Schuetz E G, Li D, Omiecinski C J, Muller-Eberhard U, Kleinman H K, Elswick B, Guzelian P S
Department of Pathology, Medical College of Virginia, Richmond 23298.
J Cell Physiol. 1988 Mar;134(3):309-23. doi: 10.1002/jcp.1041340302.
Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver-specific functions over time in culture. We incubated hepatocytes in a serum-free medium on a reconstituted basement membrane gel, "matrigel" (prepared from an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver-specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver-specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P-450b, and cytochrome P-450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P-450c by 3-methylcholanthrene and for P-450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P-450d by 3-methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P-450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte "dedifferentiation." None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H-thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of both matrices during the first 2 days in culture. However, the continuously cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver-specific functions.
新鲜分离的成年大鼠肝细胞，当在I型胶原蛋白（商品名为Vitrogen）上培养时，呈现多边形形状，在24小时内形成稳定的单层，但随着培养时间的延长会丧失表达一些肝脏特异性功能的能力。我们将肝细胞在无血清培养基中培养于重组基底膜凝胶“基质胶”（由小鼠Engelbreth-Holm-Swarm肉瘤的细胞外基质提取物制备）上，观察到细胞牢固黏附，作为单个细胞或细胞簇保持圆形，并在体外维持肝脏特异性基因表达超过1周。基质胶上的肝细胞分泌的白蛋白、转铁蛋白、触珠蛋白和血红素结合蛋白的量显著更高，对提取的细胞RNA进行Northern印迹分析，发现肝脏特异性蛋白白蛋白的mRNA表达量增加（与在Vitrogen上的细胞相比）。在用苯巴比妥、细胞色素P - 450b和细胞色素P - 450e处理的培养物中，Vitrogen上的细胞中mRNA和蛋白质几乎检测不到，但在基质胶培养物中被诱导至与体内肝脏相似的水平。同样，使用基质胶极大地增强了3 - 甲基胆蒽对P - 450c的mRNA和蛋白质的诱导以及甾体和非甾体诱导剂对P - 450p的诱导。然而，两种基质都不能使3 - 甲基胆蒽诱导P - 450d，这表明基质胶的作用即使对于细胞色素P - 450基因超家族成员在肝脏中的表达也是选择性的。在Vitrogen上培养5天内，肝细胞表达可检测量的胎肝醛缩酶活性以及波形蛋白和I型胶原蛋白的mRNA，每一项都被认为是反映肝细胞“去分化”的表型变化。这些在基质胶上的细胞中均不存在。通过将3H - 胸腺嘧啶掺入DNA来判断，在基质胶上培养的肝细胞对有丝分裂刺激的反应性也降低。最后，在培养的前两天两种基质的水平都有显著增加。然而，随着培养时间的延长，培养物中细胞骨架mRNA的水平比基质胶上圆形细胞中的增加得更多。我们得出结论，与标准胶原蛋白相比，在基质胶上培养的肝细胞表现出许多肝脏特异性功能的显著增强表达。
