Cell Differentiation ; physiology ; Cytokines ; metabolism ; Hepatocytes ; cytology ; Humans ; Stem Cells ; cytology

OBJECTIVETo investigate the effects of farnesoid X receptor (FXR) on lipid metabolism in human hepatic L02 cells.

METHODSA steatosis model and an intervention model were established by treating human hepatocyte line L02 cells with sodium oleate or sodium oleate and sodium chenodeoxycholate (a natural agonist of FXR) respectively. Non-treated L02 cells served as controls. At three time points of 24, 48 and 72 hours, the accumulation of lipid droplets in the hepatocytes was observed by optical microscopy after oil red O staining, and the the expression of FXR and SREBP-1c receptors was detected by RT-PCR and Western blot.

RESULTSCompared with the controls, expressions of FXR mRNA and protein were down-regulated gradually in the steatosis model at 24, 48 and 72 hours, FXR mRNA/beta-actin mRNA was 0.186+/-0.02, 0.182+/-0.028 and 0.181+/-0.022, FXR protein/beta-tubulin protein was 0.105+/-0.016, 0.103+/-0.012 and 0.103+/-0.018, F from 0.01 to 0.14; 24 h vs 48 h, 48 vs72 h: P more than 0.05. The expressions of SREBP-1c mRNA and protein were increased gradually. At 24, 48 and 72 hours, SREBP-1c mRNA/beta-actin mRNA was 0.495+/-0.062, 0.579+/-0.064 and 0.612+/-0.067, SREBP-1c protein/beta-tubulin protein was 0.394+/-0.044, 0.488+/-0.066 and 0.543+/-0.064, F from 0.80 to 4.66, 24 h vs 48 h, 48 vs 72 h: P less than 0.05. In the intervention model, expressions of FXR mRNA and protein were increased markedly compared with the steatosis model. At 24, 48 and 72 hours, FXR mRNA/beta-actin mRNA was 0.253+/-0.041, 0.298+/-0.042 and 0.334+/-0.051, and FXR protein/beta-tubulin protein was 0.221+/-0.022, 0.313+/-0.041 and 0.341+/-0.046, F from 6.41 to 50.93, intervention models vs steatosis models at the same time points: P less than 0.05-0.01. Expressions of SREBP-1 c mRNA and protein were significantly reduced. At 24, 48 and 72 hours, SREBP-1c mRNA/beta-actin mRNA was 0.296+/-0.038, 0.328+/-0.037 and 0.341+/-0.055, and FXR protein /beta-tubulin protein was 0.295+/-0.038, 0.334+/-0.047 and 0.355+/-0.054, F from 8.84 to 48.46; intervention models vs steatosis models at the same time point: P less than 0.01. Both in the steatosis model and the intervention model, content of TG and lipids accumulations were much more than those in the controls. Compared with the intervention model, levels of TG and lipids accumulation were markedly increased in the steatosis model at 24, 48, 72 hours. At 24, 48 and 72 hours, TG/cellular total protein in microg/mg was 173.0+/-20.5, 253.4+/-36.1 and 361.2+/-50.7 in the steatosis model, while in the intervention model the data was 84.1+/-17.2, 113.0+/-14.5 and 127.2+/-20.1, F from 38.70 to 268.13, intervention models vs steatosis models at the same time point: P less than 0.01.

CONCLUSIONExpression of FXR is closely associated with lipid homeostasis in hepatocytes. Up-regulation of the expression of FXR may improve lipidosis in L02 cells. Its possible mechanism involves reduction of SREBP-1c expression and lipogenesis in hepatocytes.

Cell Line ; Fatty Liver ; metabolism ; Hepatocytes ; cytology ; metabolism ; Humans ; Lipid Metabolism ; Receptors, Cytoplasmic and Nuclear ; Up-Regulation

Animals ; Cell Proliferation ; Chemokine CXCL12 ; metabolism ; Hepatocytes ; cytology ; metabolism ; Male ; Rats ; Rats, Wistar ; Receptors, CXCR4 ; metabolism

OBJECTIVETo investigate the effect of ginsenoside (GSS) in regulating level of sex hormone receptors in human liver cell line HL-7702.

METHODSThe growth of HL-7702 were detected by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay for choosing the available concentration of GSS; and the effect of GSS on sex hormone receptors in HL-7702 cells were detected by immuno-histochemistry, Western blot and RT-PCR.

RESULTSGSS significantly enhance the protein and mRNA expressions of estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) in HL7702 cell in a dose-dependent manner, the levels of expressions in the GSS treated group were higher than those in the solvent control group respectively (P < 0.05).

CONCLUSIONGSS can up-regulate the protein and mRNA expressions of sex hormone receptors in HL-7702.

Cell Line ; Ginsenosides ; pharmacology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Receptors, Estrogen ; metabolism

The liver is the largest internal organ in mammals, and is important for the maintenance of normal physiological functions of other tissues and organs. Hepatitis, cirrhosis, liver cancer and other chronic liver diseases are serious threats to human health, and these problems are compounded by a scarcity of liver donors for transplantation therapies. Directed differentiation of embryonic stem cells to liver cells is a promising strategy for obtaining hepatocytes that can be used for cell transplantation. In vitro hepatocyte differentiation of embryonic stem cells requires a profound understanding of normal development during embryonic hepatogenesis. Here we provide a simple description of hepatogenesis in vivo and discuss directed differentiation of embryonic stem cells into hepatocytes in vitro.
Animals ; Cell Differentiation ; Embryonic Stem Cells ; cytology ; Hepatocytes ; cytology ; metabolism ; Humans ; Liver ; cytology ; growth & development ; Signal Transduction

Cell Line ; Clone Cells ; cytology ; metabolism ; Cloning, Molecular ; Genetic Vectors ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Simian virus 40 ; genetics ; Transfection

OBJECTIVETo construct pEGFP-HBx eukaryotic expression plasmid and establish stable and effective transfected rat oval cell (LE/6) strain expressing EGFP-HBx fusion protein to explore the roles of HBx gene and oval cell in carcinogenesis of hepatocellular carcinoma (HCC).

METHODSHBx gene with EcoRI and Hind III endo-enzyme sites was obtained by using PCR from plasmid pcDNA3.1-HBx. The purified HBx gene fragment was inserted into pEGFP-N1 expression vector, and the recombinant plasmid pEGFP-HBx was identified by restriction endonuclease and DNA sequencing analysis. LE/6 cells were transfected with recombinant pEGFP-HBx by lipofectamine reagent. Resistant to G418 clones were selected, and expression of EGFP-HBx fusion protein in clones were examined directly with fluorescence microscope, and these clones were isolated and proliferated. The expression of HBx was detected by RT-PCR analysis and immunocytochemistry.

RESULTSPlasmid pEGFP-HBx has whole HBx gene base and correct reading frame as indicated by restriction endonuclease and DNA sequencing analysis. After transfecting with pEGFP-HBx plasmid, LE/6 cell clones expressing EGFP-HBx fusion protein were obtained. RT-PCR analysis and immunocytochemistry showed that HBx gene was only expression in transfected pEGFP-HBx cells.

CONCLUSIONSThe pEGFP-HBx recombinant expression vector was successfully constructed, and the stable transfected LE/6 strain expressing EGFP-HBx fusion protein was successfully established. It will be helpful in the further study on the roles of HBx and liver oval cell in carcinogenesis of HCC.

Animals ; Cell Line ; Genetic Vectors ; Hepatocytes ; cytology ; metabolism ; Plasmids ; genetics ; Rats ; Stem Cells ; cytology ; metabolism ; Trans-Activators ; genetics ; Transfection

OBJECTIVETo study the expression and distribution of hepatocyte-enriched transcriptional factors during the differentiation of hepatocyte by rat bone marrow stem cells in vitro.

METHODSMesenchymal stem cells were isolated from rat bone marrow and induced into mature hepatocyte in vitro. The mRNA expression levels of hepatocyte nuclear factor 4 alpha (HNF4 alpha), CCAAT enhancer binding protein (C/EBP) alpha and beta were compared between induced and non-induced cultures using semi-quantitative reverse transcription-polymerase chain reaction. The distribution pattern of HNF4 alpha was detected by immunofluorescence staining and observed by fluorescence microscope.

RESULTSTranscriptional factors HNF4 alpha, C/EBP alpha, and C/EBP beta were expressed in the induced cells during the culture process. The mRNA expression levels of HNF4 alpha and C/EBP alpha were significantly higher in induced cultures than those in non-induced cultures in the early stage, whereas C/EBP beta expression was significantly up-regulated in induced cultures at the late stage (P < 0.05). Immunofluorescence staining showed that HNF4 alpha was located in the cell nucleus of differentiated cells.

CONCLUSIONThe characteristic time-dependent expression of transcriptional factors HNF4 alpha, C/EBP alpha, and C/EBP beta during the hepatocyte differentiation by bone marrow stem cells demonstrates that the expressions of these transcriptional factors are closely related to the initiation and maintenance of hepatocyte differentiation.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Female ; Hepatocytes ; cytology ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transcription Factors ; metabolism

OBJECTIVETo assess the effects of nuclear translocation of tissue transglutaminase (TTG) and the release of cytochrome C on hepatocyte apoptosis and to reveal the mechanism of signal transduction of early apoptosis in injured hepatocytes.

METHODSHepatocytes isolated from tissue transglutaminase gene knock-out mice and rats were stimulated with ethanol. Proteins from whole cell, cytoplasm and nuclei were extracted for determination of TTG activity by (14)C-putrescine incorporation. Distribution of TTG throughout the entire cell, as well as just nucleus was observed under a confocal scanning microscope. The amount of cytochrome C released from mitochondria was determined by ELISA. Cell apoptosis was observed by fluorescent cytochemistry.

RESULTSTTG activity in whole cells and nuclei was significantly increased after the hepatocytes were treated with ethanol. Cytochrome C release was remarkably increased in the cells isolated from rat and wild-type mouse after treatment with ethanol but not in TTG gene knock-out mice. Cellular apoptosis appeared in hepatocytes isolated from rats and wild-type mice but not in the hepatocytes from TTG gene knock-out mice after stimulation with ethanol.

CONCLUSIONSIncreased TTG in hepatocytes can be translocated into the nucleus and promote release of mitochondrial cytochrome C into the cytoplasm. Passing through a series of signal pathways, hepatocyte apoptosis is induced eventually.

Animals ; Apoptosis ; Cell Nucleus ; metabolism ; Cytochrome c Group ; metabolism ; Hepatocytes ; cytology ; metabolism ; Male ; Mice ; Rats ; Signal Transduction ; Transglutaminases ; metabolism

Animals ; Heparin ; pharmacology ; Hepatocytes ; cytology ; metabolism ; Mice ; Promoter Regions, Genetic ; genetics ; Transforming Growth Factor beta ; genetics