Adipose Tissue Core

脂肪组织核心将提供各种具有成本效益的服务，以研究 人和小鼠的脂肪组织。服务将包括细胞分离和分选技术， 组织学、分子生物学和蛋白质化学。具体地说，新分离的人和老鼠 脂肪样本将被胶原酶消化(在最不可能改变基因的条件下 表达)将原代脂肪细胞从间质血管部分分离出来。脂肪组织 巨噬细胞随后将用抗体包被从间质血管部分分离出来。 磁珠。将分析整个脂肪组织及其细胞中的基因表达 使用实时定量逆转录聚合酶链式反应(RT-PCR， 罗氏LightCycler)。RNA拷贝数的量化将来自于质粒标准 每一个感兴趣的基因的曲线。基因表达的分析将通过分析来补充 蛋白质印迹法检测脂肪细胞关键分泌产物的蛋白表达。此外，西区 印迹将被用来研究胰岛素信号通路的各种成分的激活 在各种活体条件下采集的脂肪组织中。定量评估 巨噬细胞向脂肪组织的渗透将通过两种替代方法进行： 定量RT-PCR(使用特异性巨噬细胞标记物)和荧光激活细胞分选 (FACS)分析。由于上述许多技术都需要新获得的脂肪组织， 将这些技术集中在一个核心中应该有利于提高效率并最大限度地减少处理。 以下是Core提供的几种额外的、高度专业化的技术。脂肪细胞 大小和数量将在固定脂肪组织样本后进行量化。连载 瓦塞利博士将在活体内对大鼠脂肪组织进行活组织检查。脂蛋白脂酶活性将 由约翰逊博士在冷冻脂肪组织样本中进行检测。重组脂肪因子的生产 将由劳伦斯·夏皮罗博士提供。

The Adipose Tissue Core will provide a wide variety of cost-effective services for the study of human and mouse adipose tissue. Services will include techniques of cell separation and sorting, histology, molecular biology and protein chemistry. Specifically, freshly isolated human and mouse fat samples will be subjected to collagenase digestion (under conditions least likely to alter gene expression) to separate primary adipocytes from the stromal vascular fraction. Adipose tissue macrophages will subsequently be separated from the stromal vascular fraction with antibodycoated magnetic beads. Gene expression will be analyzed in whole adipose tissue and its cellular components using quantitative 'real-time' reverse-transcriptase polymerase chain reaction (rt-PCR, Roche LightCycler). Quantification of RNA copy number will be derived from plasmid standard curves for every gene of interest. Analysis of gene expression will be complemented by analysis of protein expression of key adipocyte secretory products by Western blotting. Additionally, Western blotting will be used to study the activation of various components of the insulin signaling pathway in adipose tissue sampled under a variety of in vivo conditions. Quantitative assessment of macrophage infiltration into adipose tissue will be performed by two alternative methods: quantitative rt-PCR (using specific macrophage markers) and Fluorescence Activated Cell Sorting (FACS) analysis. Since many of the above techniques require freshly obtained adipose tissue, the concentration of these techniques in one Core should favor efficiency and minimize handling. The following are several additional, highly specialized techniques offered by the Core. Adipocyte size and number will be quantified following osmium fixation of adipose tissue samples. Serial biopsies of rat adipose tissue will be performed in vivo by Dr. Vasselli. Lipoprotein lipase activity will be assayed in frozen adipose tissue samples by Dr. Johnson. Recombinant adipokine production will be provided by Dr. Lawrence Shapiro.