Modified culture conditions enhance expression of differentiated phenotypic properties of normal rat cholangiocytes
Modified culture conditions enhance expression of differentiated phenotypic properties of normal rat cholangiocytes
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DOI：
10.1038/labinvest.3780187
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发表时间：
2000-11-01
影响因子：
5
通讯作者：
Doctor, RB
中科院分区：
文献类型：
--
作者：
Salter, KD;Roman, RM;Doctor, RB
毛细血管细胞的分泌对肝脏胆汁的形成有重要作用。在正常情况下，细胞cAMP水平的增加会增加胆管细胞跨顶膜的Cl-和HCO 3-分泌，导致碱化和胆汁量增加。在许多疾病状态中，胆管细胞也是损伤的重要靶点，受损的细胞和上皮功能导致与原发性胆汁性肝硬化、硬化性胆管炎和移植后肝功能衰竭相关的胆汁淤积（Fitz，1996; Mennone et al，1995; Nathanson and Boyer，1991; Tavaloni，1987）。尽管其生理和病理生理的重要性，肝内的位置和有限的数量（肝细胞核质量的4%）的胆管细胞有限的直接定义参与分泌的细胞机制。最近在实验室研究中引入了培养的正常大鼠胆管细胞（NRC）模型（Vroman和LaRusso，1996）。当在存在经验定义的补充物的情况下在胶原上培养时，这些细胞保留了原位胆管细胞的许多表型特征，包括极化形态和形成适于运输和电生理学研究的单层的能力（Doctor等人，1999; Vroman和LaRusso，1996）。因此，许多实验室已使用NRC细胞来拓宽对胆管细胞功能的分子和细胞见解，包括根皮苷敏感性葡萄糖转运、Na依赖性胆汁酸转运和cAMP刺激的Cl-分泌的表征（Lazarzidine，1997 a，1997 b; Roberts et al，1994; Spirli et al，1998; Tietz et al，1997）。尽管取得了这些进展，但NRC细胞的使用仍受到部分限制：（a）在单层培养中形成相对低的跨上皮抗性，（B）需要大量昂贵的补充物，和（c）在分化的上皮表型形成之前需要延长培养时间。因此，进行本研究以严格评估NRC细胞的培养要求，使用跨上皮电阻（Rt）（Epithelial Volt-Ohm Meter; World Precision Instruments，Sarasota，佛罗里达;通过Ussing分析确认）和跨上皮H梯度（[H]）（Accu pHast; Fisher Scientific Pittsburgh，Pennsylvania）作为主要分化测定。为了评价净H通量，建立了HEPES/HCO 3缓冲培养基的pH/H滴定曲线，并将测得的pH值转换为[H]。在测试的不同补充剂中，基底外侧添加地塞米松（DEX）导致跨上皮阻力急剧增加，并通过减少细胞分化所需的时间大大降低了细胞培养费用。此外，胆管细胞功能的其他措施平行增强。NRC细胞（第9-25代）如前所述在烧瓶中增殖（Vroman和LaRusso，1996）。实验研究在铺在胶原包被的半透性支持物（Costar Corporation，剑桥，马萨诸塞州;每48小时更换一次培养基）上的NRC细胞上进行。在原始研究（Vroman和LaRusso，1996）中，NRC培养基含有大量补充剂（表1，对照）。单独评价它们对细胞生长和上皮分化的影响。显微镜下对细胞生长和融合进行评分;
C holangiocyte secretion contributes importantly to the formation of bile by the liver. Under normal conditions, increases in cellular cAMP levels increase cholangiocyte Cl-and HCO3-secretion across the apical membrane, resulting in alkalinization and increased bile volume. Cholangiocytes also represent an important target of injury in a number of disease states, and impaired cell and epithelial function contributes to the cholestasis associated with primary biliary cirrhosis, sclerosing cholangitis, and posttransplant liver failure (Fitz, 1996; Mennone et al, 1995; Nathanson and Boyer, 1991; Tavaloni, 1987). Despite their physiologic and pathophysiologic importance, the intrahepatic location and limited number (4% of the liver nuclear mass) of cholangiocytes has limited direct definition of the cellular mechanisms involved in secretion. A model of normal rat cholangiocytes (NRC) in culture was recently introduced in Laboratory Investigation (Vroman and LaRusso, 1996). When cultured on collagen in the presence of empirically defined supplements, these cells retain many of the phenotypic features of cholangiocytes in situ, including a polarized morphology and the ability to form monolayers amenable to transport and electrophysiologic studies (Doctor et al, 1999; Vroman and LaRusso, 1996). Consequently, NRC cells have been used by a number of laboratories to broaden molecular and cellular insights into cholangiocyte function, including the characterization of phlorizin-sensitive glucose transport, Na-dependent bile acid transport, and cAMP-stimulated Cl- secretion (Lazaridis, 1997a, 1997b; Roberts et al, 1994; Spirli et al, 1998; Tietz et al, 1997). Despite these advances, the use of NRC cells has been limited in part by (a) the formation in monolayer culture of relatively low transepithelial resistances,(b) the requirement for a number of expensive supplements, and (c) the need for prolonged culture before development of the differentiated epithelial phenotype. Consequently, the present studies were performed to critically assess the culture requirements of NRC cells, using transepithelial resistance (Rt)(Epithelial Volt-Ohm Meter; World Precision Instruments, Sarasota, Florida; confirmed by Ussing analysis) and transepithelial H gradient ([H])(Accu pHast; Fisher Scientific Pittsburgh, Pennsylvania) as primary assays of differentiation. To evaluate net H flux, a pH/H titration curve was established for the HEPES/HCO3-buffered media and measured pH values were converted to [H]. Among the different supplements tested, basolateral addition of dexamethasone (DEX) resulted in a dramatic increase in transepithelial resistance and greatly reduced cell culture expenses by decreasing the time required for cellular differentiation. Moreover, other measures of cholangiocyte function were enhanced in parallel. Appropriate modifications of NRC culture conditions are likely to facilitate the use of NRC cells as a model for investigation of the cellular and epithelial mechanisms responsible for cholangiocyte secretion and bile formation.NRC cells (passages 9–25) were propagated in flasks as previously described (Vroman and LaRusso, 1996). Experimental studies were performed on NRC cells plated on collagen-coated semipermeable supports (Costar Corporation, Cambridge, Massachusetts; media exchanged every 48 hours). In the original studies (Vroman and LaRusso, 1996), the NRC media contained a significant number of supplements (Table 1, Control). These were individually evaluated for their effect on cell growth and epithelial differentiation. Cell growth and confluence were scored microscopically;