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Ammonia metabolism capacity of HepG2 cells with high expression of human glutamine synthetase

Hepatobiliary & Pancreatic Diseases International 2008年第6期7卷 621-627页

作者： Tang, Nan-Hong Wang, Xiao-Qian Li, Xiu-Jin Chen, Yan-Ling Fujian Med Univ Union Hosp Hepatobiliary Surg Inst Fujian Prov Fuzhou 350001 Peoples R China

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 NH(4)(+) 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 (Pglutamine synthesis in HepG2/pLNChGS or HepG2/pLNAFhGS cells in three different concentrations of NH(4)(+

关键词： hepatocytes glutamine synthetase, human ammonia detoxification

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