FACTORS AND GENES RESPONSIBLE FOR ADIPOCYTE COMMITMENT

影响脂肪细胞承诺的因素和基因

• 批准号: 6730265
• 负责人: M DANIEL LANE
• 金额: $ 42.58万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-15 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6730265
• 关键词: SDS polyacrylamide gel electrophoresis; adipocytes; adipose tissue; athymic mouse; bone morphogenetic proteins; gene expression; immunocytochemistry; liquid chromatography; mass spectrometry; messenger RNA; northern blottings; phosphorylation; polymerase chain reaction; posttranslational modifications; protein structure function; stem cells; tissue /cell culture; transfection; western blottings

DESCRIPTION: The long-term goal is to determine the mechanisms by which pluripotent stem cells of the vascular stroma of adipose tissue undergo commitment to the adipocyte lineage. The SPECIFIC AIMS are: 1. (A) To identify BMP4-induced genes and proteins, and (B) to identify molecules that undergo phosphorylation on serine, threonine or tyrosine residues upon BMP4 treatment in C3H10TI/2 pluripotent stem cells. 2. To determine whether expression of genes "identified" above causes commitment to the adipocyte lineage in cell culture or in vivo contexts and to determine the mechanistic basis for such commitment. 3. To identify, using a proteomic approach, secreted proteins that initiate, or are involved in, the commitment process. Changes in gene expression induced by BMP4 will be examined at both the mRNA transcript and protein levels. At the transcript level, changes in expression will be monitored with oligonucleotide arrays. Since mRNA and protein levels do not always reflect regulation by post-transcriptional mechanisms, differences in protein levels will also be directly assessed by mass spectrometry-based proteomic methods. This combined approach should not only provide verification of the transcript data, but also allow identification of molecules whose mRNA levels do not change and would otherwise be missed by microarray-based methods. Other advantages of mass spectrometry-based methods include identification of proteins that undergo post-translational modifications such as BMP4-induced phosphorylation and identification of novel molecules by de novo sequencing. Our studies should result in a detailed map of the adipocyte commitment process, as we should not only be able to identify the extracellular factors that influence this process but also elucidate the intracellular signaling pathways that are involved.

产品说明：长期目标是确定脂肪组织血管基质的多能干细胞向脂肪细胞谱系定向分化的机制。具体目标是：1。(A)鉴定BMP 4诱导的基因和蛋白质，和（B）鉴定在C3 H10 TI/2多能干细胞中BMP 4处理后经历丝氨酸、苏氨酸或酪氨酸残基磷酸化的分子。 2.确定上述“鉴定”基因的表达是否导致细胞培养或体内环境中的脂肪细胞谱系定型，并确定这种定型的机制基础。 3.使用蛋白质组学方法鉴定启动或参与定型过程的分泌蛋白。 将在mRNA转录物和蛋白质水平上检查由BMP 4诱导的基因表达的变化。在转录水平，将用寡核苷酸阵列监测表达的变化。由于mRNA和蛋白质水平并不总是反映转录后机制的调节，蛋白质水平的差异也将通过基于质谱的蛋白质组学方法直接评估。这种结合的方法不仅应该提供转录数据的验证，而且还允许鉴定其mRNA水平不改变并且否则将被基于微阵列的方法遗漏的分子。基于质谱的方法的其他优点包括鉴定经历翻译后修饰的蛋白质，例如BMP 4诱导的磷酸化和通过从头测序鉴定新分子。 我们的研究应该导致脂肪细胞定型过程的详细地图，因为我们不仅应该能够识别影响这一过程的细胞外因子，而且还可以阐明所涉及的细胞内信号通路。