Regulation of gamma-glutamyltranspeptidase in rat hepatocyte monolayer cultures.
Regulation of gamma-glutamyltranspeptidase in rat hepatocyte monolayer cultures.
复制标题
大鼠肝细胞单层培养物中γ-谷氨酰转肽酶的调节。
作者：
A. Edwards
将从正常成年大鼠中分离的肝细胞作为相对纯的群体在塑料培养皿中原代单层培养4至7天。本文探讨了影响γ-谷氨酰转肽酶（GGT）活性的因素，为体外研究致癌物对GGT活性的影响奠定了基础。新鲜铺板的培养物中含有很少的GGT阳性细胞（0.4%）和非常低的总GGT活性，但在所有测试的培养基中，活性随着培养时间的推移而逐渐增加，增加的程度取决于培养基组成。对于仅改良Waymouth培养基中的肝细胞（对照培养物），在1至2天的滞后期后活性缓慢上升。对于塑料上的细胞，在3 μ M地塞米松存在下，这种增加至10倍，但对于胶原凝胶上的细胞，仅增加2至3倍。地塞米松的时间和剂量依赖性的作用被放线菌酮，放线菌素D部分阻断，并逆转后几个小时通过删除类固醇，当GGT活性下降，最大半衰期为60至80小时。这些观察结果表明，地塞米松引起可逆的酶诱导依赖于持续的RNA和蛋白质合成。用胎牛血清或N6，O2 '-二丁酰腺苷3'：5 '-单磷酸或胰高血糖素维持细胞显著降低地塞米松诱导的程度，并在对照培养物中略微降低活性。维持在7.2至7.8范围内的较低pH或2.8至28 mM范围内的较高葡萄糖浓度也导致对照或地塞米松诱导的培养物中GGT活性显著降低。葡萄糖抑制是独立的胰岛素浓度。GGT的可逆诱导和抑制可能反映了肝细胞基因表达的广泛变化，而不是GGT功能的特异性控制。由于培养的肝细胞中的GGT受多种非致癌物的调节，因此在将其用作肿瘤前标志物时需要谨慎。尽管如此，鉴定的培养基，保持低活性可能允许致癌物诱导的GGT的变化作为体外肝癌发生的早期事件的探针的研究。
Hepatocytes isolated as a relatively pure population from normal adult rats were maintained in primary monolayer culture for 4 to 7 days on plastic dishes. Factors affecting activity of gamma-glutamyltranspeptidase (GGT) in culture were investigated to establish a basis for in vitro studies on carcinogen-induced changes in GGT. Freshly plated cultures contained few GGT-positive cells (0.4%) and very low total GGT activity, but with all media tested, activity increased progressively with time in culture, the extent of increase depending on medium composition. For hepatocytes in modified Waymouth medium alone (control cultures), there was a slow rise in activity after a 1- to 2-day lag period. This increase was enhanced up to 10-fold in the presence of 3 microM dexamethasone for cells on plastic but only 2- to 3-fold for cells on collagen gels. The time- and dose-dependent action of dexamethasone was prevented by cycloheximide, partly blocked by actinomycin D, and reversed after several hr by removing the steroid, when GGT activity decreased with a maximum half-life of 60 to 80 hr. These observations suggest that dexamethasone caused reversible enzyme induction dependent on continuing RNA and protein synthesis. Cell maintenance with fetal calf serum or with N6,O2'-dibutyryl adenosine 3':5'-monophosphate or glucagon markedly reduced the extent of induction by dexamethasone and slightly lowered activity in control cultures. Maintenance at lower pH within the range of 7.2 to 7.8 or at higher glucose concentrations within the range of 2.8 to 28 mM also resulted in markedly lower GGT activities in control or dexamethasone-induced cultures. Glucose repression was independent on insulin concentration. The reversible induction and repression of GGT may reflect broad changes in hepatocyte gene expression rather than specific controls of GGT function. Since GGT in cultured hepatocytes is subject to regulation by a variety of noncarcinogens, caution is required in its use as a preneoplastic marker. Nevertheless, the identification of culture media which preserve low activity may allow studies on carcinogen-induced changes in GGT as a probe of the early events in vitro hepatocarcinogenesis.