Brown S E, Guzelian C P, Schuetz E, Quattrochi L C, Kleinman H K, Guzelian P S
University of Colorado Health Sciences Center, Hepatobiliary Research Center, Denver, USA.
Lab Invest. 1995 Dec;73(6):818-27.
Although it has been known for more than three decades that administration of lipophilic chemicals, including phenobarbital, produces liver hypertrophy, proliferation of smooth endoplasmic reticulum, and induction of liver microsomal enzymes such as cytochromes P450 (CYP) 2B1 and 2B2, the mechanism of this adaptive response remains largely unknown. An important advance was the recognition that, unlike cultures of continuously proliferating liver cell lines or cultures of primary non-proliferating adult rat hepatocytes maintained on either plastic or collagen-coated dishes, hepatocytes cultured on a basement membrane gel, Matrigel, formed rounded clusters and permitted phenobarbital-mediated induction in vitro of CYP 2B1/2 mRNAs and immunoreactive proteins (1).
We cultured adult rat hepatocytes on Type 1 collagen (Vitrogen) and allowed the cells to spread, flatten, and firmly attach to the substratum. Subsequent incubation in medium containing Matrigel as a soluble component, fully restored, in a dose-dependent manner, the ability to respond to phenobarbital with induction of CYP 2B1/2 mRNAs. Repeating this experiment with medium containing equivalent amounts of purified laminin, a major component of Matrigel, or with YIGSR or SIKVAV, two peptides known to mimic various activities of laminin, similarly restored phenobarbital responsiveness to hepatocytes cultured on Vitrogen. In contrast, use of equal amounts of SHA-23, a scrambled peptide relevant to SIKVAV, produced no such effect. None of these treatments caused a rounding or any other observable change in the flattened, cellular morphology, making it unlikely that cell-spreading or alterations in cell shape account for loss of such differentiated liver functions as phenobarbital induction of CYP 2B1/2 mRNAs in cultured hepatocytes on Vitrogen. Hepatocytes cultured on Matrigel in the presence of either colchicine, cytochalasins B and D, nocodazole, or taxol did not show induction of 2B1/2 mRNAs by phenobarbital specifically, while the amounts of both albumin and glucose-6-phosphate dehydrogenase (G6PD) mRNAs were unaffected.
We conclude that the process by which phenobarbital induced 2B1/2 mRNAs in hepatocytes appears to require highly concerted effects of specific extracellular components prominently involving laminin. This likely occurs through a signal transduction process requiring probably both microfilament and microtubular integrity.
尽管三十多年来已知给予包括苯巴比妥在内的亲脂性化学物质会导致肝脏肥大、滑面内质网增殖以及诱导肝脏微粒体酶如细胞色素P450（CYP）2B1和2B2，但这种适应性反应的机制在很大程度上仍不清楚。一个重要进展是认识到，与在塑料或胶原包被培养皿上培养的持续增殖肝细胞系或原代非增殖成年大鼠肝细胞不同，在基底膜凝胶基质胶上培养的肝细胞形成圆形簇，并允许苯巴比妥在体外诱导CYP 2B1/2 mRNA和免疫反应性蛋白（1）。
我们在I型胶原（Vitrogen）上培养成年大鼠肝细胞，使细胞铺展、变平并牢固附着于基质。随后在含有基质胶作为可溶性成分的培养基中孵育，以剂量依赖方式完全恢复了对苯巴比妥诱导CYP 2B1/2 mRNA的反应能力。用含有等量纯化层粘连蛋白（基质胶的主要成分）的培养基，或用已知模拟层粘连蛋白各种活性的两种肽YIGSR或SIKVAV重复此实验，同样恢复了在Vitrogen上培养的肝细胞对苯巴比妥的反应性。相比之下，使用等量与SIKVAV相关的随机肽SHA - 23则没有这种效果。这些处理均未导致扁平细胞形态出现变圆或任何其他可观察到的变化，这使得细胞铺展或细胞形状改变不太可能是在Vitrogen上培养的肝细胞中苯巴比妥诱导CYP 2B1/2 mRNA等分化肝功能丧失的原因。在秋水仙碱、细胞松弛素B和D、诺考达唑或紫杉醇存在的情况下，在基质胶上培养的肝细胞未显示苯巴比妥特异性诱导2B1/2 mRNA，而白蛋白和葡萄糖 - 6 - 磷酸脱氢酶（G6PD）mRNA的量未受影响。
我们得出结论，苯巴比妥在肝细胞中诱导2B1/2 mRNA的过程似乎需要特定细胞外成分的高度协同作用，其中层粘连蛋白起主要作用。这可能通过一个可能需要微丝和微管完整性的信号转导过程发生。
