Structural polarity and functional bile canaliculi in rat hepatocyte spheroids
Structural polarity and functional bile canaliculi in rat hepatocyte spheroids
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DOI：
10.1006/excr.2001.5467
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发表时间：
2002-03-10
影响因子：
3.7
通讯作者：
Hu, WS
中科院分区：
文献类型：
--
作者：
Abu-Absi, SF;Friend, JR;Hu, WS
原代肝细胞自组装成具有紧密连接和微绒毛内衬通道的球状体。我们假设极性逐渐发展，通道的结构和功能类似于胆小管。免疫荧光标记的顶侧和基底侧的蛋白质表现出重组成极化分布的膜蛋白在球体培养。通过荧光葡聚糖扩散和共聚焦显微镜，一个广泛的网络通道被发现在球体的内部。这些通道连接在几个平面上，并在表面上打开孔隙。为了检查通道膜中顶端蛋白的含量，使用荧光底物Ala-Pro-甲酚紫定位胆小管酶二肽基肽酶IV（DPPIV）。结果表明，DPPIV活性在球状体中呈不均匀分布，部分位于通道中。然后研究胆汁酸排泄以证明功能极性。一种荧光胆汁酸类似物，异硫氰酸荧光素标记的甘氨胆酸盐，被吸收到球状体中并分泌到胆小管通道中。由于球状体的结构极性和它们将胆汁排泄到通道中的能力，它们是体外肝组织自组装的独特三维模型。（一些结果的视频动画可在http：//hugroup.cems.umn.edu/research_movies上获得）。(C)2002 Elsevier Science（美国）。
Primary hepatocytes self-assemble into spheroids that possess tight junctions and microvilli-lined channels. We hypothesized that polarity develops gradually and that the channels structurally and functionally resemble bile canaliculi. Immunofluorescence labeling of apical and basolateral proteins demonstrated reorganization of the membrane proteins into a polarized distribution during spheroid culture. By means of fluorescent dextran diffusion and confocal microscopy, an extensive network of channels was revealed in the interior of the spheroids. These channels connected over several planes and opened to pores on the surface. To examine the content of apical proteins in the channel membranes, the bile canalicular enzyme dipeptidyl peptidase IV (DPPIV) was localized using a fluorogenic substrate, Ala-Pro-cresyl violet. The results show that DPPIV activity is heterogeneously distributed in spheroids and localized in part to channels. Bile acid excretion was then investigated to demonstrate functional polarity. A fluorescent bile acid analogue, fluorescein isothiocyanate-labeled glycocholate, was taken up into the spheroids and excreted into bile canalicular channels. Due to the structural polarity of spheroids and their ability to excrete bile into channels, they are a unique three-dimensional model of in vitro liver tissue self-assembly. (Video animations of some results are available at http://hugroup.cems.umn.edu/research_movies). (C) 2002 Elsevier Science (USA).