Decaens C, Rodriguez P, Bouchaud C, Cassio D
UMR 177 CNRS, Institut Curie, Centre Universitaire, Orsay, France.
J Cell Sci. 1996 Jun;109 ( Pt 6):1623-35. doi: 10.1242/jcs.109.6.1623.
By immunofluorescence and freeze fracture methods, we have studied the establishment of hepatic cell polarity in WIF-B9 cells, a subclone of the WIF-B rat hepatoma-derived hybrid cell line. As previously shown (Ihrke et al. (1993) J. Cell Biol. 123, 1761-1775; Shanks et al. (1994) J. Cell Sci. 107, 813-825), these cells are a suitable model for in vitro studies of various hepatic functions, particularly polarity: in confluent cultures, the majority of cells form bile canaliculus-like structures; membrane domains are settled, according to plasma membrane protein localization similar to rat hepatocytes in situ. We here report that the establishment of WIF-B9 cell polarity is a slow progressive biphasic phenomenon. During the first days of culture, the majority of cells do not make bile canaliculus-like structures. However, they display a polarity similar to that of simple epithelial cells: apical membrane proteins and villin are found at the cell apex; basolateral ones, excluded from this area, are expressed in the remaining membrane area; the tight junction-associated protein ZO-1 and actin are concentrated at the boundary of these two poles, whereas E-cadherin is present at the lateral pole just under the apex. With time in culture, the number of cells expressing this simple epithelial polarized phenotype decreases progressively and, after 10-15 days, depending on the plating density, nearly all the cells express the typical hepatic polarized phenotype. The expression of these two phenotypes is mutually exclusive. Freeze-fracture replicas of both types of polarized cells show either macula occludens, fascia occludens (simple epithelial polarity) or zonula occludens (hepatic polarity), associated with gap junctions. In this last case, two or three continuous strands are generally present all around the bile canaliculus-like structures.
通过免疫荧光和冷冻蚀刻方法，我们研究了WIF-B9细胞中肝细胞极性的建立，WIF-B9细胞是源自WIF-B大鼠肝癌的杂交细胞系的一个亚克隆。如先前所示（Ihrke等人，（1993年）《细胞生物学杂志》123卷，1761 - 1775页；Shanks等人，（1994年）《细胞科学杂志》107卷，813 - 825页），这些细胞是体外研究各种肝功能，特别是极性的合适模型：在汇合培养物中，大多数细胞形成胆小管样结构；膜结构域根据质膜蛋白定位而定，类似于原位大鼠肝细胞。我们在此报告，WIF-B9细胞极性的建立是一个缓慢进展的双相现象。在培养的最初几天，大多数细胞不形成胆小管样结构。然而，它们表现出与简单上皮细胞相似的极性：顶端膜蛋白和绒毛蛋白位于细胞顶端；基底外侧的蛋白被排除在该区域之外，在其余膜区域表达；紧密连接相关蛋白ZO-1和肌动蛋白集中在这两个极的边界，而E-钙黏蛋白存在于顶端正下方的侧极。随着培养时间的推移，表达这种简单上皮极化表型的细胞数量逐渐减少，并且在10 - 15天后，取决于接种密度，几乎所有细胞都表达典型的肝极化表型。这两种表型的表达是相互排斥的。两种类型极化细胞的冷冻蚀刻复制品显示出与间隙连接相关的闭锁小带、闭锁带（简单上皮极性）或紧密连接（肝极性）。在最后一种情况下，通常在胆小管样结构周围存在两到三条连续的链。
