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Time-serial analysis of position of LDL and infiltration of monocyte in vascular wall by laser scanning confocal microscope

激光扫描共聚焦显微镜对血管壁LDL位置及单核细胞浸润的时间序列分析

基本信息

批准号：
06558129
负责人：
TSUJIOKA Katsuhiko
金额：
$ 6.78万
依托单位：
Kawasaki Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06558129/
关键词：
localization atherosclerosis local flow normal LDL modified LDL monocyte macrophage

项目摘要

Recently vascular wall attracts many research workers, as vascular endothelial cell has been found to regulate vascular tone by production and release of physiologically active substances, such as endothelin and nitric oxide. In addition, vascular wall is not only regulator of flow distribution but also place where atherosclerosis develops. Phagocyte takes up modified low density lipoprotein (LDL) in the vascular wall to become fatty streak. The purpose of this study was to three-dimensionally observe movement of normal LDL and modified LDL into vascular wall and attachment and invasion of monocyte by using laser scanning confocal microscope, which can reconstruct three-dimensional image by optical sectioning, and analyze the difference in dynamics of normal and modified LDLs infiltration and monocyte movement at three different places where local flow condition is different.(1) Development of a system for quantitative analysis of three-dimensional image : we developed a system which can analyze three-dimensional image of nuclei of vascular endothelial cell and monocyte, and dynamic distribution of normal and modified LDLs.(2) Experiment : fluorescence-labeled normal or modified LDL was injected into tail vein of anesthetized rats. Aortorenal bifurcation was perfusion fixed at 75 mmHg, and cell nucleus was stained by bisBenzimide. We examined adhesion and infiltration of monocyte and movement of normal and modified LDLs at atherosclerosis-prone and non-prone areas in three-dimensional fashion.Distribution of both normal or modified LDL was more dense at intima than media and was the most dense at the atherosclerosis-prone area. Normal LDL scarcely distributed at media. Attachment and infiltration of monocyte was time-serially observed by three-dimensional analysis of images obtained by laser scanning confocal microscope.

近年来，血管壁的研究引起了人们的广泛关注，因为血管内皮细胞通过产生和释放内皮素和一氧化氮等生理活性物质来调节血管张力。此外，血管壁不仅是血流分布的调节器，也是动脉粥样硬化发生发展的场所。吞噬细胞摄取血管壁中修饰的低密度脂蛋白（LDL），形成脂纹。本研究旨在利用激光扫描共聚焦显微镜，通过光学切片重建三维图像，对正常LDL和修饰LDL向血管壁内的运动及单核细胞的附着和侵袭进行三维观察，分析在局部血流条件不同的三个不同部位，正常LDL和修饰LDL的浸润及单核细胞运动的动力学差异。(1)三维图像定量分析系统的研制：我们研制了一套能分析血管内皮细胞和单核细胞核的三维图像，以及正常和修饰LDL的动态分布的系统。(2)实验：麻醉大鼠尾静脉注射荧光标记的正常或修饰LDL。将主动脉肾分叉部灌注固定在75 mmHg下，并用双苯甲亚胺染色细胞核。我们检测了单核细胞的粘附和浸润以及正常和修饰的LDL在动脉粥样硬化易发区和非易发区的三维运动。正常或修饰的LDL在内膜的分布比中膜更密集，在动脉粥样硬化易发区最密集。正常LDL几乎不分布于中膜。通过激光扫描共聚焦显微镜获得的图像的三维分析，观察单核细胞的附着和浸润的时间序列。