;S9638&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Potassium chloride&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;P9333&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Glucose&lt;/p&gt; </td><td> &lt;p&gt;Acros Organics&lt;/p&gt; </td><td> &lt;p&gt;410950010&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Adenosine&lt;/p&gt; </td><td> &lt;p&gt;Acros Organics&lt;/p&gt; </td><td> &lt;p&gt;164040250&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Bovine Serum Albumin&lt;/p&gt; </td><td> &lt;p&gt;GE Healthcare Bio-Sciences&lt;/p&gt; </td><td> &lt;p&gt;SH30574.02&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Penicillin/Streptomycin&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;15070063&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;&lt;strong&gt;Tissue Digestion&lt;/strong&gt;&lt;/p&gt; </td><td></td><td></td><td></td></tr><tr><td> &lt;p&gt;20 mL Syringe&lt;/p&gt; </td><td> &lt;p&gt;Global Medical&lt;/p&gt; </td><td> &lt;p&gt;67-2020&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Nylon Mesh, 250 &amp;micro;m&lt;/p&gt; </td><td> &lt;p&gt;Sefar&lt;/p&gt; </td><td> &lt;p&gt;03-250/50&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Pipetting Needles&lt;/p&gt; </td><td> &lt;p&gt;Popper&lt;/p&gt; </td><td> &lt;p&gt;7934&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Fine Scissors&lt;/p&gt; </td><td> &lt;p&gt;Fine Science Tools&lt;/p&gt; </td><td> &lt;p&gt;14058-11&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Tissue Forceps&lt;/p&gt; </td><td> &lt;p&gt;George Tiemann &amp;amp; Co&lt;/p&gt; </td><td> &lt;p&gt;160-20&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Collagenase, Type I&lt;/p&gt; </td><td> &lt;p&gt;Worthington Biochemical&lt;/p&gt; </td><td> &lt;p&gt;LS004196&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Petri Dishes, 100 mm&lt;/p&gt; </td><td> &lt;p&gt;USA Scientific&lt;/p&gt; </td><td> &lt;p&gt;5666-4160&lt;/p&gt; </td><td> &lt;p&gt;TC Treated&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Eppendorf Tubes, 1.5 mL&lt;/p&gt; </td><td> &lt;p&gt;USA Scientific&lt;/p&gt; </td><td> &lt;p&gt;1615-5500&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Conical Tubes, 15 mL&lt;/p&gt; </td><td> &lt;p&gt;Nest Scientific&lt;/p&gt; </td><td> &lt;p&gt;601052&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Conical Tubes, 50 mL&lt;/p&gt; </td><td> &lt;p&gt;Nalgene&lt;/p&gt; </td><td> &lt;p&gt;3119-0050&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Scintillation Vials&lt;/p&gt; </td><td> &lt;p&gt;Kimble&lt;/p&gt; </td><td> &lt;p&gt;74505-20&lt;/p&gt; </td><td> &lt;p&gt;Tissue Dicing&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;&lt;strong&gt;Cell Isolation&lt;/strong&gt;&lt;/p&gt; </td><td></td><td></td><td></td></tr><tr><td> &lt;p&gt;Cellometer&lt;/p&gt; </td><td> &lt;p&gt;Nexcelom&lt;/p&gt; </td><td> &lt;p&gt;Auto 2000&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Cellometer Slides&lt;/p&gt; </td><td> &lt;p&gt;Nexcelom&lt;/p&gt; </td><td> &lt;p&gt;CHT4-SD100-002&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Cellometer Viability Stain&lt;/p&gt; </td><td> &lt;p&gt;Nexcelom&lt;/p&gt; </td><td> &lt;p&gt;CS2-0106-5mL&lt;/p&gt; </td><td> &lt;p&gt;Acridine Orange/Propidium Iodine&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-CD34 Magnetic Beads&lt;/p&gt; </td><td> &lt;p&gt;StemCell Technologies&lt;/p&gt; </td><td> &lt;p&gt;18056&lt;/p&gt; </td><td> &lt;p&gt;Kit&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;EasySep Magnet&lt;/p&gt; </td><td> &lt;p&gt;StemCell Technologies&lt;/p&gt; </td><td> &lt;p&gt;18000&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Anti-CD31 Plastic Beads&lt;/p&gt; </td><td> &lt;p&gt;pluriSelect USA&lt;/p&gt; </td><td> &lt;p&gt;19-03100-10&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;pluriSelect 10x Wash Buffer&lt;/p&gt; </td><td> &lt;p&gt;pluriSelect USA&lt;/p&gt; </td><td> &lt;p&gt;60-00080-10&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;pluriSelect Connector Ring&lt;/p&gt; </td><td> &lt;p&gt;pluriSelect USA&lt;/p&gt; </td><td> &lt;p&gt;41-50000-03&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;pluriSelect Detachment Buffer&lt;/p&gt; </td><td> &lt;p&gt;pluriSelect USA&lt;/p&gt; </td><td> &lt;p&gt;60-00046-12&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;pluriSelect Incubation Buffer&lt;/p&gt; </td><td> &lt;p&gt;pluriSelect USA&lt;/p&gt; </td><td> &lt;p&gt;60-00060-12&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;pluriSelect S Cell Strainer&lt;/p&gt; </td><td> &lt;p&gt;pluriSelect USA&lt;/p&gt; </td><td> &lt;p&gt;43-50030-03&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;&lt;strong&gt;Cell Culture&lt;/strong&gt;&lt;/p&gt; </td><td></td><td></td><td></td></tr><tr><td> &lt;p&gt;6-well Plates&lt;/p&gt; </td><td> &lt;p&gt;USA Scientific&lt;/p&gt; </td><td> &lt;p&gt;CC7682-7506&lt;/p&gt; </td><td> &lt;p&gt;TC Treated&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;4-well chambered slides&lt;/p&gt; </td><td> &lt;p&gt;Corning Life Sciences&lt;/p&gt; </td><td> &lt;p&gt;354559&lt;/p&gt; </td><td> &lt;p&gt;Fibronectin coated&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;4-well chambered slides&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;154526PK&lt;/p&gt; </td><td> &lt;p&gt;Uncoated glass&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Human Adipose Microvascular Endothelial Cells (HAMVEC)&lt;/p&gt; </td><td> &lt;p&gt;Sciencell Research Laboratories&lt;/p&gt; </td><td> &lt;p&gt;7200&lt;/p&gt; </td><td> &lt;p&gt;Primary cell line&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Endothelial Cell Media (ECM)&lt;/p&gt; </td><td> &lt;p&gt;ScienCell Research Laboratories&lt;/p&gt; </td><td> &lt;p&gt;1001&lt;/p&gt; </td><td> &lt;p&gt;Complete Kit&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;DMEM/F12 Basal Media&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;11320082&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Fetal Bovine Serum (FBS)&lt;/p&gt; </td><td> &lt;p&gt;Rocky Mountain Biologicals&lt;/p&gt; </td><td> &lt;p&gt;FBS-BBT&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Insulin&lt;/p&gt; </td><td> &lt;p&gt;Lilly&lt;/p&gt; </td><td> &lt;p&gt;U-100&lt;/p&gt; </td><td> &lt;p&gt;Humalog&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Rosiglitazone&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;R2408&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;&lt;strong&gt;Cell Analysis&lt;/strong&gt;&lt;/p&gt; </td><td></td><td></td><td></td></tr><tr><td> &lt;p&gt;Oil Red O Dye&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;O0625&lt;/p&gt; </td><td> &lt;p&gt;Prepared in isopropanol&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;96 well plates&lt;/p&gt; </td><td> &lt;p&gt;USA Scientific&lt;/p&gt; </td><td> &lt;p&gt;1837-9600&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;96 well PCR plates&lt;/p&gt; </td><td> &lt;p&gt;Genesee Scientific&lt;/p&gt; </td><td> &lt;p&gt;24-300&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;RNA Extraction&lt;/p&gt; </td><td> &lt;p&gt;Zymo Research&lt;/p&gt; </td><td> &lt;p&gt;R2072&lt;/p&gt; </td><td> &lt;p&gt;Kit&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;cDNA Synthesis&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;1708841&lt;/p&gt; </td><td> &lt;p&gt;Supermix&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;JumpStart PCR Polymerase&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;D9307-250UN&lt;/p&gt; </td><td> &lt;p&gt;Hot start, with PCR Buffer N&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Magnesium Chloride Solution&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;M8787-5ML&lt;/p&gt; </td><td> &lt;p&gt;3 mM final in PCR reaction&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;dNTPs&lt;/p&gt; </td><td> &lt;p&gt;Promega&lt;/p&gt; </td><td> &lt;p&gt;U1515&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;TaqMan AdipoQ&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;Hs00605917_m1&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;TaqMan CIDEA&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;Hs00154455_m1&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;TaqMan RPL27&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;Hs03044961_g1&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;TaqMan UCP1&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;Hs00222453_m1&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;BCA Assay&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;QPBCA-1KT&lt;/p&gt; </td><td> &lt;p&gt;Kit&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Bis-acrylamide&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;1610146&lt;/p&gt; </td><td> &lt;p&gt;40% stock solution&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Ammonium Persulfate&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;1610700&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;TEMED&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;1610800&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Tris&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;T1503&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Glycine&lt;/p&gt; </td><td> &lt;p&gt;BIO-RAD&lt;/p&gt; </td><td> &lt;p&gt;1610718&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Sodium Dodecal Sulfate&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;L3771&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;EDTA&lt;/p&gt; </td><td> &lt;p&gt;Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;S311-100&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Bromophenol Blue&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;B8026&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Blot Membrane&lt;/p&gt; </td><td> &lt;p&gt;EMD Millipore&lt;/p&gt; </td><td> &lt;p&gt;IPFL00010&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Methanol&lt;/p&gt; </td><td> &lt;p&gt;Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;A452-SK4&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Odyssey Blocking Buffer, Tris&lt;/p&gt; </td><td> &lt;p&gt;LI-COR&lt;/p&gt; </td><td> &lt;p&gt;927-50000&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Anti-AKT antibody&lt;/p&gt; </td><td> &lt;p&gt;Cell Signaling Technology&lt;/p&gt; </td><td> &lt;p&gt;2920S&lt;/p&gt; </td><td> &lt;p&gt;Mouse monoclonal&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-pAKT antibody&lt;/p&gt; </td><td> &lt;p&gt;Cell Signaling Technology&lt;/p&gt; </td><td> &lt;p&gt;9271S&lt;/p&gt; </td><td> &lt;p&gt;Rabbit polyclonal&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-UCP1 antibody&lt;/p&gt; </td><td> &lt;p&gt;Abcam&lt;/p&gt; </td><td> &lt;p&gt;ab10983&lt;/p&gt; </td><td> &lt;p&gt;Rabbit polyclonal&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-Mouse IgG antibody&lt;/p&gt; </td><td> &lt;p&gt;LI-COR&lt;/p&gt; </td><td> &lt;p&gt;926-68070&lt;/p&gt; </td><td> &lt;p&gt;Goat Polyclonal, IRDye 680RD&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-Rabbit IgG antibody&lt;/p&gt; </td><td> &lt;p&gt;LI-COR&lt;/p&gt; </td><td> &lt;p&gt;926-32211&lt;/p&gt; </td><td> &lt;p&gt;Goat Polyclonal, IRDye 800CW&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Anti-Rabbit IgG antibody&lt;/p&gt; </td><td> &lt;p&gt;Jackson ImmunoResearch&lt;/p&gt; </td><td> &lt;p&gt;111-025-003&lt;/p&gt; </td><td> &lt;p&gt;Goat Polyclonal, TRITC&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Phosphate Buffer Saline&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;10010049&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;37% Formaldehyde Solution&lt;/p&gt; </td><td> &lt;p&gt;Electron Microscopy Sciences&lt;/p&gt; </td><td> &lt;p&gt;15686&lt;/p&gt; </td><td> &lt;p&gt;4% solution for cell fixation&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Normal Goat Serum&lt;/p&gt; </td><td> &lt;p&gt;Vector Laboratories&lt;/p&gt; </td><td> &lt;p&gt;S-1000&lt;/p&gt; </td><td> &lt;p&gt;10% blocking solution&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Triton X-100&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;X-100&lt;/p&gt; </td><td> &lt;p&gt;0.1% permeabilization solution&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;DAPI&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;D1306&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Calcein AM&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;65-0853-39&lt;/p&gt; </td><td> &lt;p&gt;Cell fluorescent visualization&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;Matrigel Basement Membrane Matrix&lt;/p&gt; </td><td> &lt;p&gt;Corning Life Sciences&lt;/p&gt; </td><td> &lt;p&gt;356231&lt;/p&gt; </td><td> &lt;p&gt;Growth factor reduced&lt;/p&gt; </td></tr><tr><td> &lt;p&gt;DiI labeled Acetylated LDL&lt;/p&gt; </td><td> &lt;p&gt;Thermo-Fisher Scientific&lt;/p&gt; </td><td> &lt;p&gt;L3484&lt;/p&gt; </td><td></td></tr></tbody></table>

isolation, expansion, and adipogenic induction of cd34+cd31+ endothelial cells from human omental and subcutaneous adipose tissue

肥胖伴随着脂肪组织的广泛重塑, 主要是通过脂肪细胞肥大。极端脂肪细胞的生长导致胰岛素、局部缺氧和炎症反应不良。通过刺激祖细胞功能性白细胞的分化, 可以预防脂肪细胞的自由基肥大, 从而可以改善脂肪组织的代谢健康, 同时减少炎症。另外, 通过刺激米色/褐色脂肪细胞的分化, 总能量消耗可以增加, 导致体重减轻。这种方法可以防止肥胖症的发病率, 如2型糖尿病和心血管疾病的发展。
本文描述了 CD31 和 CD34 标记的人脂肪组织内皮细胞的一个子集对白色和米色脂肪体的分离、扩增和分化。该方法相对便宜, 不劳动密集型。它需要进入人体脂肪组织和皮下仓库是适合取样。对于这个协议, 肥胖症患者的新鲜脂肪组织样本 [身体质量指数 (BMI) > 35] 是在减肥手术过程中收集的。使用序贯 immunoseparation 从基质血管的分数, 足够的细胞产生的小到 2–3 g 的脂肪。这些细胞可以在10–14天内进行培养, 可以冷冻保存, 并保留其脂肪的性质, 传代直至通过5–6。细胞被治疗14天与脂肪鸡尾酒使用人胰岛素和 PPARγ激动剂-罗格列酮的组合。
这种方法可用于获取分子机制的概念实验的证明, 推动脂肪内皮细胞的脂肪反应, 或筛选新的药物, 可以提高脂肪反应, 无论是对白色或米色/棕色脂肪细胞分化。使用小皮下活检, 这种方法可用于筛选非应答者的临床试验目的是刺激米色/棕色和白色脂肪细胞的治疗肥胖和共同疾病。

我们的协议旨在提供一种体外方法, 以确定 CD34+CD31+ 血管细胞从不同的储存库的人脂肪组织的脂肪电位。图 1A显示了简化流程图。第一步使用阳性选择的 CD34 表达细胞导致 > 95% CD34+ 细胞在人口的新鲜隔离细胞 (图 1A)。重要的是, 这个标记是丢失后, 细胞培养了几个段落。由于 CD34 是多种造血和非造血祖细胞的常见标记, 因?...

Watch this Scientific Journal Video about Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue at JoVE.com

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

脂肪组织血管祖细胞中白、米色脂肪体的分化具有改善肥胖代谢的潜力。我们描述了 CD34+CD31+ 内皮细胞与人的脂肪分离的协议, 并为随后的体外扩增和分化为白色和米色脂肪。讨论了几种下游应用。

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 ...

isolation-expansion-adipogenic-induction-cd34-cd31-endothelial-cells

Research

JoVE Journal

Immunology and Infection

11.7K 次观看.  Eastern Virginia Medical School. 脂肪组织血管祖细胞中白、米色脂肪体的分化具有改善肥胖代谢的潜力。我们描述了 CD34+CD31+ 内皮细胞与人的脂肪分离的协议, 并为随后的体外扩增和分化为白色和米色脂肪。讨论了几种下游应用。

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

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

隔离和人力富集脂肪基质细胞的成骨增强

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

科研

发育生物学

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 ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

免疫与感染

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

生物学

<meta charset="utf8"></head><body>人 SATMVEC：分离和脂肪细胞串扰研究

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

<meta charset="utf8"></head><body>作者聚焦：通过皮下脂肪组织微血管研究洞察心脏代谢疾病

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. ...

医学

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

从不同的脂肪库分离和鉴定血管内皮细胞，用于下游应用

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

脂肪组织来源的干细胞分离和表征

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

作者聚焦:分离和鉴定源自 Sprague Dawley 大鼠脂肪组织的间充质干细胞  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

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

摘要

探索更多视频

此视频中的章节

隔离和人力富集脂肪基质细胞的成骨增强

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

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰

从不同的脂肪库分离和鉴定血管内皮细胞，用于下游应用

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定