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

近年来，由于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，血管壁吸引了许多研究工作者。此外，血管壁不仅是血流分布的调节剂，也是动脉粥样硬化发生的地方。吞噬细胞摄取血管壁上修饰过的低密度脂蛋白（LDL），形成脂肪条纹。本研究的目的是利用激光扫描共聚焦显微镜，三维观察正常LDL和修饰LDL进入血管壁的运动以及单核细胞的附着和侵袭，通过光学切片重建三维图像，分析正常LDL和修饰LDL在局部血流条件不同的三种不同部位浸润和单核细胞运动的动力学差异。(1)三维图像定量分析系统的开发：开发了血管内皮细胞和单核细胞核三维图像分析系统，正常和修饰ldl动态分布分析系统。(2)实验：将荧光标记的正常LDL或修饰LDL注入麻醉大鼠尾静脉。主动脉肾分叉灌注固定75mmhg，双苯并胺染色细胞核。我们以三维方式检测了动脉粥样硬化易发区和非易发区单核细胞的粘附和浸润以及正常和修饰ldl的运动。正常或修饰LDL在内膜的分布均比中膜密度大，且在动脉粥样硬化易发区域密度最大。正常LDL在培养基中几乎不分布。通过激光扫描共聚焦显微镜三维图像的三维分析，时间序列观察单核细胞的附着和浸润。

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.