Article
Recent advances in the isolation of liver cells.
Center for Basic Research in Digestive Diseases, Mayo Medical School, Clinic and Foundation, Rochester, Minnesota 55905.
Hepatology (impact factor:
11.66).
09/1994;
20(2):494-514.
pp.494-514
Source: PubMed
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Citations (0)
- Cited In (8)
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Article: Cyclosporine enhances liver regeneration: the role of hepatocyte MHC expression and PGE2--a study relevant to graft immunogenicity.
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ABSTRACT: We have investigated CsA induced liver hyperplasia to explore the potential effects on the immunogenicity of the regenerating liver within the clinical context of rejection after transplantation. Flow cytometry analysis of hepatocytes, isolated 48 hours after 2/3 partial hepatectomy (PH2/3) or sham operation in rats, was performed to determine the effect of CsA on DNA synthesis and MHC molecule expression. The possible role of PGE2 was evaluated by the administration of SC-19220, an EP1-PGE2 receptor antagonist. CsA augmented liver regeneration and this was partially attenuated by SC-19220. The moderate expression of class I MHC expression, as well as the very low class II MHC expression detected in normal hepatocytes by flow cytometry was augmented after PH2/3 and reduced by CsA. The CsA-mediated decrease of hepatocyte immunogenicity was not SC-19220 dependent. It is proposed that the enhancing effect of CsA on hepatocyte proliferation is by means of an indirect mechanism that can be attributed to a) reduced immunogenicity of the regenerating liver as a result of inhibition of class I and II MHC hepatocyte expression and b) increased PGE2 synthesis in the liver mediated by its action on EP1 receptor.European journal of medical research 05/2008; 13(4):154-62. · 1.13 Impact Factor -
Article: The hepatic vagus nerve stimulates hepatic stellate cell proliferation in rat acute hepatitis via muscarinic receptor type 2
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ABSTRACT: Background & aims: We have previously shown that the hepatic vagus nerve stimulates the activation of hepatic progenitor cells (HPC), via muscarinic acetylcholine receptor type 3. Given the coproliferation of HPC and hepatic stellate cells (HSC) in acute hepatitis, we determined whether HSC proliferation is also modulated by vagal activity.Methods: We induced acute hepatitis in Wistar rats by injection of galactosamine and lipopolysaccharides. Hepatitis was preceded by hepatic branch vagotomy or sham vagotomy, by electrical stimulation or sham stimulation and by muscarinic receptor antagonist atropine, nicotinic receptor antagonist mecamylamine or saline injection. Rats were sacrificed after 12 and 48 h and HSC numbers were quantified on immunohistochemical stainings. Furthermore, we performed reverse transcriptase-polymerase chain reaction with receptor-specific primers on total RNA from isolated HSC and determined the in vitro proliferation of HSC in response to acetylcholine, atropine and mecamylamine.Results: HSC numbers were significantly lower after vagotomy than after sham vagotomy. Conversely, more HSC were seen after electrical stimulation than after sham stimulation. Atropine resulted in less HSC than saline at both time points, while mecamylamine treatment only diminished HSC after 12 h, suggesting a predominant involvement of muscarinic receptors. Moreover, HSC express muscarinic receptor type 2 mRNA and protein, as well as nicotinic receptor α1, α5, β1 and vasoactive intestinal peptide receptor 1 mRNA. Furthermore, acetylcholine enhanced the in vitro proliferation of HSC, which was inhibited by atropine, but not by mecamylamine.Conclusions: We show here that the hepatic vagus nerve stimulates HSC proliferation, most likely through binding of acetylcholine on muscarinic receptor type 2.Liver international: official journal of the International Association for the Study of the Liver 04/2010; 30(5):693 - 702. · 3.82 Impact Factor -
Article: Comparative nucleic acid transfection efficacy in primary hepatocytes for gene silencing and functional studies.
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ABSTRACT: Primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as non-alcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. There is a need to identify simple methods to genetically manipulate primary hepatocytes and conduct functional studies with plasmids, small interfering RNA (siRNA) or microRNA (miRNA). New lipofection reagents are available that have the potential to yield higher levels of transfection with reduced toxicity. We have tested several liposome-based transfection reagents used in molecular biology research. We show that transfection efficiency with one of the most recently developed formulations, Metafectene Pro, is high with plasmid DNA (>45% cells) as well as double stranded RNA (>90% with siRNA or microRNA). In addition, negligible cytotoxicity was present with all of these nucleic acids, even if cells were incubated with the DNA:lipid complex for 16 hours. To provide the proof of concept that these conditions can be used not only for overexpression of a gene of interest, but also in RNA interference applications, we targeted two liver expressed genes, Sterol Regulatory Element-Binding Protein-1 and Fatty Acid Binding Protein 5 using plasmid-mediated short hairpin RNA expression. In addition, similar transfection conditions were used to optimally deliver siRNA and microRNA. We have identified a lipid-based reagent for primary hepatocyte transfection of nucleic acids currently used in molecular biology laboratories. The conditions described here can be used to expedite a large variety of research applications, from gene function studies to microRNA target identification.BMC Research Notes 01/2011; 4:8.
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Keywords
CFE
cholangiocytes
conventional purification methods
detect subtle differences
FFE
Immunological approaches
isopyknic centrifugation
key development
large yields
limited preparative value
liver cells
lymphocytes
myofibroblasts
profound impact
purified preparations
reliable
specific cell types
specific liver cell types