Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration
Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration
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DOI：
10.1152/ajpgi.1999.277.1.g12
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发表时间：
1999-07-01
影响因子：
4.5
通讯作者：
Brouwer, KLR
中科院分区：
文献类型：
--
作者：
Liu, XR;LeCluyse, EL;Brouwer, KLR
本研究的目的是研究在两层凝胶化胶原蛋白（三明治结构）之间培养的新鲜分离肝细胞中极性的功能重建。免疫印迹分析表明，在第5天的三明治结构培养的肝细胞中，小管多特异性有机阴离子转运蛋白（多药耐药相关蛋白，Mrp2）被部分维持。荧光素标记的牛磺胆酸盐和羧基二氯荧光素被排泄到第5天三明治培养的肝细胞的胆管腔内并浓缩，在标准缓冲液（完整的胆管）中形成荧光网络。共聚焦显微镜研究表明：1)羧基二氯荧光素；在无Ca2+缓冲液（破坏胆管）存在的情况下释放到孵卵缓冲液中，2)罗丹明-葡聚糖，一种细胞外空间标记物，只有在无Ca2+缓冲液存在的情况下才能扩散到小管腔中。在第5天的三明治培养肝细胞中，标准缓冲液中[H-3]牛磺胆酸的累积摄取明显高于无Ca2+缓冲液，这是由于牛磺胆酸在小管空间中的积累。当在第5天的三明治培养肝细胞中预加载[H-3]牛磺胆酸盐时，与标准缓冲液相比，无Ca2+的牛磺胆酸盐外排更大。在三明治培养的肝细胞中，牛磺酸胆酸盐的胆道排泄指数，相当于在小管网络中保留的牛磺酸胆酸盐的百分比，从第0天的8%增加到第5天的60%。总之，在胶原夹层结构中培养5天的肝细胞建立了完整的小管网络，维持了Mrp2，重建了有机阴离子和胆汁酸的极化排泄，并且。代表一个有用的体外模型系统来研究底物的肝胆处置。
The objective of the present investigation was to examine the functional reestablishment of polarity in freshly isolated hepatocytes cultured between 2 layers of gelled collagen (sandwich configuration). Immunoblot analysis demonstrated that the canalicular multispecific organic anion transport protein (multidrug resistance-associated protein, Mrp2) was partially maintained in day 5 hepatocytes cultured in a sandwich configuration. Fluorescein-labeled taurocholate and carboxydichloro-fluorescein were excreted into and concentrated in the bile canalicular lumen of day 5 sandwich-cultured hepatocytes, resulting in formation of fluorescent networks in standard buffer (intact bile canaliculi). Confocal microscopy studies demonstrated that 1) carboxydichlorofluorescein that; had concentrated in the canalicular lumen was released into the incubation buffer in the presence of Ca2+-free buffer (disrupted bile canaliculi), and 2) rhodamine-dextran, an extracellular space marker, was only able to diffuse into the canalicular lumen in the presence of Ca2+ free buffer. The cumulative uptake of [H-3]taurocholate in day 5 sandwich-cultured hepatocytes was significantly higher in standard buffer compared with Ca2+-free buffer, due to accumulation of taurocholate in canalicular spaces. When [H-3]taurocholate was preloaded in the day 5 sandwich-cultured hepatocytes, taurocholate efflux was greater in Ca2+-free compared with standard buffer. The biliary excretion index of taurocholate, equivalent to the percentage of retained taurocholate in the canalicular networks, increased from similar to 8% at day 0 to similar to 60% at day 5 in sandwich-cultured hepatocytes. In summary, hepatocytes cultured in a collagen-sandwich configuration for up to 5 days establish intact canalicular networks, maintain Mrp2, reestablish polarized excretion of organic anions and bile acids, and. represent a useful in vitro model system to investigate the hepatobiliary disposition of substrates.