AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.
Background/Aim We investigated the relationship between ethanol exposure and heme oxygenase (HO-1) in human hepatocytes in order to ascertain if induction of HO-1 can prevent ethanol induced cellular damage. Methods...Background/Aim We investigated the relationship between ethanol exposure and heme oxygenase (HO-1) in human hepatocytes in order to ascertain if induction of HO-1 can prevent ethanol induced cellular damage. Methods Dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) ethanol exposure were used in the present study. HO-1 mRNA and protein expression were detected by PT-PCR and Western blot respectively. HO-1 activity was indicated by bilirubin and Fe2+ formation. Cytotoxicity was investigated by means of lactate dehydrogenate (LDH) and aspartate transaminase (AST) level in culture supernatants, as well as the intracellular formation of malondialdehyde (MDA), cellular glutathione (GSH) status and CYP 2E1 activity. Results We first demonstrated a dose-dependent response between ethanol exposure and HO-1 mRNA and protein expression in human hepatocytes. We further observed a time-dependent increase of HO-1 mRNA expression using 100 mmol/L ethanol starting 30 minutes after ethanol exposure, reaching its maximum between 3 h and 9 h. Being similar to what had been demonstrated with the mRNA level, increased protein expression started at 6 h after ethanol exposure, and kept continuous elevated over 18 h. In addition, we found that ethanol exposure to hepatocytes markedly increased HO-1 enzyme activity in a time-dependent manner measured as bilirubin and Fe2+ formation in human hepatocytes. Our results clearly showed that ethanol exposure caused a significant increase of LDH, AST, and MDA levels, while the antioxidant GSH was time-dependently reduced. Furthermore, we demonstrated that pre-administration of cobalt protoporphyrin (CoPP) induced HO-1 in human hepatocytes, and prevented an increase of MDA and a decrease of GSH. These effects could be partially reversed by zinc protoporphyrin (ZnPP), an antagonist of HO-1 induction. Conclusion HO-1 expression in cells or organs could lead to new strategies for better prevention and treatment of ethanol-induced oxidative damage in human liver.展开更多
AIM： To find the relationship between hepatitis B virus （HBV） and hepatocytes during the initial state of infection by cDNA microarray. METHODS： Primary normal human hepatocytes （PNHHs） were isolated and infecte...AIM： To find the relationship between hepatitis B virus （HBV） and hepatocytes during the initial state of infection by cDNA microarray. METHODS： Primary normal human hepatocytes （PNHHs） were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA （cDNA） with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS： From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes： LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, HuhT, and Chang liver cells, which were transfected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV- mediated NF-κB activation. CONCLUSION： During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK.展开更多
AIM:Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expr...AIM:Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GMCSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome dedifferentiation, which occurs during continuous stimulation by means of growth factors.展开更多
BACKGROUND: The bioartificial liver is anticipated to be a promising alternative choice for patients with liver failure. Toxic substances which accumulate in the patients' plasma exert deleterious effects on hepat...BACKGROUND: The bioartificial liver is anticipated to be a promising alternative choice for patients with liver failure. Toxic substances which accumulate in the patients' plasma exert deleterious effects on hepatocytes in the bioreactor, and potentially reduce the efficacy of bioartificial liver devices. This study was designed to investigate the effects of plasma from patients with acute on chronic liver failure (AoCLF) on immortalized human hepatocytes in terms of cytochrome P450 gene expression, drug metabolism activity and detoxification capability. METHODS: Immortalized human hepatocytes (HepLi-2 cells) were cultured in medium containing fetal calf serum or human plasma from three patients with AoCLF. The cytochrome P450 (CYP3A5, CYP2E1, CYP3A4) expression, drug metabolism activity and detoxification capability of HepLi-2 cells were assessed by RT-PCR, lidocaine clearance and ammonia elimination assay. RESULTS: After incubation in medium containing AoCLF plasma for 24 hours, the cytochrome P450 mRNA expression of HepLi-2 cells was not significantly decreased compared with control culture. Ammonia elimination and lidocaine clearance assay showed that the ability of ammonia removal and drug metabolism remained stable. CONCLUSIONS: Immortalized human hepatocytes can be exposed to AoCLF plasma for at least 24 hours with no significant reduction in the function of cytochrome P450. HepLi-2 cells appear to be effective in metabolism and detoxification and can be potentially used in the development of bioartificial liver. (Hepatobiliary Pancreat Dis Int 2010; 9:611-614)展开更多
Cryopreserved human hepatocytes were used to investigate the role of arylamine N-acetyltransferase 2 (NAT2; EC 2.3.1.5) polymorphism on the N-acetylation of isoniazid (INH). NAT2 genotype was determined by Taqman alle...Cryopreserved human hepatocytes were used to investigate the role of arylamine N-acetyltransferase 2 (NAT2; EC 2.3.1.5) polymorphism on the N-acetylation of isoniazid (INH). NAT2 genotype was determined by Taqman allelic discrimination assay and INH N-acetylation was measured by high performance liquid chromatography. INH N-acetylation rates in vitro exhibited a robust and highly significant (P<0.005) NAT2 phenotype-dependent metabolism. N-acetylation rates in situ were INH concentration- and time-dependent. Following incubation for 24 h with 12.5 or 100 µmol/L INH, acetyl-INH concentrations varied significantly (P = 0.0023 and P = 0.0002) across cryopreserved human hepatocytes samples from rapid, intermediate, and slow acetylators, respectively. The clear association between NAT2 genotype and phenotype supports use of NAT2 genotype to guide INH dosing strategies in the treatment and prevention of tuberculosis.展开更多
Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage...Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.展开更多
Functional human hepatocytes xenografted into the liver of mice can be used as a model system to study pharmacokinetics,infection of hepatitis viruses,and the efficacy of hepatitis vaccines.Significant levels of liver...Functional human hepatocytes xenografted into the liver of mice can be used as a model system to study pharmacokinetics,infection of hepatitis viruses,and the efficacy of hepatitis vaccines.Significant levels of liver xeno-repopulation have been reported in Fah-/-Rag2-/-Il2rg-/-mice.However,the high mortality and low breeding rate of this model may hinder its application.A new model,termed Fah-/-Nod/Scid mice,which combines the advantages of liver repopulation in Fah-/-mice with the ease of xenotransplantation in Nod/Scid mice was obtained by gradual cross-breeding.Fah-/-Nod/Scid mice were easily maintained in breeding colonies and in adult animal care facilities.FK506 treatment combined with gradual withdrawal of NTBC before cell transplantation ensured that Fah-/-Nod/Scid mice were susceptible to liver xeno-repopulation by human hepatocytes;the proportion of engrafted human hepatocytes reached 33.6%.The function of the expanded human hepatocytes within the chimeric liver was confirmed by weight curve analysis,the expression of characteristic proteins,and the biochemical analysis of liver function.These results show that Fah-/-Nod/Scid mice are an ideal humanized liver mouse model with many useful applications.展开更多
AIM： To observe the relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and cytochrome P450 2E1 （CYP2E1） activity, in order to address if inhibition of CYP2E1 could attenuate...AIM： To observe the relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and cytochrome P450 2E1 （CYP2E1） activity, in order to address if inhibition of CYP2E1 could attenuate ethanol- induced cellular damage. METHODS： The dose-dependent （25-100 mmol/L） and time-dependent （0-24 h） exposures of primary human cultured hepatocytes to ethanol were carried out. CYP2E1 activity and protein expression were detected by spectrophotometer and Western blot analysis respectively. Hepatotoxicity was investigated by determination of lactate dehydrogenase （LDH） and aspartate transaminase （AST） level in hepatocyte culture supernatants, as well as the intracellular formation of malondialdehyde （MDA）. RESULTS： A dose-and time-dependent response between ethanol exposure and CYP2E1 activity in human hepatocytes was demonstrated. Moreover, there was a time-dependent increase of CYP2E1 protein after 100 mmol/L ethanol exposure. Meanwhile, ethanol exposure of hepatocytes caused a time-dependent increase of cellular MDA level, LDH, and AST activities in supernatants. Furthermore, the inhibitor of CYP2E1, diallyl sulfide （DAS） could partly attenuate the increases of MDA, LDH, and AST in human hepatocytes. CONCLUSION： A positive relationship between ethanolinduced oxidative damage in human primary cultured hepatocytes and CYP2E1 activity was exhibited, and the inhibition of CYP2E1 could partly attenuate ethanol-induced oxidative damage.展开更多
In order to investigate the effect of salvia miltiorrhiza hunge(SMB)on the plasma membrane fluidity and the relationship between the lipid peroxidation and the Plasma membrane fluidityin cultured human fetdal hepatocy...In order to investigate the effect of salvia miltiorrhiza hunge(SMB)on the plasma membrane fluidity and the relationship between the lipid peroxidation and the Plasma membrane fluidityin cultured human fetdal hepatocytes,the plasma membrane fluidity,using 1,6-dipheny-1,3,5-hexatriene(DPH)as a fluorescence probe, malondialdehyde(MDA)production as well as alanine aminotransferase(ALT)release of human fetal hepatocytes cultured in Presence of carbon tetrachloride(CCl4)or SMB puls CCl4 were estimated. In the cultured hepatocytes injured by CCl4,significant increments of the MDA production and the ALT release,and significant decrease in the plasma membrane fluidity were observed.when the culture medium was supplied with SMB prior to the additionof CCl4,the CCl4 induced increments in MDA production and ALT release was suppressed signifi cantly and a concomitant raise of plasma membrane fluidity towards normal occurred.The resultssuggested that SMB could suppress the lipid peroxidation in bepatocytes,thereby normal membranefluidity might be retained.展开更多
AIM:To investigate effects of hepatotropic growth factors on radical production in rat hepatocytes during sepsis.METHODS:Rat hepatocytes,isolated by collagenase perfusion,were incubated with a lipopolysaccharide(LPS)-...AIM:To investigate effects of hepatotropic growth factors on radical production in rat hepatocytes during sepsis.METHODS:Rat hepatocytes,isolated by collagenase perfusion,were incubated with a lipopolysaccharide(LPS)-containing cytokine mixture of interleukin-1β,tumor necrosis factor-α and interferon-γ to simulate sepsis and either co-incubated or pre-incubated with hepatotropic growth factors,e.g.hepatocyte growth factor,epidermal growth factor and/or transforming growth factor-α.Cells were analyzed for glutathione levels.Culture supernatants were assayed for produc-tion of reactive oxygen intermediates(ROIs) as well as NO2-,NO3-and S-nitrosothiols.To determine cellular damage,release of aspartate aminotransferase(AST) into the culture medium was analyzed.Activation of nuclear factor(NF)-κB was measured by electrophoretic mobility shift assay.RESULTS:Rat hepatocytes treated with the LPS-containing cytokine mixture showed a significant increase in ROI and nitrogen oxide intermediate formation.AST leakage was not significantly increased in cells treated with the LPS-containing cytokine mixture,independent of growth-factor co-stimulation.However,pretreatment with growth factors significantly reduced AST leakage and ROI formation while increasing cellular glutathione.Application of growth factors did not result in increased NF-κB activation.Pretreatment with growth factors further increased formation of NO2-,NO3-and S-nitrosothiols in hepatocytes stimulated with LPS-containing cytokine mixture.Thus,we propose that,together with an increase in glutathione increased NO2-,NO3-formation might shift their metabolism towards non-toxic products.CONCLUSION:Our data suggest that hepatotropic growth factors positively influence sepsis-induced hepatocellular injury by reducing cytotoxic ROI formation via induction of the cellular protective antioxidative systems.展开更多
BACKGROUND： Differentiation of liver progenitor cells（LPCs） to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. H...BACKGROUND： Differentiation of liver progenitor cells（LPCs） to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system.METHODS： Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line（HSCLi） we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS： Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, lowdensity lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity.CONCLUSIONS： Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.展开更多
Human hepatocyte growth factor can be used to treat cerebral infarction, administered by lateral ventricular, cerebellomedullary cistern or subarachnoid injections. However, the target gene ex-pression product is scar...Human hepatocyte growth factor can be used to treat cerebral infarction, administered by lateral ventricular, cerebellomedullary cistern or subarachnoid injections. However, the target gene ex-pression product is scarcely found in the ischemic penumbra, but extensively distributes in other regions, increasing the risks of gene therapy. The present study directly transfected hepatocyte growth factor gene into the ischemic penumbra of rats with transient middle cerebral artery occlusion. Immunohistochemical analysis revealed that infarct volume was significantly decreased, hepatocyte growth factor protein expression level and vessel quantity in the ischemic penumbra were significantly increased, and learning and memory were significantly improved.展开更多
AIM: To construct and evaluate the functionality of a choanoid-fluidized bed bioreactor (CFBB) based on microencapsulated immortalized human hepatocytes.
AIM: To construct a hepatitis B virus (HBV)-based vector with a reporter gene and to establish an HBV infection system to evaluate the availability of the vector. METHODS: The HBV-based vectors with green fluorescence...AIM: To construct a hepatitis B virus (HBV)-based vector with a reporter gene and to establish an HBV infection system to evaluate the availability of the vector. METHODS: The HBV-based vectors with green fluorescence protein (GFP) were packaged into the liver of immunodeficient mice through transfer and helper plasmid using hydrodynamic technology. Wild type HBV (wt HBV) was provided by plasmid MC2009. Primary human hepatocytes (PHH) were isolated and infected with recombinant HBV (rHBV) or wt HBV. GFP expression was monitored by confocal and flow cytometry. HBV DNA and HBV surface antigen (HBSAg) were analyzed by PCR and ELISA. RESULTS: 3 × 107 wt HBV copies/mL and 5 × 106 rHBV copies/mL were collected from mice serum. In the wt HBV infected group, HBV progeny was 2 × 107 copies/mL and HBSAg was 770 ng/mL. In the rHBV infected group, GFP fluorescence was detected on d 3 post-infection and over 85% of the parenchymal cells expressed green fluorescence on d 12 post-infection. Compared with wt HBV in the PHH infection system, no rHBV DNA or HBSAg were detected in PHH culture media. CONCLUSION: An effective HBV based vector was developed, which proved to be a useful HBV infection system. This vector and infection system can be applied to develop a therapeutic vector and study the HBV life cycle and viral pathogenesis.展开更多
基金Supported by Major Scientific and Technological Project of Shandong Province,No.201221019Cisco Clinical Oncology Research Fund and Bayer Schering Cancer Research Fund,No.Y-B2012-011
文摘AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.
基金This work was supported by the National Natural Science Foundation of China (NSFC) (30271130).
文摘Background/Aim We investigated the relationship between ethanol exposure and heme oxygenase (HO-1) in human hepatocytes in order to ascertain if induction of HO-1 can prevent ethanol induced cellular damage. Methods Dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) ethanol exposure were used in the present study. HO-1 mRNA and protein expression were detected by PT-PCR and Western blot respectively. HO-1 activity was indicated by bilirubin and Fe2+ formation. Cytotoxicity was investigated by means of lactate dehydrogenate (LDH) and aspartate transaminase (AST) level in culture supernatants, as well as the intracellular formation of malondialdehyde (MDA), cellular glutathione (GSH) status and CYP 2E1 activity. Results We first demonstrated a dose-dependent response between ethanol exposure and HO-1 mRNA and protein expression in human hepatocytes. We further observed a time-dependent increase of HO-1 mRNA expression using 100 mmol/L ethanol starting 30 minutes after ethanol exposure, reaching its maximum between 3 h and 9 h. Being similar to what had been demonstrated with the mRNA level, increased protein expression started at 6 h after ethanol exposure, and kept continuous elevated over 18 h. In addition, we found that ethanol exposure to hepatocytes markedly increased HO-1 enzyme activity in a time-dependent manner measured as bilirubin and Fe2+ formation in human hepatocytes. Our results clearly showed that ethanol exposure caused a significant increase of LDH, AST, and MDA levels, while the antioxidant GSH was time-dependently reduced. Furthermore, we demonstrated that pre-administration of cobalt protoporphyrin (CoPP) induced HO-1 in human hepatocytes, and prevented an increase of MDA and a decrease of GSH. These effects could be partially reversed by zinc protoporphyrin (ZnPP), an antagonist of HO-1 induction. Conclusion HO-1 expression in cells or organs could lead to new strategies for better prevention and treatment of ethanol-induced oxidative damage in human liver.
基金Supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea, No. A050145
文摘AIM： To find the relationship between hepatitis B virus （HBV） and hepatocytes during the initial state of infection by cDNA microarray. METHODS： Primary normal human hepatocytes （PNHHs） were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA （cDNA） with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS： From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes： LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, HuhT, and Chang liver cells, which were transfected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV- mediated NF-κB activation. CONCLUSION： During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK.
基金Supported by the "Matthias Lackas-Stiftung", "Paul und Ursula Klein-Stiftung", "Heinrich und Erna Schaufler-Stiftung", "Gisela Stadelmann-Stiftung", and study grants from the Johann Wolfgang Goethe-Universitatsklinikum,Universitatsklinikum Essen (IFORES),and Deutsche Forschungsgemeinschaft (AU 117/4-1)
文摘AIM:Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GMCSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome dedifferentiation, which occurs during continuous stimulation by means of growth factors.
基金supported by grants from the NationalS&T Major Project for Infectious Disease Control of China(2008ZX10002-005)the National High Technology Research and Development Program of China(2006AA02A140)+1 种基金the National Natural Science Foundation of China(30630023)Zhejiang Health Science Foundation(2009A076)
文摘BACKGROUND: The bioartificial liver is anticipated to be a promising alternative choice for patients with liver failure. Toxic substances which accumulate in the patients' plasma exert deleterious effects on hepatocytes in the bioreactor, and potentially reduce the efficacy of bioartificial liver devices. This study was designed to investigate the effects of plasma from patients with acute on chronic liver failure (AoCLF) on immortalized human hepatocytes in terms of cytochrome P450 gene expression, drug metabolism activity and detoxification capability. METHODS: Immortalized human hepatocytes (HepLi-2 cells) were cultured in medium containing fetal calf serum or human plasma from three patients with AoCLF. The cytochrome P450 (CYP3A5, CYP2E1, CYP3A4) expression, drug metabolism activity and detoxification capability of HepLi-2 cells were assessed by RT-PCR, lidocaine clearance and ammonia elimination assay. RESULTS: After incubation in medium containing AoCLF plasma for 24 hours, the cytochrome P450 mRNA expression of HepLi-2 cells was not significantly decreased compared with control culture. Ammonia elimination and lidocaine clearance assay showed that the ability of ammonia removal and drug metabolism remained stable. CONCLUSIONS: Immortalized human hepatocytes can be exposed to AoCLF plasma for at least 24 hours with no significant reduction in the function of cytochrome P450. HepLi-2 cells appear to be effective in metabolism and detoxification and can be potentially used in the development of bioartificial liver. (Hepatobiliary Pancreat Dis Int 2010; 9:611-614)
基金supported by National Institutes of Health grants R25-CA134283 and P20-GM113226(USA)
文摘Cryopreserved human hepatocytes were used to investigate the role of arylamine N-acetyltransferase 2 (NAT2; EC 2.3.1.5) polymorphism on the N-acetylation of isoniazid (INH). NAT2 genotype was determined by Taqman allelic discrimination assay and INH N-acetylation was measured by high performance liquid chromatography. INH N-acetylation rates in vitro exhibited a robust and highly significant (P<0.005) NAT2 phenotype-dependent metabolism. N-acetylation rates in situ were INH concentration- and time-dependent. Following incubation for 24 h with 12.5 or 100 µmol/L INH, acetyl-INH concentrations varied significantly (P = 0.0023 and P = 0.0002) across cryopreserved human hepatocytes samples from rapid, intermediate, and slow acetylators, respectively. The clear association between NAT2 genotype and phenotype supports use of NAT2 genotype to guide INH dosing strategies in the treatment and prevention of tuberculosis.
基金The work was supported by grants from the National Nature Science Foundation of China (No. 30271155) China national key basic research and development program (No. 2022CB512908).
文摘Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.
基金supported by the National High Technology Research and Development Program of China(Grant No. 2006AA02Z474)the National Natural Science Foundation of China(Grant No. 30801115)+1 种基金the China Postdoctoral Science Foundation(Grant No. 20070410743)the National Basic Research Program of China(Grant No. 2010CB945600)
文摘Functional human hepatocytes xenografted into the liver of mice can be used as a model system to study pharmacokinetics,infection of hepatitis viruses,and the efficacy of hepatitis vaccines.Significant levels of liver xeno-repopulation have been reported in Fah-/-Rag2-/-Il2rg-/-mice.However,the high mortality and low breeding rate of this model may hinder its application.A new model,termed Fah-/-Nod/Scid mice,which combines the advantages of liver repopulation in Fah-/-mice with the ease of xenotransplantation in Nod/Scid mice was obtained by gradual cross-breeding.Fah-/-Nod/Scid mice were easily maintained in breeding colonies and in adult animal care facilities.FK506 treatment combined with gradual withdrawal of NTBC before cell transplantation ensured that Fah-/-Nod/Scid mice were susceptible to liver xeno-repopulation by human hepatocytes;the proportion of engrafted human hepatocytes reached 33.6%.The function of the expanded human hepatocytes within the chimeric liver was confirmed by weight curve analysis,the expression of characteristic proteins,and the biochemical analysis of liver function.These results show that Fah-/-Nod/Scid mice are an ideal humanized liver mouse model with many useful applications.
基金Supported by the National Science Foundation of China, No. 30271130
文摘AIM： To observe the relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and cytochrome P450 2E1 （CYP2E1） activity, in order to address if inhibition of CYP2E1 could attenuate ethanol- induced cellular damage. METHODS： The dose-dependent （25-100 mmol/L） and time-dependent （0-24 h） exposures of primary human cultured hepatocytes to ethanol were carried out. CYP2E1 activity and protein expression were detected by spectrophotometer and Western blot analysis respectively. Hepatotoxicity was investigated by determination of lactate dehydrogenase （LDH） and aspartate transaminase （AST） level in hepatocyte culture supernatants, as well as the intracellular formation of malondialdehyde （MDA）. RESULTS： A dose-and time-dependent response between ethanol exposure and CYP2E1 activity in human hepatocytes was demonstrated. Moreover, there was a time-dependent increase of CYP2E1 protein after 100 mmol/L ethanol exposure. Meanwhile, ethanol exposure of hepatocytes caused a time-dependent increase of cellular MDA level, LDH, and AST activities in supernatants. Furthermore, the inhibitor of CYP2E1, diallyl sulfide （DAS） could partly attenuate the increases of MDA, LDH, and AST in human hepatocytes. CONCLUSION： A positive relationship between ethanolinduced oxidative damage in human primary cultured hepatocytes and CYP2E1 activity was exhibited, and the inhibition of CYP2E1 could partly attenuate ethanol-induced oxidative damage.
文摘In order to investigate the effect of salvia miltiorrhiza hunge(SMB)on the plasma membrane fluidity and the relationship between the lipid peroxidation and the Plasma membrane fluidityin cultured human fetdal hepatocytes,the plasma membrane fluidity,using 1,6-dipheny-1,3,5-hexatriene(DPH)as a fluorescence probe, malondialdehyde(MDA)production as well as alanine aminotransferase(ALT)release of human fetal hepatocytes cultured in Presence of carbon tetrachloride(CCl4)or SMB puls CCl4 were estimated. In the cultured hepatocytes injured by CCl4,significant increments of the MDA production and the ALT release,and significant decrease in the plasma membrane fluidity were observed.when the culture medium was supplied with SMB prior to the additionof CCl4,the CCl4 induced increments in MDA production and ALT release was suppressed signifi cantly and a concomitant raise of plasma membrane fluidity towards normal occurred.The resultssuggested that SMB could suppress the lipid peroxidation in bepatocytes,thereby normal membranefluidity might be retained.
基金Supported by The Federal Ministry of Research (BMBF-01 GN0984)
文摘AIM:To investigate effects of hepatotropic growth factors on radical production in rat hepatocytes during sepsis.METHODS:Rat hepatocytes,isolated by collagenase perfusion,were incubated with a lipopolysaccharide(LPS)-containing cytokine mixture of interleukin-1β,tumor necrosis factor-α and interferon-γ to simulate sepsis and either co-incubated or pre-incubated with hepatotropic growth factors,e.g.hepatocyte growth factor,epidermal growth factor and/or transforming growth factor-α.Cells were analyzed for glutathione levels.Culture supernatants were assayed for produc-tion of reactive oxygen intermediates(ROIs) as well as NO2-,NO3-and S-nitrosothiols.To determine cellular damage,release of aspartate aminotransferase(AST) into the culture medium was analyzed.Activation of nuclear factor(NF)-κB was measured by electrophoretic mobility shift assay.RESULTS:Rat hepatocytes treated with the LPS-containing cytokine mixture showed a significant increase in ROI and nitrogen oxide intermediate formation.AST leakage was not significantly increased in cells treated with the LPS-containing cytokine mixture,independent of growth-factor co-stimulation.However,pretreatment with growth factors significantly reduced AST leakage and ROI formation while increasing cellular glutathione.Application of growth factors did not result in increased NF-κB activation.Pretreatment with growth factors further increased formation of NO2-,NO3-and S-nitrosothiols in hepatocytes stimulated with LPS-containing cytokine mixture.Thus,we propose that,together with an increase in glutathione increased NO2-,NO3-formation might shift their metabolism towards non-toxic products.CONCLUSION:Our data suggest that hepatotropic growth factors positively influence sepsis-induced hepatocellular injury by reducing cytotoxic ROI formation via induction of the cellular protective antioxidative systems.
基金supported by grants from the Chinese High-Tech Research&Development(863)Program(2013AA020102 and 2012AA020204)Science Fund for Creative Research Groups of the National Natural Science Foundation of China(81121002)+3 种基金Fundamental Research Funds for the Central Universities(2014XZZX008 and 2014FZA7010)Zhejiang CTM Science and Technology Project(2011ZB061)Zhejiang Health Science Foundation(2016KYA148)the National Health and Medical Research Council of Australia and Cancer Council of Western Australia
文摘BACKGROUND： Differentiation of liver progenitor cells（LPCs） to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system.METHODS： Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line（HSCLi） we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS： Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, lowdensity lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity.CONCLUSIONS： Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.
基金the Out-standing Middle-aged and Young Talents, Education Department of Hubei Province, No. Q20082403
文摘Human hepatocyte growth factor can be used to treat cerebral infarction, administered by lateral ventricular, cerebellomedullary cistern or subarachnoid injections. However, the target gene ex-pression product is scarcely found in the ischemic penumbra, but extensively distributes in other regions, increasing the risks of gene therapy. The present study directly transfected hepatocyte growth factor gene into the ischemic penumbra of rats with transient middle cerebral artery occlusion. Immunohistochemical analysis revealed that infarct volume was significantly decreased, hepatocyte growth factor protein expression level and vessel quantity in the ischemic penumbra were significantly increased, and learning and memory were significantly improved.
基金Supported by The Grants from the National Scientific and Technological Major Project of China,No.2011ZX10004-901,No.2013ZX10004904the National Science and Technology Major Project,No.2012ZX10002006
文摘AIM: To construct and evaluate the functionality of a choanoid-fluidized bed bioreactor (CFBB) based on microencapsulated immortalized human hepatocytes.
基金the grants from the National Science Foundation of China, No. 30271177 and No. 39870676the Natural Science Foundation of Guangdong Province, No. 021903
文摘AIM: To construct a hepatitis B virus (HBV)-based vector with a reporter gene and to establish an HBV infection system to evaluate the availability of the vector. METHODS: The HBV-based vectors with green fluorescence protein (GFP) were packaged into the liver of immunodeficient mice through transfer and helper plasmid using hydrodynamic technology. Wild type HBV (wt HBV) was provided by plasmid MC2009. Primary human hepatocytes (PHH) were isolated and infected with recombinant HBV (rHBV) or wt HBV. GFP expression was monitored by confocal and flow cytometry. HBV DNA and HBV surface antigen (HBSAg) were analyzed by PCR and ELISA. RESULTS: 3 × 107 wt HBV copies/mL and 5 × 106 rHBV copies/mL were collected from mice serum. In the wt HBV infected group, HBV progeny was 2 × 107 copies/mL and HBSAg was 770 ng/mL. In the rHBV infected group, GFP fluorescence was detected on d 3 post-infection and over 85% of the parenchymal cells expressed green fluorescence on d 12 post-infection. Compared with wt HBV in the PHH infection system, no rHBV DNA or HBSAg were detected in PHH culture media. CONCLUSION: An effective HBV based vector was developed, which proved to be a useful HBV infection system. This vector and infection system can be applied to develop a therapeutic vector and study the HBV life cycle and viral pathogenesis.
