Sakisaka S, Ng O C, Boyer J L
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut.
Gastroenterology. 1988 Sep;95(3):793-804. doi: 10.1016/s0016-5085(88)80030-1.
Isolated rat hepatocyte couplets in short-term culture (6 h) were labeled for 3 min with horseradish peroxidase (HRP) to characterize the transcytotic vesicle transport pathway in this cell culture system that retained an "apical" canalicular membrane polarity. Microtubules were identified with monoclonal antibodies to beta-tubulin and fluorescein iso-thiocyanate-labeled goat-antimouse antibody and were concentrated in the apical domain, a structural polarity that was eliminated by pretreatment with colchicine. In control cells, HRP immediately labeled vesicles and tubules in the submembrane regions of the periphery of the cell. Within 10 min tubules and vesicles were prominently labeled in pericanalicular regions, a process blocked by colchicine but not by lumicolchine or taurocholate administration. A quantitative morphometric analysis utilizing a Zeiss Videoplan-2 image analyzer established that (a) HRP-containing structures increased in density, area, length, and diameter in the pericanalicular region by 10 min; (b) colchicine, but not lumicolchicine, pretreatment diminished their density, area, and length; and (c) taurocholate (50 microM), a choleretic and biliary lipid-stimulating bile acid, had no effect on HRP density or percentage of area in the pericanalicular region, but decreased the diameter of the pericanalicular HRP-containing structures and increased the percentage of tubules containing HRP from 29% to 40%. Tubules were particularly prominent in thick sections (400 nm) in both peripheral and pericanalicular regions and were viewed as continuous anastomosing linear arrays in stereo-paired micrographs. These studies established that isolated rat hepatocyte couplets maintain a highly polarized tubulovesicular transcytotic pathway in short-term culture that is micro-tubule-dependent. Taurocholate stimulates the transformation of tubules from vesicles in this isolated rat hepatocyte couplet system.
对短期培养（6小时）的分离大鼠肝细胞双联体用辣根过氧化物酶（HRP）标记3分钟，以表征该保留“顶端”胆小管膜极性的细胞培养系统中的转胞吞囊泡运输途径。用抗β-微管蛋白单克隆抗体和异硫氰酸荧光素标记的山羊抗小鼠抗体鉴定微管，其集中在顶端区域，这种结构极性可通过秋水仙碱预处理消除。在对照细胞中，HRP立即标记细胞周边膜下区域的囊泡和小管。10分钟内，胆小管周围区域的小管和囊泡被显著标记，这一过程被秋水仙碱阻断，但不受光秋水仙碱或牛磺胆酸盐给药的影响。利用蔡司Videoplan-2图像分析仪进行的定量形态计量分析表明：（a）含HRP的结构在胆小管周围区域的密度、面积、长度和直径在10分钟时增加；（b）秋水仙碱预处理而非光秋水仙碱预处理可降低其密度、面积和长度；（c）牛磺胆酸盐（50微摩尔），一种利胆和刺激胆汁脂质的胆汁酸，对胆小管周围区域的HRP密度或面积百分比无影响，但减小了胆小管周围含HRP结构的直径，并使含HRP小管的百分比从29%增加到40%。小管在周边和胆小管周围区域的厚切片（400纳米）中特别突出，并在立体配对显微照片中被视为连续吻合的线性阵列。这些研究表明，分离的大鼠肝细胞双联体在短期培养中维持高度极化的微管依赖性管泡转胞吞途径。牛磺胆酸盐在这个分离的大鼠肝细胞双联体系统中刺激囊泡向小管的转化。
