QUANTITATION OF EXPRESSION OF ENDOTHELIAL CAM IN VIVO

描述（摘自研究者摘要）：内皮细胞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，有说服力的证据表明，它们通过表达CAM在炎症的产生、调节和终止中起着关键作用。本提案描述了一种独特的创新技术的发展，旨在建立和验证一种实验方案，用于检测和测量内皮细胞表面的血管CAM（即p -选择素，E-选择素）。他们的具体目标集中在开发一种基于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中，并将通过高分辨率场发射扫描电镜的反向散射电子成像（原子序数对比）检测到。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，内皮表面的CAM/microM（sup2）的数量将被确定。三个相关的具体目标是：1，建立一种在体内复制内皮细胞表面的创新方法，以便形成含有CAM免疫金标记的血管模型，并使用STERECON程序通过立体分析（来自高分辨率场发射扫描电镜的图像）量化表面CAM的密度；2、明确和定量体内个体内皮细胞表面两种血管CAM （P-选择素和e -选择素）在组成分泌过程和内毒素（LPS）上调后在选定时间间隔内的空间关系；3、明确和量化小动脉-毛细血管-毛细血管后轴在构成分泌过程中p -选择素和E-选择素的关系，并检测LPS上调后不同时间点的表达变化。

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.