Luttringer Olivier, Theil Frank Peter, Lavé Thierry, Wernli-Kuratli Karin, Guentert Theodor W, de Saizieu Antoine
F. Hoffmann-La Roche Ltd., Pharmaceutics, CH-4070, Basel, Switzerland.
Biochem Pharmacol. 2002 Dec 1;64(11):1637-50. doi: 10.1016/s0006-2952(02)01382-5.
The influence of the isolation procedure of hepatocytes, extracellular matrix (ECM) configuration and incubation medium supplementation by dexamethasone (DEX) on the cell morphology and on the gene expression of membrane transporters was examined in rat hepatocytes. The mRNA levels were determined using oligonucleotide microarrays, in liver, in suspension and in primary culture in monolayer (CPC), and in collagen gels sandwich (SPC) in absence and presence of DEX (100 and 1000 nM). The results indicated pronounced morphological differences between CPC and SPC in response to DEX demonstrating that the hepatocytes re-formed, as in vivo, multicellular arrays with extensive bile canalicular network only in SPC in presence of DEX. The mRNA levels of membrane transporters were not affected significantly during isolation procedure. However, plating hepatocytes in CPC resulted in a decrease of major basolateral transporters mRNA level whereas mRNA levels of mdr1b and mrp3 were increased (>100-fold). Similar observations were made in SPC in the absence of DEX demonstrating that the ECM configuration alone did not play a critical role in the regulation of membrane transporters. However, adding DEX to the incubation medium in SPC resulted in an up-regulation of mdr2, oatp2 and mrp2 in a concentration-dependent way for the two latter genes, whereas mdr1b and mrp3 expression were maintained to their baseline liver levels. These data suggested therefore that the combination of ECM and DEX supplementation is essential for the formation of the bile canalicular network and is a determinant factor in the regulation of membrane transporters in cultured rat hepatocytes.
在大鼠肝细胞中，研究了肝细胞分离程序、细胞外基质（ECM）构型以及地塞米松（DEX）对培养培养基的补充对细胞形态和膜转运蛋白基因表达的影响。使用寡核苷酸微阵列测定肝脏、悬浮液、单层原代培养（CPC）以及在有无DEX（100和1000 nM）情况下胶原凝胶夹心培养（SPC）中的mRNA水平。结果表明，CPC和SPC对DEX的反应存在明显的形态学差异，表明仅在存在DEX的SPC中，肝细胞如在体内一样重新形成了具有广泛胆小管网络的多细胞阵列。在分离过程中，膜转运蛋白的mRNA水平未受到显著影响。然而，将肝细胞接种在CPC中会导致主要基底外侧转运蛋白的mRNA水平降低，而mdr1b和mrp3的mRNA水平则升高（>100倍）。在不存在DEX的SPC中也有类似观察结果，表明单独的ECM构型在膜转运蛋白的调节中不发挥关键作用。然而，在SPC的培养培养基中添加DEX会导致mdr2、oatp2和mrp2上调，后两个基因呈浓度依赖性，而mdr1b和mrp3的表达维持在其肝脏基线水平。因此，这些数据表明，ECM和DEX补充的组合对于胆小管网络的形成至关重要，并且是培养的大鼠肝细胞中膜转运蛋白调节的决定性因素。
