STUDY ON MICRO-BIOMECHANICS OF PERMEABILITY OF ENDOTHELIAL CELLS

大分子跨内皮细胞转运在动脉粥样硬化性病变的发生发展中起着重要作用，因为它是向动脉壁转运的主要屏障。大分子的运输途径包括吞饮小泡和细胞间连接，其剪切依赖性尚不清楚。激光共聚焦扫描显微镜的优点是可以在细胞内原位观察到大分子的传输。在渗透率宏观测量的基础上，进行了定性微观观察和定量评价。通过激光共聚焦扫描显微镜观察血管内皮细胞转运的微观特征，并用带有流动系统的扩散腔测量宏观通透性，以揭示大分子转运的特征。实验中使用的细胞是在第五代到第七代。用激光共聚焦扫描显微镜(MRC-600，Bio Rad Co.Ltd.)观察FITC-葡聚糖70和FITC-白蛋白转运的显微图像。盖玻片上的细胞在含有FITC-葡聚糖70的Krebs Ringer葡萄糖溶液中孵育15min，然后用KRG液冲洗3次。用相差显微镜和激光共聚焦扫描显微镜观察。用扩散室测量了FITC-葡聚糖跨细胞层转运的宏观特征，细胞内出现了大量的亮点，但细胞内连接处没有明显的亮点。此外，垂直扫描还得到了由小泡形成的通道状结构。土壤吸收对剪应力的依赖性增强，在低剪切应力下吸收增加，而在高剪切应力下吸收减少。这一显著的结果对探讨硫杆菌毒素的形成机制具有重要意义。

Macromolecular transport across the endothelial cells play an important role for the development of atherosclerotic lesionbecaouse of its principal barrier to transport to arterial vessel wall. The transport routes of macromolecule includes pinocytotic vesicles and intercellular junctions and its shear dependent property remains unknown. The advantage of confocal laser scanning microscopy is to observe the macromolecule transport in the cell in situ. we made both qualitative microscopic observation and quantitative evaluation based on the macroscopic measurements of permeability. To reveal the feature of macromolecular transport through the cultured endothelial cells, the microscopic feature of transport was observed by confocal laser scanning microscopy and the macroscopic permeability was measured by diffusion chamber with flow system.Pig aortic endothelial cells were harvested and cultured in incubator. Cells used in experiments were in the fifth to seventh passage. Microscopic views of transport of FITC-Dextran 70 and FITC-albumin were observed by the confocal laser scanning microscopy (MRC-600, Bio Rad Co. Ltd.). The cells on cover slips were incubated in the Krebs Ringer Glucose solution containing FITC-Dextran 70 for 15 minutes, and were rinsed by KRG solution for three times. Then they were observed by the phase contrast and confocal laser scanning microscopy. Macroscopic feature of transport of FITC-Dextran across the cell layr was measured by the diffusion chamberNumerous bright spots appeared inside the cells, but did not remarkably appear at the intracellular junctions. Furthermore the vertical scanning gave the channel-like structure formed by the vesicles. The dependency of uptake on the shear stress in peculiar and the uptake at the low shear stress increases, on the other land the uptake at the high shear stress decreases. This striking result gives an important significance to the mechanism of atheloscrelotic formation.