QUANTITATION OF EXPRESSION OF ENDOTHELIAL CAM IN VIVO

描述（改编自研究者摘要）：内皮细胞战略性地位于心血管系统的血管内环境和每个器官的实质元素之间，并且已经积累了有说服力的证据，表明它们通过表达CAM在炎症的产生、调节和终止中发挥关键作用。该提案描述了一种独特的创新技术的开发，该技术旨在建立和验证用于检测和测量血管CAM的实验方案（即，P-选择素、E-选择素）在体内内皮细胞表面的表达。他们的具体目标集中在开发一种基于使用免疫金染色检测血管CAM的方法。CAM的免疫金标记物将被捕获在不溶性血管铸型的聚合基质内，并将通过具有高分辨率场发射SEM的反向散射电子成像（原子序数对比）进行检测。胶体金的定量将通过使用STERECON计算机程序分析立体图像来完成，并将确定内皮表面的CAM/microM（sup 2）数量。本研究的三个具体目标是：1、建立一种新的方法，用于在体内复制内皮细胞表面，以形成含有CAM免疫金标记的血管铸型，并通过立体分析定量表面CAM的密度（来自高分辨率场发射SEM的图像）; 2、明确和量化两个血管CAM的空间关系（P-选择素和E-选择素）在组成性分泌期间和用内毒素（LPS）上调后的选定时间间隔在体内个体内皮细胞表面上的表达; 3.阐明和定量组成性分泌过程中小动脉-毛细血管-毛细血管后轴中P-选择素和E-选择素的关系，并检测LPS上调后不同时间点表达的变化。

DESCRIPTION (Adapted from Investigator's Abstract): Endothelial cells are strategically located between the intravascular milieu of the cardiovascular system and the parenchyma elements of every organ, and persuasive evidence has accumulated that they play a pivotal role in the creation, modulation, and termination of inflammation through their expression of CAM. This proposal describes the development of a unique and innovative technique designed to establish and verify an experimental protocol for the detection and measurement of vascular CAM (i.e., P-selectin, E- selectin) on the surface of endothelial cells in vivo. Their specific aims are focused on the development of a methodology based on the use of immunogold staining to detect vascular CAM. The immunogold marker for the CAM will be trapped within the polymerized matrix of an insoluble vascular cast and will be detected by back scatter electron imaging (atomic number contrast) with high resolution field emission SEM. Quantitation of the colloidal gold will be accomplished by means of analysis of stereographic images using the STERECON computer program and the number of CAM/microM(sup2) of endothelial surface will be determined. The three related specific aims are: 1, to establish an innovative method for replicating the surface of endothelial cells in vivo so as to permit formation of vascular casts that contain immunogold markers for CAM and to quantitate the density of surface CAM by stereographic analysis (images from high resolution field emission SEM) using the STERECON program; 2, to clarify and quantitate the spatial relationships of two vascular CAM (P- selectin and E-selectin) on the surface of individual endothelial cells in vivo during constitutive secretion and at selected time intervals after up regulation with endotoxin (LPS); and 3, to clarify and quantitate the relationship of P-selectin and E- selectin in the arteriole-capillary-postcapillary axis during constitutive secretion and to examine alterations in expression at different time points after up regulation with LPS.