Li Wan-Chun, Ralphs Kate L, Slack Jonathan M W, Tosh David
Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.
Int J Biochem Cell Biol. 2007;39(3):541-54. doi: 10.1016/j.biocel.2006.10.017. Epub 2006 Oct 28.
Freshly isolated hepatocytes rapidly lose their differentiated properties when placed in culture. Therefore, production of a simple culture system for maintaining the phenotype of hepatocytes in culture would greatly facilitate their study. Our aim was to identify conditions that could maintain the differentiated properties of hepatocytes for up to 28 days of culture. Adult rat hepatocytes were isolated and attached in Williams' medium E containing 10% serum. The medium was changed to either fresh Williams' medium E or keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract. The hepatic phenotype was then analysed using RT-PCR, immunohistochemistry, Western blotting and assays of liver function. Cells cultured in keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract maintained their phenotype for 3-4 weeks, based on expression of liver proteins, ureagenesis and response to xenobiotics. In contrast, hepatocytes cultured in Williams' medium E rapidly lost the expression of liver proteins after 3 days. Cells cultured in keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract maintained their expression of liver-enriched transcription factors (C/EBPalpha and beta, HNF4alpha and RXRalpha) while expression was either lost or reduced in cells cultured in Williams' medium E. These results suggest that keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract can maintain the hepatic phenotype for a prolonged period and that this is probably related to the continued expression of the liver-enriched transcription factors.
刚分离出的肝细胞在培养时会迅速丧失其分化特性。因此，建立一种能在培养中维持肝细胞表型的简单培养系统将极大地促进对它们的研究。我们的目标是确定能在长达28天的培养过程中维持肝细胞分化特性的条件。分离成年大鼠肝细胞并将其接种于含10%血清的威廉姆斯E培养基中。然后将培养基更换为新鲜的威廉姆斯E培养基或添加了地塞米松、表皮生长因子和垂体提取物的角质形成细胞无血清培养基。接着使用逆转录聚合酶链反应（RT-PCR）、免疫组织化学、蛋白质印迹法以及肝功能检测来分析肝细胞表型。基于肝蛋白的表达、尿素生成以及对外源生物的反应，在添加了地塞米松、表皮生长因子和垂体提取物的角质形成细胞无血清培养基中培养的细胞在3 - 4周内维持了其表型。相比之下，在威廉姆斯E培养基中培养的肝细胞在3天后迅速丧失了肝蛋白的表达。在添加了地塞米松、表皮生长因子和垂体提取物的角质形成细胞无血清培养基中培养的细胞维持了其肝富集转录因子（C/EBPα和β、HNF4α和RXRα）的表达，而在威廉姆斯E培养基中培养的细胞中这些转录因子的表达要么丧失要么减少。这些结果表明，添加了地塞米松、表皮生长因子和垂体提取物的角质形成细胞无血清培养基能够长时间维持肝细胞表型，这可能与肝富集转录因子的持续表达有关。
