LaRusso N F, Ishii M, Vroman B T
Center for Basic Research in Digestive Diseases, Mayo Clinic, Medical School and Foundation, Rochester, Minnesota 55905.
Trans Am Clin Climatol Assoc. 1991;102:245-58; discussion 258-9.
We have developed a novel technique for the isolation from normal rat liver of morphologically polar, intrahepatic bile duct epithelial cells which exhibit clathrin-coated pits. Using electron microscopic cytochemistry, we demonstrated receptor-mediated endocytosis of EGF by cultured IBDEC. Also, using freshly isolated polar couplets of IBDEC, we demonstrated that these cells participate in fluid-phase endocytosis. Finally, using a novel fluorescence unquenching assay and our isolated bile duct epithelial cell model, we showed that secretin stimulates exocytosis in IBDEC, a finding compatible with the possibility that secretin-induced changes in ductular bile flow may occur by an exocytic process. The availability of a reproducible and reliable technique to prepare liver cell fractions highly enriched in intrahepatic bile duct epithelial cells with morphologic polarity has made it possible to do direct experiments on the functions of intrahepatic bile duct epithelial cells, including the study of plasma membrane movement (i.e., endocytosis and exocytosis). With the availability of this technique, other studies previously impossible to carry out in IBDEC are now feasible. Such studies are too numerous to mention, but would include experiments on ligand binding, transport of macromolecules, assessment of metabolic activities and toxicity studies, to name just a few. Indeed, virtually any question that has been asked about hepatocytes and addressed using isolated hepatocytes can now be directed toward isolated intrahepatic bile duct epithelial cells. Finally, the methodology described here is theoretically applicable to human liver. Indeed, intrahepatic bile duct epithelial cells are considered to be involved in the pathogenesis of several kinds of drug and immunologically induced liver diseases, including allograft rejection, primary biliary cirrhosis, and primary sclerosing cholangitis. The availability of the technology described here should make feasible direct experimental approaches to questions in all of these areas.
我们已经开发出一种新技术，可从正常大鼠肝脏中分离出形态极化的肝内胆管上皮细胞，这些细胞具有网格蛋白包被小窝。利用电子显微镜细胞化学技术，我们证明培养的肝内胆管上皮细胞（IBDEC）能通过受体介导对表皮生长因子（EGF）进行内吞作用。此外，利用新鲜分离的极化的IBDEC双联体，我们证明这些细胞参与液相内吞作用。最后，利用一种新型荧光去淬灭测定法和我们分离的胆管上皮细胞模型，我们发现促胰液素能刺激IBDEC的胞吐作用，这一发现符合促胰液素诱导的胆小管胆汁流动变化可能通过胞吐过程发生的可能性。有了一种可重复且可靠的技术来制备高度富集具有形态极性的肝内胆管上皮细胞的肝细胞组分，就有可能直接对肝内胆管上皮细胞的功能进行实验，包括对质膜运动（即内吞作用和胞吐作用）的研究。有了这项技术，以前无法在IBDEC中进行的其他研究现在也可行了。这类研究多得不胜枚举，包括配体结合实验、大分子运输、代谢活性评估和毒性研究等等。实际上，几乎任何关于肝细胞并利用分离的肝细胞进行研究的问题，现在都可以针对分离的肝内胆管上皮细胞来进行。最后，这里描述的方法理论上适用于人类肝脏。的确，肝内胆管上皮细胞被认为与几种药物和免疫诱导性肝病的发病机制有关，包括同种异体移植排斥、原发性胆汁性肝硬化和原发性硬化性胆管炎。这里描述的技术的可用性应该会使针对所有这些领域的问题采取直接实验方法成为可能。
