Talamini M A, Kappus B, Hubbard A
Johns Hopkins University, School of Medicine, Baltimore, MD 21287, USA.
Hepatology. 1997 Jan;25(1):167-72. doi: 10.1002/hep.510250131.
We have evaluated the biochemical, morphological, and functional redevelopment of polarity in freshly isolated hepatocytes cultured using a double layer collagen gel sandwich technique. Western blot analysis showed increased cellular levels of the cell adhesion protein uvomorulin as cultured hepatocytes repolarized. Immunofluorescence studies using antibodies against domain-specific membrane proteins showed polarity as early as 48 hours, although the pattern of the polymeric Immunoglobulin-A receptor (pIgA-R) differed from in vivo liver. Electron microscopy showed developing bile canaliculi at 1 day. However, the functional presence of tight junctions was absent at 1 day, but present at 5 days. We further showed functional polarity to be present at 4 days by documenting the ability of cultured hepatocytes to metabolize and excrete fluorescein diacetate into visible bile canaliculi. We conclude that hepatocytes cultured appropriately develop morphological and functional polarity. Hepatocyte culture is therefore a useful tool for the study of mechanisms responsible for the development of polarized function.
我们使用双层胶原凝胶夹心技术对新鲜分离的培养肝细胞极性的生化、形态和功能重建进行了评估。蛋白质免疫印迹分析显示，随着培养肝细胞重新极化，细胞黏附蛋白埃-钙黏蛋白的细胞水平升高。使用针对结构域特异性膜蛋白的抗体进行的免疫荧光研究显示，早在48小时就出现了极性，尽管聚合免疫球蛋白A受体（pIgA-R）的模式与体内肝脏不同。电子显微镜显示在第1天出现了发育中的胆小管。然而，紧密连接在第1天不存在功能性存在，但在第5天存在。通过记录培养肝细胞将荧光素二乙酸酯代谢并排泄到可见胆小管中的能力，我们进一步证明在第4天存在功能极性。我们得出结论，适当培养的肝细胞会发育出形态和功能极性。因此，肝细胞培养是研究负责极化功能发展机制的有用工具。
