DeSante D C, Little L, Peavy D E, Vinicor F
Biochem J. 1984 Oct 1;223(1):39-46. doi: 10.1042/bj2230039.
An improved non-perfusion method for the preparation of cultured foetal-rat hepatocytes is described. Digestion of the liver with collagenase and deoxyribonuclease I gave yields of 40 X 10(6) hepatocytes/g of liver. The plating efficiency of hepatocytes in medium with 10 microM-cortisol was 50%. Cell morphology and metabolism were maintained through 3 days of monolayer culture, with minimal contamination by haematopoietic cells or fibroblasts. The cultured cells bound and degraded 125I-insulin in a time- and dose-dependent manner. The estimated ED50 for competitive binding at 37 degrees C was 1.1 nM. Curvilinear Scatchard plots were observed, with estimates of 16 500 high-affinity sites (Kd = 813 pM) and 53 000 low-affinity sites (Kd = 23 nM) per cell. The cultured cells demonstrated a glycogenic response to insulin, with an estimated ED50 of 120 pM. The degree of glycogenic response to insulin varied with time in culture: 500% above basal on day 1, 200% on day 2, and only 150% on day 3. Cultured foetal cells also exhibited a time-dependent uptake of 2-aminoisobutyric acid, which, in contrast with previous reports with adult cells, was not stimulated by the presence of 10 nM-insulin. Cultured foetal hepatocytes may provide an interesting model with which to study the relationship between insulin-receptor binding and insulin action.
本文描述了一种改良的非灌注法，用于制备培养的胎鼠肝细胞。用胶原酶和脱氧核糖核酸酶I消化肝脏，每克肝脏可获得40×10⁶个肝细胞。肝细胞在含有10微摩尔皮质醇的培养基中的接种效率为50%。在单层培养的3天时间里，细胞形态和代谢得以维持，造血细胞或成纤维细胞的污染极少。培养的细胞以时间和剂量依赖的方式结合并降解¹²⁵I标记的胰岛素。在37℃下竞争性结合的估计半数有效浓度（ED50）为1.1纳摩尔。观察到曲线型Scatchard图，每个细胞估计有16500个高亲和力位点（解离常数Kd = 813皮摩尔）和53000个低亲和力位点（Kd = 23纳摩尔）。培养的细胞对胰岛素表现出糖原生成反应，估计ED50为120皮摩尔。糖原生成反应对胰岛素的程度随培养时间而变化：第1天比基础水平高500%，第2天高200%，第3天仅高150%。培养的胎肝细胞还表现出对2-氨基异丁酸的时间依赖性摄取，与先前关于成年细胞的报道不同，10纳摩尔胰岛素的存在并未刺激这种摄取。培养的胎肝细胞可能为研究胰岛素受体结合与胰岛素作用之间的关系提供一个有趣的模型。
