Behnia K, Bhatia S, Jastromb N, Balis U, Sullivan S, Yarmush M, Toner M
Centre for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School and Shriners Hospitals for Children, Boston, Massachusetts 02114, USA.
Tissue Eng. 2000 Oct;6(5):467-79. doi: 10.1089/107632700750022125.
Considering the large yield of viable cells comparable to human liver, primary porcine hepatocytes offer a valuable resource for constructing a bioartificial liver device. In this study, the ability of cultured primary porcine hepatocytes to detoxify xenobiotics has been examined using various known substrates of cytochrome P450 isoenzymes and UDP-glucuronosyltransferases. Present investigation demonstrated the stability of the isoenzymes responsible for the metabolism of diazepam in native state and stabilization of other isoenzymes, as judged by ethoxycoumarin o-dealkylase (ECOD), ethoxyresorufin o-dealkylase (EROD), benzyloxyresorufin o-dealkylase (BROD), and pentoxyresorufin o-dealkylase (PROD) activities following induction in culture environment, for a period of 8 days. Resorufin O-dealkylase activities were found to be the most unstable and deteriorated within first 5 days in culture. These activities were restored following induction with 3-methylcholanthrene (3-MC) or sodium phenobarbital (PB) to 20-fold of 1 activity for EROD, and 60 and 174% of day 1 activity for PROD and BROD on day 8, respectively. Metabolism of methoxyresorufin was most strikingly increased following induction with 3-MC to approximately 60-fold of day 1 activity, on day 8. UDP-glucuronosyltransferase-dependent glucuronidation of phenol red, however, stayed intact during the course of our study without induction. Our study indicated that porcine hepatocytes in vitro maintain many important liver-specific functions including detoxification (steady state and inducibility).
鉴于原代猪肝细胞的活细胞产量与人类肝脏相当,其为构建生物人工肝装置提供了宝贵资源。在本研究中,已使用细胞色素P450同工酶和UDP-葡萄糖醛酸基转移酶的各种已知底物,检测了培养的原代猪肝细胞对异生物素的解毒能力。目前的研究表明,通过乙氧香豆素O-脱烷基酶(ECOD)、乙氧试卤灵O-脱烷基酶(EROD)、苄氧试卤灵O-脱烷基酶(BROD)和戊氧试卤灵O-脱烷基酶(PROD)活性判断,负责地西泮代谢的同工酶在天然状态下具有稳定性,而其他同工酶在培养环境中诱导8天后得到稳定。发现试卤灵O-脱烷基酶活性最不稳定,在培养的前5天内就会下降。在用3-甲基胆蒽(3-MC)或苯巴比妥钠(PB)诱导后,这些活性得以恢复,EROD在第8天恢复到1活性的20倍,PROD和BROD在第8天分别恢复到第1天活性的60%和174%。在用3-MC诱导后,甲氧基试卤灵的代谢在第8天显著增加至第1天活性的约60倍。然而,在我们的研究过程中,酚红的UDP-葡萄糖醛酸基转移酶依赖性葡萄糖醛酸化在未诱导的情况下保持完整。我们的研究表明,体外培养的猪肝细胞维持许多重要的肝脏特异性功能,包括解毒(稳态和诱导性)。