Shenvi Swapna V, Dixon Brian M, Petersen Shay Kate, Hagen Tory M
Molecular and Cellular Biology Program, Oregon State University, Corvallis, Oregon, USA.
Curr Protoc Toxicol. 2008 Aug;Chapter 14:Unit 14.7. doi: 10.1002/0471140856.tx1407s37.
The purpose of this protocol is to establish a primary hepatocyte culture system as a suitable model to examine age-related changes in Phase II detoxication gene expression. Hepatocytes are isolated using a two-step collagenase perfusion technique from young (3 to 6 months) and old (24 to 28 months) rats and placed in primary culture using collagen (Type I)-coated plates as the extracellular matrix. A supplemented William's E Medium is used as the medium. This culture system maintains hepatocyte viability from both young and old rats for ∼60 hr, as measured by lactate dehydrogenase activity, while also maintaining their respective phenotypes relative to Phase II detoxification. We thus conclude that a collagen-based cell culture system is suitable to study age-associated deficits in Nrf2/ARE-mediated Phase II gene regulation provided that experiments can be conducted within 60 hr after cell isolation.
本实验方案的目的是建立一个原代肝细胞培养系统，作为研究Ⅱ相解毒基因表达中与年龄相关变化的合适模型。使用两步胶原酶灌注技术从年轻（3至6个月）和老年（24至28个月）大鼠中分离肝细胞，并使用I型胶原包被的培养板作为细胞外基质进行原代培养。添加了营养成分的威廉姆斯E培养基用作培养液。通过乳酸脱氢酶活性测定，该培养系统可使年轻和老年大鼠的肝细胞活力维持约60小时，同时还能维持它们相对于Ⅱ相解毒的各自表型。因此，我们得出结论，基于胶原的细胞培养系统适合用于研究Nrf2/ARE介导的Ⅱ相基因调控中与年龄相关的缺陷，前提是实验可以在细胞分离后60小时内进行。
