ラット培養肝細胞における高比重リポ蛋白 (HDL<sub>3</sub>) の合成

Abstract

It is indicated that liver plays an important role in lipoproteins metabolism. Recently relationship between plasma concentrations of high density lipoprotein (HDL) and coronary heart disease has been reported. The present study was to investigate lipoproteins synthesis, especially HDL3 or Apo A-I, using a culture system of rat liver parenchymal cells. The cell culture system has several advantages such as cell homogeneity and the possibility of longer incubation than liver perfusion system that has been used by many investigators.The isolation of rat hepatocytes was performed accordint to the method of Berry and Friend with some modifications. The hepatocytes were cultured in the complete synthetic culture media HI/WO5/BA2000 (International Scientific Ind.) under 5% CO2/95% air at 37°C. Twenty-four and 48 hours after cell inoculation, light and electron microscopic study and the following metabolic function of the cells were examined: 1) gluconeogenesis from pyruvate and lactate (179.5 nmoles/mg cell protein/hour), 2) induction of tyrosine aminotransferase by 10-5M dexamethasone (43.8mU/mg cell protein), 3) membrane transport of C14-α-aminoisobutyric acid (AIB)(9.5 AIB distribution ratio) and 4) albumin synthesis (156μg/mg cell protein/hour). The results of metabolic and morphological studies showed that integrity of cultured cells was well maintained at least for 48 hours after inoculation.Rat HDL3 (1.110<d<1.210g/ml) was isolated by sequential ultracentrifugation. Blood was obtained from male rats weighting 250 to 300g who had been fasted for 24 hours. The density of plasma was adjusted to d=1.110g/ml with solid KBr and centrifuged in a Type 40.3 rotor on the LS-50 preparative ultracentrofuge (Beckamn Instruments Inc.) for 22 hours at 105, 000×g. After 22 hours, the top 2ml was removed by tube slicing technique. Then, the density of the infranatant solution was adjusted with solid KBr to d=1.210g/ml and centrifuged at 105, 000×g for 44 hours. HDL3 was washed at d=1.210g/ml to remove contaminating serum albumin and was dialyzed exhaustively against 0.154M NaCl, pH 7.5 or double distilled water at 4°C. The rationale for using HDL3 is that this density fraction contains predominantly Apo A-I and a minimal amount of other apolipoproteins, as compared to other density classes. Anti-rat HDL3 serum was prepared by several intraperitoneal injections of rat HDL3 with equal volumes of Freund's complete adjuvant at intervals of 7 days. The anti-sera were examined by immunodiffusion and immunoelectrophoresis with various antigens such as VLDL (d<1.006g/ml), LDL2 (1.019<d<1.050g/ml), HDL3 and peak II (containing only Apo A-I) and peak III (containing mainly C peptides) fractions of apo-HDL3 from Sephadex G-100 column chromatography. Anti-rat HDL3 serum used for current study reacted only with HDL3 or Apo A-I. The concentrated culture media in which hepatocytes were cultured for 20 hours, were examined immunologically. A single precipitin line was observed between culture media and anti-rat HDL3 or anti-rat Apo A-I on immunodiffusion and immunoelectrophoresis, indicating the presence of Apo A-I in cultured media. HDL3 fraction (1.110<d<1.210g/ml) isolated from pooled culture media was analyzed for identification of apolipoproteins. The electrophoretic patterns of totally delipidized HDL3 fractionated from culture-media on 7% polyacrylamide gel with 8M urea were comparable with electrophoretic patterns of rat plasma Apo-HDL3. Not only the Apo A-I band but also the Apo C bands were observed. These findings indicated that HDL3 or Apo A-I synthesis and secretion by cultured hepatocytes.