The hepatoblastoma cell line HepG2 has been a matter of many investigations; most of them include biochemical studies of lipoprotein and other hepatic protein metabolism. However, the accurate cellular features of these cells have not been emphasized. We studied the cellular histologic, histochemical, and ultrastructural characteristics of this cell line. In addition, we investigated by immunoenzymatic methods the cellular biosynthesis of several proteins: apolipoproteins-AI, -B, -D, and -E, albumin, alpha-fetoprotein, transferrin, alpha-1-antitrypsin, C-reactive protein, fibronectin, and collagens I, III and IV. The rates of accumulation, in the medium of HepG2 cells, of albumin, alpha-1-antitrypsin, transferrin, and alpha-fetoprotein were 13.2 +/- 1.9; 4.9 +/- 1.5; 3.2 +/- 0.4; and 10.7 +/- 1.7 micrograms/10(6) cells/24 h, respectively. Our results show that HepG2 cells exhibited most cellular features of normal human hepatocytes. Bile canaliculi as well as Golgi apparatus complexes were particularly developed. Except for the C-reactive protein, HepG2 cells have all retained the ability to synthesize hepatic proteins but with some variable intensity from cell to cell. This hepatoblastoma cell line seems to represent a useful tool in the understanding of hepatic protein biosynthesis, particularly for the investigation on the secretory pathway of plasma proteins.
Abstract-Anderson's disease is a rare, hereditary hypocholesterolemic syndrome characterized by chronic diarrhea, steatorrhea, and failure to thrive associated with the absence of apo B48 -containing lipoproteins. To further define the molecular basis of the disease, we studied 8 affected subjects in 7 unrelated families of North African origin after treatment with a low-fat diet. Lipid loading of intestinal biopsies persisted, but the pattern and extent of loading was variable among the patients. Electron microscopy showed lipoprotein-like particles in membrane-bound compartments, the densities (0.65 to 7.5 particles/ 2 ) and the mean diameters (169 to 580 nm) of which were, in general, significantly larger than in a normal fed subject (0.66 particles/ 2 , 209 nm mean diameter). There were also large lipid particles having diameters up to 7043 nm (average diameters from 368 to 2127 nm) that were not surrounded by a membrane. Rarely, lipoprotein-like particles 50 to 150 nm in diameter were observed in the intercellular spaces. Intestinal organ culture showed that apo B and apo AIV were synthesized with apparently normal molecular weights and that small amounts were secreted in lipid-bound forms (density Ͻ1.006 g/mL). Normal microsomal triglyceride transfer protein (MTP) and activity were also detected in intestinal biopsies. Segregation analyses of 4 families excluded, as a cause of the disease, significant regions of the genome surrounding the genes for apo AI, AIV, B, CI, CII, CIII, and E, as were the genes encoding 3 proteins involved in intracellular lipid transport, MTP, and fatty acid binding proteins 1 and 2. The results suggest that a factor other than apoproteins and MTP are important for human intestinal chylomicron assembly and secretion.
The creation of an auxiliary liver by autotransplantation of liver parenchymal cells into the spleen has mainly been studied in rats for the treatment of acute liver failure. In order to apply this procedure to humans with chronic liver insufficiency the aim of this work was: (1) To demonstrate that hepatocytes can survive for long periods after autotransplantation into the spleen; (2) to increase the yield of the isolation of hepatocytes obtained from pig livers since this animal has a more fibrous liver than rats or normal humans and consequently one which is more difficult to dissociate. In 21 pigs isolated hepatocytes were obtained with in collagenase dissociation technique, the yield being 1–3 × 107 cells per gram of liver and the viability 70–95%. The hepatocytes survived and maintained normal morphological and histochemical characteristics up to 7 months after transplantation, the date of sacrifice of the last animal.
The aim of this work was to study hepatocyte multiplication after transplantation into the spleen, in order to apply this technique to the treatment of chronic liver disease. Hepatocytes isolated by an in situ collagenase perfusion technique in Wistar Furth rats were injected into the splenic parenchyma of three groups of syngeneic rats: controls with normal liver (group 1), 75% hepatectomies (group 2), and end-to-side portacaval shunts (group 3). The proliferation of transplanted hepatocytes was studied by autohistoradiography after the intraperitoneal administration of 0.6 µCi/g body weight of [3H]-thymidine, at 1, 3, 7 and 15 days after transplantation of hepatocytes. Significant incorporation of [3H]-thymidine by the transplanted hepatocytes during the study period was observed mostly in groups 2 and 3. The incorporation, although delayed was sustained and of greatest magnitude in the portacaval-shunted animals. The ability of transplanted hepatocytes to proliferate in the spleen, particularly after a portacaval shunt, indicates that this procedure may have therapeutic applications in the treatment of chronic liver disease.
In this study we prepared a pure apolipoprotein D and obtained a specific antiserum to it. The purified apolipoprotein D migrated as a single band of Mr = 29,000 but appeared as five isoforms on isoelectrofocusing. The antiserum did not cross-react with other apolipoproteins. Immunoenzymatic staining revealed the presence of apolipoprotein D in the perinuclear area of the cytoplasm of isolated normal hepatocytes and HepG2 cells. Apolipoprotein D was also localized in intestinal epithelium and in liver cells. The intracellular distribution of apolipoprotein D was similar to that of apolipoprotein B. Our results indicated that apolipoprotein D, like many other circulating apolipoproteins, is synthesized in enterocytes and hepatocytes.
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