Hutson S M, Stinson-Fisher C, Shiman R, Jefferson L S
Am J Physiol. 1987 Mar;252(3 Pt 1):E291-8. doi: 10.1152/ajpendo.1987.252.3.E291.
Culture conditions necessary for optimizing albumin secretion were studied in rat hepatocytes maintained in a chemically defined, serum-free medium. Amino acid analysis of the culture medium, which was based on a 1:1 mixture of Ham's F12:Dulbecco's modified Eagle's medium (unsupplemented medium), revealed that certain essential amino acids were depleted from this medium over a 24-h incubation. Rates of albumin secretion were significantly higher and better maintained when the medium was supplemented with additional amino acids (supplemented medium). Moreover, selective removal of an essential amino acid resulted in an immediate decrease in total protein and albumin synthesis and after 48 h a further selective decrease in albumin synthesis. Linear rates of albumin secretion were observed over a wide variety of experimental conditions, but secretion was not strictly proportional to cell number. Maximal rates of secretion were obtained at plating densities of 2-3 X 10(6) cells/60 mm culture dish. Albumin secretion also increased with time in culture reaching a maximum on days 3 and 4. When added singly, either insulin or dexamethasone increased rates of albumin secretion in a dose-dependent manner, but both hormones and an adequate supply of amino acids were necessary for maximal rates of secretion as well as long-term maintenance of the hepatocytes (greater than 3-4 days). In the presence of dexamethasone the dose-response curve for insulin was shifted toward physiological insulin concentrations. Changes in rates of albumin secretion in response to added hormones in supplemented media were found to parallel changes in albumin synthesis and relative amounts of albumin mRNA. Changes in gene transcription were probably involved.
在化学成分明确的无血清培养基中培养的大鼠肝细胞中，研究了优化白蛋白分泌所需的培养条件。基于Ham's F12：Dulbecco改良 Eagle培养基（未补充培养基）1:1混合物的培养基氨基酸分析表明，在24小时孵育过程中，该培养基中的某些必需氨基酸被耗尽。当培养基补充额外的氨基酸（补充培养基）时，白蛋白分泌率显著更高且维持得更好。此外，选择性去除一种必需氨基酸会导致总蛋白和白蛋白合成立即减少，48小时后白蛋白合成进一步选择性减少。在各种实验条件下均观察到白蛋白分泌的线性速率，但分泌与细胞数量并不严格成比例。在接种密度为2 - 3×10(6)个细胞/60毫米培养皿时获得最大分泌速率。白蛋白分泌也随培养时间增加，在第3天和第4天达到最大值。单独添加时，胰岛素或地塞米松均以剂量依赖性方式增加白蛋白分泌率，但两种激素和充足的氨基酸供应对于最大分泌速率以及肝细胞的长期维持（大于3 - 4天）都是必需的。在地塞米松存在的情况下，胰岛素剂量反应曲线向生理胰岛素浓度偏移。发现在补充培养基中添加激素后白蛋白分泌速率的变化与白蛋白合成和白蛋白mRNA相对量的变化平行。可能涉及基因转录的变化。
