Memon R A, Mohan C, Geiger P J, Bessman S P, Rogers K S
Department of Pharmacology and Nutrition, University of Southern California, Los Angeles 90033.
Biochem Med Metab Biol. 1989 Dec;42(3):216-9. doi: 10.1016/0885-4505(89)90058-3.
Rat liver hepatocytes isolated from a 30-31% percoll density gradient at 10,000g are refractory toward insulin stimulation of 14CO2 formation and 14C-incorporation into protein from [2,3-14C]succinate. Basal hepatocyte oxidation of succinate was not impaired by the presence of 5% percoll in the incubation medium nor was it impaired when percoll-free hepatocytes were used that had been isolated after centrifugation at 9000g; however, in both instances the stimulatory effect of insulin was lost. Hepatocyte damage may have occurred in these processes. This is in contrast to previous work which shows that insulin (10 mU/ml) will stimulate [2,3-14C]succinate oxidation and [2,3-14C]succinate carbon incorporation into protein in non-percoll-treated hepatocytes (isolated by centrifugation at 10g) by about 29%. We conclude that the latter procedure although more time consuming is the more gentle method of choice and leaves the hepatocyte in a form more closely related to an in vivo state than does treatment with a percoll density gradient at 10,000g.
从10,000g下30 - 31% Percoll密度梯度中分离出的大鼠肝脏肝细胞，对胰岛素刺激[2,3 - 14C]琥珀酸生成14CO2以及将14C掺入蛋白质的过程具有抗性。在孵育培养基中存在5% Percoll时，肝细胞对琥珀酸的基础氧化未受损害，使用在9000g离心后分离的无Percoll肝细胞时也未受损害；然而，在这两种情况下，胰岛素的刺激作用均丧失。在这些过程中可能发生了肝细胞损伤。这与之前的研究结果相反，之前的研究表明胰岛素（10 mU/ml）可刺激非Percoll处理的肝细胞（通过10g离心分离）中[2,3 - 14C]琥珀酸氧化以及[2,3 - 14C]琥珀酸碳掺入蛋白质，刺激幅度约为29%。我们得出结论，后一种方法虽然更耗时，但却是更温和的选择方法，与10,000g的Percoll密度梯度处理相比，它使肝细胞保持更接近体内状态的形式。
