Braet F, De Zanger R, Crabbé E, Wisse E
Laboratory for Cell Biology and Histology, Free University of Brussels (VUB), Laarbeeklaan, Belgium.
J Gastroenterol Hepatol. 1995;10 Suppl 1:S3-7. doi: 10.1111/j.1440-1746.1995.tb01792.x.
Fenestrae control the exchange of fluids, dissolved compounds and small particles between the blood and the space of Disse, and are primarily limited at one side by parenchymal cells. We recently described a simple and rapid method for the isolation, purification and cultivation of rat liver sinusoidal endothelial cells. With regard to the purity and morphology of liver endothelial cells, a detailed microscopic study was performed. Purity and viability after selective adherence was 74 and 95%, respectively. Liver endothelial cell purity was further enhanced to about 95% during adherence and spreading on collagen after 8 h of culture. Liver endothelial cells isolated by this method provide a viable cell population, enabling the study of structure and function of these cells in vitro. We investigated the cytoskeleton associated with fenestrae and sieve plates of liver endothelial cells. Cultured cells were slightly fixed and treated with cytoskeleton extraction buffer containing 0.1% Triton. Whole mounts of extracted liver endothelial cells were prepared for scanning and transmission electron microscopy. Extracted liver endothelial cells show an integral, intricate cytoskeleton. Sieve plates and fenestrae are clearly delineated by cytoskeleton elements. Fenestrae are surrounded by a filamentous, fenestrae-associated cytoskeleton ring with an average filament thickness of 16 nm. Additionally, sieve plates are surrounded and delineated by microtubuli, which form a network together with additional branching cytoskeletal elements. Microtubuli are sometimes found delineating linear arrangements of fenestrae. In conclusion, liver endothelial cells possess a cytoskeleton, that defines and supports sieve plates and fenestrae. Fenestrae-associated cytoskeleton rings are involved in determining the size of fenestrae. The fenestrae-associated cytoskeleton therefore probably controls the important hepatic function of endothelial filtration.
窗孔控制着血液与狄氏间隙之间液体、溶解化合物和小颗粒的交换，并且在一侧主要受到实质细胞的限制。我们最近描述了一种分离、纯化和培养大鼠肝窦内皮细胞的简单快速方法。关于肝内皮细胞的纯度和形态，进行了详细的显微镜研究。选择性贴壁后的纯度和活力分别为74%和95%。培养8小时后，肝内皮细胞在胶原上贴壁和铺展过程中，纯度进一步提高到约95%。通过这种方法分离的肝内皮细胞提供了一个有活力的细胞群体，能够在体外研究这些细胞的结构和功能。我们研究了与肝内皮细胞窗孔和筛板相关的细胞骨架。将培养的细胞轻度固定，并用含有0.1% Triton的细胞骨架提取缓冲液处理。制备提取的肝内皮细胞整装片用于扫描电子显微镜和透射电子显微镜观察。提取的肝内皮细胞显示出完整、复杂的细胞骨架。筛板和窗孔由细胞骨架元件清晰界定。窗孔被一个丝状的、与窗孔相关的细胞骨架环包围，平均丝厚度为16 nm。此外，筛板被微管包围和界定，微管与额外的分支细胞骨架元件一起形成网络。有时会发现微管界定窗孔的线性排列。总之，肝内皮细胞拥有一种细胞骨架，它定义并支撑筛板和窗孔。与窗孔相关的细胞骨架环参与确定窗孔的大小。因此，与窗孔相关的细胞骨架可能控制着内皮过滤这一重要的肝脏功能。
