抄録
The present study was designed to investigate the synthesis of lipoproteins in liver, using human adult hepatocytes in primary monolayer culture.
The hepatocytes were isolated by collagenase and Dispase (Godo Shusei Co.) digestion from the liver specimens taken for a biopsy from patients. Approximately 85% or more of total isolated cells excluded trypan blue dye. They were inoculated in serum-free media, DM-170 (Kyokuto Seiyaku Co., Ltd.), supplemented with 10-6M insulin, 10-6M dexamethasone, human fibronectin and bovine albumin on collagen precoated culture dishes. These dishes were placed in a humidified incubator at 37°C under 5% CO2/95% air and the medium was changed every 24 hours. Twenty four hours after cell inoculation, the initially round shape of the hepatocytes changed to a cuboidal form as they contacted neighboring cells. The fine structure of cultured hepatocytes such as Golgi apparatus, rough and smooth endoplasmic reticulum in the cytoplasm, and desmosomes and bile canaliculi in the intercellular bordary were observed on day 5 by electron microscopic pictures. A constant rate of total protein synthesis, determined by [14C]-leucine incorporation into trichloroacetic acid (TCA) insoluble material, was observed up to 96 hours of the cultured periods. Albumin and several serum proteins in the concentrated culture media were detected by double immunodiffusion and immuno-electrophoresis between anti human-serum antibody and concentrated conditioning culture media in which the hepatocytes were cultured for 24 hours.
Human plasma HDL3 1.110<d<1.210g/ml and LDL2 1, 019<d<1, 050g/ml were fractionated from pooled normal human plasma by sequential ultracentrifugation. Each lipoprotein fraction was dialyzed, and immunized into rabbits by injection with Freund's complete adjuvant. Each anti-sera was used as anti Apo A-I or Apo B antibody for immunological studies.
A single precipitin line was observed between anti Apo B sera and conc. culture media (approximately 25 times), in which the hepatocytes were cultured for 48 hours. However, Apo A-I was not detected in the same culture media (concentrated by approximately 50 times).
The hepatocytes were cultured with [14C]-leucine in DM-170 under the same condition, incorporation of [14C]-leucine into Apo A-I or Apo B in the media was investigated. After four days of culture, the culture media was pooled and analyzed by anti Apo A-I or anti Apo B sera and anti-rabbit IgG goat sera. The incorporated radioactivities to Apo A-I and Apo B was 371 (92*) and 2886 (183*) dpm/dish/4 days, respectively (control*).
In conclusion, we showed directly synthesis and secretion of Apo A-I and Apo B by the cultured adult hepatocytes of human. This culture system can be utilized for the metabolic study of not only lipoproteins but also hormones and drugs.
