Bissell D M, Caron J M, Babiss L E, Friedman J M
Department of Medicine, University of California, San Francisco.
Mol Biol Med. 1990 Apr;7(2):187-97.
The expression of tissue-specific functions by hepatocytes in primary culture is enhanced in the presence of an extracellular matrix. A basement membrane-like substratum, derived from the Engelbreth-Holm-Swarm mouse sarcoma (EHS) and termed EHS gel, supports synthesis and secretion of albumin for at least three weeks, in contrast to a conventional substratum (plastic or collagen-coated plastic), on which cells rapidly lose this function. The presence of an EHS matrix (as a substratum or added to the medium as a dilute gel) supports transcriptional activity at 30 to 35% and specific mRNA at 70 to 80% of initial values after five days of culture, at a time when transcription in cells plated in conventional culture is undetectable. For examining the cis elements required for transcriptional regulation by EHS matrix, we are utilizing recombinant adenoviruses to introduce DNA into hepatocytes, as an alternative to transfection of DNA fragments. Initial studies are presented, in which hepatocytes are cultured on either collagen-coated plastic or on EHS gel. At various times after plating, the cultures are infected with an adenovirus containing the proximal 5' regulatory region (to -441 base-pair) of the albumin gene. The results indicate no effect of EHS gel on this proximal promoter region, implying that matrix-responsive element(s) lie further upstream, possibly within the previously described enhancer at about -10,000 base-pairs.
在原代培养中，肝细胞组织特异性功能的表达在细胞外基质存在时会增强。一种源自恩格尔布雷特 - 霍尔姆 - 斯旺小鼠肉瘤（EHS）的基底膜样基质，称为EHS凝胶，可支持白蛋白的合成与分泌至少三周，相比之下，在传统基质（塑料或胶原包被的塑料）上，细胞会迅速丧失此功能。在培养五天后，当接种于传统培养基中的细胞转录无法检测到时，EHS基质（作为基质或作为稀释凝胶添加到培养基中）可使转录活性维持在初始值的30%至35%，特异性mRNA维持在70%至80%。为了研究EHS基质转录调控所需的顺式元件，我们正在利用重组腺病毒将DNA导入肝细胞，作为转染DNA片段的替代方法。本文展示了初步研究，其中肝细胞在胶原包被的塑料或EHS凝胶上培养。接种后的不同时间，用含有白蛋白基因近端5'调控区（至 - 441碱基对）的腺病毒感染培养物。结果表明EHS凝胶对该近端启动子区域无影响，这意味着基质反应元件位于更上游，可能在先前描述的约 - 10,000碱基对处的增强子内。
