QUANTITATION OF EXPRESSION OF ENDOTHELIAL CAM IN VIVO

描述(摘自研究人员摘要)：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成分之间，有说服力的证据表明，它们通过表达细胞间黏附分子在炎症的产生、调节和终止中发挥关键作用。这项提案描述了一种独特的创新技术的发展，旨在建立和验证体内血管内皮细胞表面血管CAM(即P-选择素、E-选择素)的检测和测量的实验方案。他们的具体目标是开发一种基于免疫金染色法检测血管CAM的方法学。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子显微镜的背散射电子成像(原子序数对比)进行检测。胶体金的定量将通过使用STERECON计算机程序对立体图像的分析来完成，并将确定内皮表面的CAM/Microm(SUP2)的数量。这三个相关的具体目标是：1，建立一种体内复制内皮细胞表面的创新方法，以允许形成含有CAM免疫金标记物的血管铸型，并使用STERECON程序通过极光分析(来自高分辨率场发射扫描电子显微镜的图像)来定量表面CAM的密度；2，澄清和定量两种血管CAM(P-选择素和E-选择素)在体内单个内皮细胞的构成分泌过程中和内毒素(LPS)上调后的选定时间间隔上的空间关系；3、明确和定量P-选择素和E-选择素在构成分泌过程中微动脉-毛细血管-毛细血管后轴的关系，并检测内毒素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.