Uncovering the routes of distribution and metabolism of extracellular sphingosine 1-phosphate in vivo and in vitro by imaging and analyzing fluorescently-labelled sphingolipids

通过对荧光标记的鞘脂进行成像和分析，揭示细胞外 1-磷酸鞘氨醇在体内和体外的分布和代谢途径

• 批准号: 92820999
• 负责人: Professor Dr. Markus Gräler, Ph.D.
• 金额: --
• 依托单位: Sektion Experimentelle Sepsisforschung
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/92820999?language=en
• 关键词: Uncovering; routes; distribution; metabolism; extracellular

Sphingosine 1-phosphate (S1P) in blood is stored by erythrocytes, and it is inducibly released into plasma, where it acts as an extracellular stimulus for S1P-receptors. Based on preliminary data we hypothesize that blood serves as a major source for S1P, which is distributed to lymphoid and peripheral tissues and lymph. In order to define the pathways that are involved in the distribution of S1P, fluorescently-labelled sphingolipids will be used to track the release and distribution of S1P in vivo and in vitro. The release and distribution of S1P from erythrocytes into plasma and surrounding cells and tissues will be monitored in tissue sections by confocal laser scanning microscopy and in co-culture experiments by flow cytometry. Mice deficient for the sphingosine-phosphorylating enzyme sphingosine kinase 2 and for the S1P-degrading enzyme S1P-lyase will be used as recipient mice to determine the role of de- and re-phosphorylation for tissue distribution of blood-borne S1P, and for determining the routes and origin of accumulating S1P in tissues of S1P-lyase deficient mice. To uncover the metabolic fate of extracellular S1P, fluorescently-labelled sphingolipid metabolites will be identified and analyzed using fluorescence detection in high performance liquid chromatography (HPLC) combined with tripple-quadrupole mass spectrometry (Q-Trap). The subcellular localization of fluorescently-labelled sphingolipids will be analyzed by confocal laser scanning microscopy in vitro using tissue cell lines and primary cells. We hypothesize that differences in subcellular sphingolipid localization separate incorporated extracellular and endogenous sphingolipids, and determine their fate whether they are secreted, stored, transported, or degraded.

血液中的1-磷酸鞘氨酸（S1P）由红细胞储存，并诱导地释放到血浆中，在该血浆中，它充当S1P受体的细胞外刺激。基于初步数据，我们假设血液是S1P的主要来源，S1P分布在淋巴机和外周组织和淋巴上。为了定义与S1P分布相关的途径，荧光标记的鞘脂将用于跟踪体内和体外S1P的释放和分布。通过共聚焦激光扫描显微镜以及通过流式细胞仪，在组织切片中监测S1P从红细胞中的S1P释放和分布将在组织切片中监测和周围细胞和组织。缺乏鞘氨醇 - 磷酸化酶鞘氨醇激酶2和S1P降解酶S1P-乙烯酶的小鼠将用作受体小鼠，以确定脱磷酸化和再磷酸化在血液 - 培养基S1P的组织分布中的作用，并确定S1P的均值途径s1p的途径。为了揭示细胞外S1P的代谢命运，将在高性能液相色谱（HPLC）中与Tripple-Quadrupole质谱法（Q-Trap）结合使用荧光检测来鉴定并使用荧光检测来鉴定并分析荧光标记的鞘脂代谢物。使用组织细胞系和原代细胞的共聚焦激光扫描显微镜在体外进行共聚焦激光扫描显微镜分析荧光标记的鞘脂的亚细胞定位。我们假设亚细胞鞘脂定位的差异单独掺入细胞外和内源性鞘脂，并确定它们是否被分泌，存储，运输或退化，它们是否是分泌，存储，运输或退化。