Adipose Tissue Core - Columbia

脂肪组织核心 - 哥伦比亚

基本信息

批准号：
7418671
负责人：
XAVIER PI-SUNYER
金额：
$ 12.83万
依托单位：
ST. LUKE'S-ROOSEVELT INST FOR HLTH SCIS
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-04-01 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7418671
关键词：
Academic Medical Centers Adipocytes Adipose tissue Biological Assay Biopsy Blood Vessels Cell Separation Cells Cellularity Complement Condition Digestion Dissection Elements Fatty acid glycerol esters Fluorescence-Activated Cell Sorting Freezing Funding Gene Expression Genes Histology Hospitals Human Individual Infiltration Instruction Insulin Signaling Pathway Laboratories Measurement Measures Medicine Methodology Methods Modeling Molecular Biology Morphology Mus Numbers Obesity Osmium Plasmids Postdoctoral Fellow Production Protein Chemistry Proteins Purpose RNA Rattus Recombinants Research Research Personnel Retroperitoneal Space Reverse Transcriptase Polymerase Chain Reaction Rodent Model Sampling Scientist Services Signal Transduction Site Sorting - Cell Movement Standards of Weights and Measures Students Techniques Time Tissue Sample Training Western Blotting adipokines collagenase college cost in vivo insulin signaling interest lipid metabolism lipoprotein lipase macrophage magnetic beads programs protein expression sample fixation size

项目摘要

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.

脂肪组织核心将为研究提供多种成本效益的服务人和小鼠脂肪组织。服务将包括细胞分离和分类技术，组织学，分子生物学和蛋白质化学。具体而言，新鲜孤立的人和老鼠脂肪样品将进行胶原酶消化（在最不可能改变基因的条件下表达式）将原发性脂肪细胞与基质血管分数分开。脂肪组织巨噬细胞随后将与基质血管分数分开，并用抗体涂料分离磁珠。将在整个脂肪组织及其细胞中分析基因表达使用定量“实时”逆转录酶聚合酶链反应（RT-PCR，RT-PCR， Roche Lightcycler）。 RNA拷贝数的量化将从质粒标准中得出每个感兴趣的基因的曲线。基因表达的分析将通过分析通过蛋白质印迹的关键脂肪细胞分泌产物的蛋白质表达。另外，西方印迹将用于研究胰岛素信号通路的各种成分的激活在各种体内条件下采样的脂肪组织中。定量评估巨噬细胞浸润到脂肪组织中将通过两种替代方法进行：定量RT-PCR（使用特定的巨噬细胞标记）和荧光激活的细胞分选（FACS）分析。由于以上许多技术需要新鲜获得的脂肪组织，因此这些技术在一个核心中的浓度应有利于效率并最大程度地减少处理。以下是核心提供的其他几种高度专业化的技术。脂肪细胞脂肪组织样品的oSmium固定后，大小和数量将进行量化。系列大鼠脂肪组织的活检将由Vasselli博士在体内进行。脂蛋白脂肪酶活性将约翰逊博士在冷冻脂肪组织样品中测定。重组脂肪因子产生将由Lawrence Shapiro博士提供。