ISOLATION AND MORPHOLOGIC CHARACTERIZATION OF BILE-DUCT EPITHELIAL-CELLS FROM NORMAL RAT-LIVER
ISOLATION AND MORPHOLOGIC CHARACTERIZATION OF BILE-DUCT EPITHELIAL-CELLS FROM NORMAL RAT-LIVER
复制标题
DOI：
10.1016/0016-5085(89)91695-8
复制
发表时间：
1989-11-01
期刊：
影响因子：
29.4
通讯作者：
LARUSSO, NF
中科院分区：
文献类型：
--
作者：
ISHII, M;VROMAN, B;LARUSSO, NF
为了直接研究肝内胆管上皮细胞的功能，我们建立了一种从正常大鼠肝脏中分离肝内胆管上皮细胞（IBDECs）的新技术。通过酶消化和机械破碎从整个肝脏中分离出实质细胞和非实质细胞;然后通过连续逆流淘洗、等密度离心和免疫亲和分离分离单个非实质细胞的亚群，所述免疫亲和分离用针对IBDEC质膜上的抗原的特异性单克隆抗体。使用这种方法，我们分离出1.2 .+-。0.2. times. 106（平均值±）SE）活的（> 95%台盼蓝排除）细胞，其中> 95%通过形态学外观和特异性细胞化学标记鉴定为IBDEC。IBDEC的平均值为7.4 .+-。0.16μ m的直径，并保持其原位外观，包括形态极性。它们表现为单细胞或通过排除钌红的紧密连接连接的细胞双峰。微绒毛丰富且仅限于顶端（即，管腔）区域。在顶侧和基底侧细胞表面均观察到被膜凹陷。在内部，IBDEC包含一个发达的细胞器系统，包括线粒体，高尔基体和离散类型的囊泡，如包被囊泡，多泡体和溶酶体。这些结果表明，一个高度纯化的悬浮液的活的，形态完整的，和极性的IBDEC可以从正常大鼠肝脏usign一种新的方法，分离肝细胞的大小，密度和特定的膜组件的基础上制备。这种模型的可用性允许直接对IBDEC进行实验研究，这是一种以前不可能的方法。
To study directly the functions of the cells that line the bile ducts inside the liver, we developed a new technique for isolating intrahepatic bile duct epithelial cells (IBDECs) from normal rat liver. Parenchymal and nonparenchymal cells were separated from whole liver by enzymatic digestion and mechanical disruption; subpopulations of individual nonparenchymal cells then were isolated by serial counter-flow elutriation, isopycnic centrifugation, and immunoaffinity separation with a specific monoclonal antibody against an antigen on the plasma membrane of IBDECs. Using this approach, we isolated 1.2 .+-. 0.2 .times. 106 (mean .+-. SE) viable (> 95% trypan blue exclusion) cells, > 95% of which were identified as IBDECs by morphologic appearance and specific cytochemical markers. The IBDECs averaged 7.4 .+-. 0.16 .mu.m in diameter and retained their in situ appearance, including morphologic polarity. They appeared as single cells or as cell doublets attached by tight junctions that excluded ruthenium red. Microvilli were abundant and were restricted to the apical (i.e., luminal) domain of the plasma membrane. Coated pits were observed on both apical and basolateral cell surfaces. Internally, IBDECs contained a well-developed system of organelles, including mitochondria, Golgi, and discrete types of vesicles, such as coated vesicles, multivesicular bodies, and lysosomes. These results indicate that a highly purified suspension of viable, morphologically intact, and polar IBDECs can be prepared from normal rat liver usign a novel approach that separates liver cells on the basis of size, density, and specific membrane components. The availability of such a model with allow experimental studies to be performed directly on IBDECs, an approach that has not previously been possible.