Vanhaecke T, Elaut G, Rogiers V
Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.
Toxicol In Vitro. 2001 Aug-Oct;15(4-5):387-92. doi: 10.1016/s0887-2333(01)00041-8.
Cultures of perivenous (PV) and periportal (PP) hepatocytes could provide suitable in vitro models for studying the zone-specific hepatotoxic potential of xenobiotics. However, it is not known whether cultured PP and PV hepatocytes keep their phenotypes when the microcirculation of the liver changes. This question has been studied by culturing rat hepatocytes at 13 and 4% (v/v) O(2), respectively, mimicking the acinar oxygen gradient. PP and PV adult rat hepatocytes were isolated by digitonin-collagenase in situ perfusion and cultured on plastic Falcon and gas-permeable Petriperm dishes in Williams' E medium and kept at 13 and 4% (v/v) O(2), respectively. Cultures at 20% (v/v) O(2) on plastic dishes served as a control. Two types of cultures were studied, namely conventional cultures either unsupplemented or supplemented with 30 mM pyruvate. The activities of glutamine synthetase (GS) and glutathione S-transferase (GST) were measured in freshly isolated PP and PV hepatocytes and all cultures. The heterogeneous expression of GS (PV>PP), observed in freshly isolated hepatocytes, was kept for at least 4 days in culture. Total, Mu and Alpha class GST activities were predominantly expressed in PV freshly isolated cells. However, no beneficial effect could be observed in culture by exposing the cells to their specific in vivo oxygen concentration. The best maintenance of GST PV predominance in culture was observed in Petriperm dishes at 20% (v/v) O(2), as well in pyruvate-supplemented as unsupplemented cultures. PV GST predominance was thus kept in particular when the highest oxygen concentration was used and made available to the cells through the gas-permeable membranes. The results on GS PV predominance support these findings.
肝静脉周围（PV）和门静脉周围（PP）肝细胞的培养可为研究外源性物质的区域特异性肝毒性潜力提供合适的体外模型。然而，尚不清楚当肝脏微循环发生变化时，培养的PP和PV肝细胞是否能保持其表型。通过分别在13%和4%（v/v）O₂条件下培养大鼠肝细胞来研究这个问题，模拟腺泡氧梯度。采用洋地黄皂苷 - 胶原酶原位灌注法分离成年大鼠的PP和PV肝细胞，并在塑料Falcon培养皿和透气的Petriperm培养皿中于Williams' E培养基中培养，分别保持在13%和4%（v/v）O₂条件下。在塑料培养皿中20%（v/v）O₂条件下的培养作为对照。研究了两种类型的培养物，即未添加或添加30 mM丙酮酸的传统培养物。在新鲜分离的PP和PV肝细胞以及所有培养物中测量了谷氨酰胺合成酶（GS）和谷胱甘肽S - 转移酶（GST）的活性。在新鲜分离的肝细胞中观察到的GS异质性表达（PV>PP）在培养中至少保持4天。总、Mu和Alpha类GST活性主要在新鲜分离的PV细胞中表达。然而，将细胞暴露于其特定的体内氧浓度在培养中未观察到有益效果。在20%（v/v）O₂条件下的Petriperm培养皿中，无论是添加丙酮酸还是未添加丙酮酸的培养物中，均观察到培养物中GST PV优势的最佳维持。因此，特别是当使用最高氧浓度并通过透气膜使细胞获得该氧浓度时，PV GST优势得以保持。关于GS PV优势的结果支持了这些发现。
