Isolation and characterization of cholangiocyte primary cilia
Isolation and characterization of cholangiocyte primary cilia
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DOI：
10.1152/ajpgi.00064.2006
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发表时间：
2006-09-01
影响因子：
4.5
通讯作者：
LaRusso, Nicholas F.
中科院分区：
文献类型：
--
作者：
Huang, Bing Q.;Masyuk, Tatyana V.;LaRusso, Nicholas F.
初级纤毛是许多脊椎动物细胞（包括胆管细胞）表达的独特细胞器;然而，它们的功能仍然不清楚。为了开始探索这些细胞器在肝脏中的生理作用，我们描述了胆管细胞纤毛的形态和结构，并开发了分离它们的新方法。初级纤毛仅存在于胆管中，在肝细胞或肝动脉或门静脉内皮细胞中未观察到。每个胆管细胞都有一根纤毛，从顶膜延伸到胆管腔。纤毛长度与胆管直径成正比。我们可重复地分离富集组分的纤毛从正常大鼠和小鼠胆管细胞通过两种不同的方法，通过扫描电子，透射电子显微镜和共聚焦显微镜进行评估。分离的睫状体组分的纯度通过蛋白质印迹分析进一步分析，使用乙酰化微管蛋白作为睫状体标记物和P2 Y（2）作为非睫状体细胞膜标记物。这些新的技术产生了丰富的纤毛馏分足够的纯度和数量的光学和电子显微镜和生化分析。它们将允许进一步评估初级纤毛在正常和病理条件下的作用。
Primary cilia are distinct organelles expressed by many vertebrate cells, including cholangiocytes; however, their functions remain obscure. To begin to explore the physiological role of these organelles in the liver, we described the morphology and structure of cholangiocyte cilia and developed new approaches for their isolation. Primary cilia were present only in bile ducts and were not observed in hepatocytes or in hepatic arterial or portal venous endothelial cells. Each cholangiocyte possesses a single cilium that extends from the apical membrane into the bile duct lumen. In addition, the length of the cilia was proportional to the bile duct diameter. We reproducibly isolated enriched fractions of cilia from normal rat and mouse cholangiocytes by two different approaches as assessed by scanning electron, transmission electron, and confocal microscopy. The purity of isolated ciliary fractions was further analyzed by Western blot analysis using acetylated tubulin as a ciliary marker and P2Y(2) as a nonciliary cell membrane marker. These novel techniques produced enriched ciliary fractions of sufficient purity and quantity for light and electron microscopy and for biochemical analyses. They will permit further assessment of the role of primary cilia in normal and pathological conditions.