在胶原蛋白夹心结构中培养的成年大鼠肝细胞对牛磺胆酸盐摄取的部分维持。
Partial maintenance of taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich configuration.
作者信息
Liu X, Brouwer K L, Gan L S, Brouwer K R, Stieger B, Meier P J, Audus K L, LeCluyse E L
机构信息
Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina at Chapel Hill, 27599, USA.
出版信息
Pharm Res. 1998 Oct;15(10):1533-9. doi: 10.1023/a:1011994831139.
PURPOSE
This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions.
METHODS
Hepatocytes isolated from male Wistar rats (230-280 g) were cultured on a simple collagen film, on a substratum of gelled collagen or between two layers of gelled collagen (sandwich configuration). Hepatocyte morphology, taurocholate uptake properties, and expression of the sinusoidal transport protein. Na+/taurocholate-cotransporting polypeptide (Ntcp) were examined in these cultures at day 0 and day 5.
RESULTS
By day 5, monolayer integrity had deteriorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At day 5, taurocholate accumulation at 5 min in hepatocytes cultured on a simple collagen film, on a substratum of gelled collagen, and in a sandwich configuration was approximately 13%, 20% and 35% of day-0 levels, respectively, and occurred predominately by a Na+-dependent mechanism. The initial taurocholate uptake rate vs. concentration (1-200 microM) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent Km values were comparable for day-0 and day-5 hepatocytes (3241 microM). In contrast, the Vmax values of hepatocytes cultured on a simple collagen film, on gelled collagen and in a sandwich configuration were approximately 5, 6 and 14% of the values at day 0, respectively; values for the first-order rate constant were 5-, 3- and 2-fold lower, respectively. Immunoblot analysis indicated that at day 5 Ntcp expression in hepatocytes cultured in a sandwich configuration was greater than in hepatocytes cultured on a simple collagen film.
CONCLUSIONS
A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid uptake properties in primary cultures of rat hepatocytes.
目的
本研究旨在表征在不同基质条件下维持的大鼠原代肝细胞培养物中牛磺胆酸盐的摄取特性。
方法
从雄性Wistar大鼠（230 - 280 g）分离的肝细胞，培养在简单胶原膜上、凝胶化胶原基质上或两层凝胶化胶原之间（三明治结构）。在第0天和第5天检测这些培养物中肝细胞的形态、牛磺胆酸盐摄取特性以及窦状隙转运蛋白钠/牛磺胆酸盐共转运多肽（Ntcp）的表达。
结果
到第5天，简单胶原培养物中的单层完整性已恶化。相比之下，维持在三明治结构中的肝细胞形态得以保留。在第5天，培养在简单胶原膜上、凝胶化胶原基质上以及三明治结构中的肝细胞在5分钟时牛磺胆酸盐的积累量分别约为第0天水平的13%、20%和35%，且主要通过钠依赖性机制发生。初始牛磺胆酸盐摄取速率与浓度（1 - 200 microM）的关系曲线最适合用米氏方程和一级函数组合来描述。在所有情况下，第0天和第5天肝细胞的估计表观Km值相当（3241 microM）。相比之下，培养在简单胶原膜上、凝胶化胶原上以及三明治结构中的肝细胞的Vmax值分别约为第0天值的5%、6%和14%；一级速率常数的值分别低5倍、3倍和2倍。免疫印迹分析表明，在第5天，培养在三明治结构中的肝细胞中Ntcp的表达高于培养在简单胶原膜上的肝细胞。
结论
胶原三明治结构可重建大鼠原代肝细胞培养物中的正常形态，并部分恢复胆汁酸摄取特性。
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