Document Detail


Ammonia metabolism capacity of HepG2 cells with high expression of human glutamine synthetase.
MedLine Citation:
PMID:  19073408     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
BACKGROUND: Currently, one of the tough problems for the application of bioartificial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxification. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated. METHODS: hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and amplified. Then hGS mRNA was measured by semi-quantitative RT-PCR; hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH4+ were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry. RESULTS: The expression of hGS mRNA in HepG2/pLNChGS cells (8.306+/-0.336) and HepG2/pLNAFhGS cells (21.358+/-1.716) was much stronger than in control cells (P<0.05), and that in HepG2/pLNAFhGS cells was markedly stronger than in HepG2/pLNChGS cells (P<0.05). The hGS enzymatic activities of HepG2/pLNChGS cells (3.279+/-0.328 U/mg prot) and HepG2/pLNAFhGS cells (4.557+/-0.253 U/mg prot) were higher than those of control cells (P<0.05), and those of HepG2/pLNAFhGS cells were also higher than the activities of HepG2/pLNChGS cells (P<0.05). In addition, the effect of hGS introduction on HepG2 cell proliferation was not significant. The amount of glutamine synthesis in HepG2/pLNChGS or HepG2/pLNAFhGS cells in three different concentrations of NH4+ was higher than in the two control cells (P<0.05). The amount of glutamine synthesis and cell proliferation in the higher concentrations of NH4+ (5 or 10 mmol/L) in HepG2/pLNAFhGS cells increased more than those in HepG2/pLNChGS cells (P<0.05). NH4+ at a high concentration (10 mmol/L) was toxic to HepG2 and HepG2/pLNCX cells, but less toxic to HepG2/pLNChGS and HepG2/pLNAFhGS cells. CONCLUSION: The constructed hepatocytes (HepG2 cells) with specific high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.
Authors:
Nan-Hong Tang; Xiao-Qian Wang; Xiu-Jin Li; Yan-Ling Chen
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Hepatobiliary & pancreatic diseases international : HBPD INT     Volume:  7     ISSN:  1499-3872     ISO Abbreviation:  HBPD INT     Publication Date:  2008 Dec 
Date Detail:
Created Date:  2008-12-16     Completed Date:  2009-04-13     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101151457     Medline TA:  Hepatobiliary Pancreat Dis Int     Country:  China    
Other Details:
Languages:  eng     Pagination:  621-7     Citation Subset:  IM    
Affiliation:
Hepato-Biliary Surgery Institute of Fujian Province, Union Hospital, Fujian Medical University, Fuzhou 350001, China.
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MeSH Terms
Descriptor/Qualifier:
Ammonia / metabolism*
Carcinoma, Hepatocellular*
Cell Division / physiology
Cell Line, Tumor
Gene Expression Regulation, Enzymologic
Gene Expression Regulation, Neoplastic
Glutamate-Ammonia Ligase / genetics*,  metabolism*
Hepatocytes / cytology,  metabolism
Humans
Liver Neoplasms*
Liver, Artificial*
Plasmids
RNA, Messenger / metabolism
Transfection
Chemical
Reg. No./Substance:
0/RNA, Messenger; 7664-41-7/Ammonia; EC 6.3.1.2/Glutamate-Ammonia Ligase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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