Hepatic stellate cells (HSCs), also called Ito cells or lipocytes, are one of inherent liver nonparenchymal cell types located in the Dissé space between hepatocytes and sinusoidal endothelial cells, and account for up to 50%-80% of vitamin A in the form of lipid drops. The methods of primary HSCs isolation mainly focus on density gradient centrifugation combined with centrifugal elutriation, side scatter-activated cell sorting, UV-excited autofluorescence or antibody-based flow cytometry, etc., and will provide solid foundation for the research on physiological and pathological HSCs function. The research of this vitamin A-storing cells has developed and expanded vigorously. In physiological conditions, HSCs are quiescent and play pivotal roles in the synthesis of extracellular matrix (ECM) to maintain its stability with broad uptake and storage of vitamin A, and also regulate liver regeneration. But in pathological conditions, HSCs are activated by constant stimulations or liver injury, then with activated proliferation, reduced lipid drops, and increased ECM synthesis. Morphology of these cells also changes from the star-shaped stellate cells to that of fibroblasts or myofibroblasts with obvious contractibility and secretion of cytokines and chemokines including a variety of proinflammatory factors and adhesion molecules, suggesting that the activation of HSCs is one of the key events in the development of liver fibrosis. Study on the isolation and function of HSCs is always one of the hot topics for liver biology. In this review, we systematically summarize and discuss the recent advances in our understanding of the isolation methods and improvements of HSCs, and functional research of HSCs biology in health and disease, as well as potential directions.
Extracellular Matrix ; metabolism ; Hepatic Stellate Cells ; cytology ; Humans ; Liver ; cytology ; Regeneration ; Vitamin A ; metabolism

Objective To identify and verify the distribution of Telocytes derived from heterogeneous interstitial cells in the vital organs of ApoE mice.Methods Heart,kidney,and liver tissues were harvested from ApoE adult mice. Immunohistochemical assays were performed by using different immunobiological markers.Results Telocytes were found in these vital organs. The expressions of immunobiological markers differed among different organs. CD34,CD117,and CD28 were positively expressed in Telocytes in cardiac tissue;CD117 and plateled-derived growth factor-Α were negatively expressed in Telocytes in renal tissue;and CD117 and plateled-derived growth factor receptor-Α had negative expression in Telocytes in hepatic tissue. Furthermore,the distribution of Telocytes also differed in the same organ.Conclusions Telocytes exist in the vital organs of ApoE mice,as demonstrated by immunohistochemisty assay. The expressions of immunobiological markers differ among Telocytes in different organs.
Animals ; Antigens, CD34 ; metabolism ; CD28 Antigens ; metabolism ; Kidney ; cytology ; Liver ; cytology ; Mice ; Mice, Knockout, ApoE ; Myocardium ; cytology ; Proto-Oncogene Proteins c-kit ; metabolism ; Telocytes ; cytology

OBJECTIVETo study the effect of N-glycosylation on the modification of microvilli on the surface of rat liver epithelial cell WB-F344 and the growth of the cells in culture.

METHODSRecombinant adeno-associated virus (rAAV) expression vector pAGX (+) containing an antisense or a sense fragment of 6A8 cDNA encoding a human alpha-mannosidase was constructed. The recombinant vectors or the mock were transfected into WB-F344 cells by means of lipofectAmine. The transfected cells were selected in G418 medium and cloned by means of limiting dilution. Integration of the transfected DNA into host DNA was detected by neo PCR. Rat liver ER alpha-mannosidase activity in cell supernatant was measured by using P-nitrophenyl-alpha-D-mannopyranoside as a substrate. Microvilli on cell surface were observed upon a scan electron microscope. The growth curves of the cells in culture were drawn.

RESULTSThe cell clones transfected with antisense 6A8 showed reduction of ER alpha-mannosidase activity with various degrees. Clone AS1 and AS2 cell showed a pronounced reduction of the enzymatic activity. In the study on AS1 cells, Con A binding to the cells was found to be enhanced, cell growth in culture became slow from day 5. The microvilli on the cells were reduced and blunted.

CONCLUSIONSTransfection with antisense 6A8 resulted in reduction and blunting of microvilli on the surface of growing WB-F344 cells, which might be related to N-glycosylation modification.

Animals ; Cloning, Molecular ; Epithelial Cells ; cytology ; Glycosylation ; Liver ; cytology ; Microvilli ; Rats ; Transfection ; alpha-Mannosidase ; genetics ; metabolism

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 ; Humans ; Hydrogen Peroxide ; metabolism ; Liver ; cytology ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; 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

Connective Tissue Growth Factor ; metabolism ; Epithelial-Mesenchymal Transition ; Hepatocytes ; cytology ; Humans ; Liver ; pathology ; Liver Cirrhosis ; pathology

OBJECTIVETo explore the separation and culture method of adult hepatocytes.

METHODSThe isolated adult hepatocytes were cultivated by RPMI 1640 medium at 37 degrees C in vitro. The characteristics of the growing hepatocytes were observed. Their synthesis of urea was detected. The transformation efficiency and density's change of lidocaine were analyzed.

RESULTSHepatocytes were successful separated from adult liver. And they were cultivated in common condition and hollow fiber reactor. The functional capacity of hepatocytes was for lidocaine metabolism and urea excretion.

CONCLUSIONThe adult hepatocytes have been successful separated from liver. And they can be cultivated in common condition and hollow fiber reactor. And it could provide a great quantity and high activity of hepatocytes for bioartificial liver.

Animals ; Cell Culture Techniques ; Cells, Cultured ; Hepatocytes ; metabolism ; Humans ; Liver ; cytology ; Liver, Artificial ; trends ; Research

Humans ; Liver ; cytology ; metabolism ; Liver Diseases ; metabolism ; pathology ; therapy ; Receptors, Angiotensin ; metabolism ; Receptors, Cell Surface ; metabolism ; Receptors, Endothelin ; metabolism ; Receptors, Vasopressin ; metabolism

OBJECTIVETo investigate the morphologic and functional characteristics of the immortalized human liver sinusoidal endothelial cell line (LSEC line).

METHODSImmunofluorescence staining and fluorescence microscopy were used to detect the classic endothelial cell markers in LSEC line, and flow cytometry was used to analyze the purity of the human LSEC line. The morphology (including W-P bodies and surface fenestrations) and phagocytotic capacity of the human LSEC line were observed by transmission and scanning electron microscope. The proliferation curve of the human LSEC line was analyzed by MTT assay. The functional differences between the human LSEC line and human primary LSEC in expression of ELAM-1 and ICAM-1, activities of fibrinolysis (PAI-1, t-PA, u-PA), releasing of IL-6 and IL-8 were compared respectively by enzyme linked immunosorbent assay. Comparison of the susceptibility to hypoxia-reoxygenation induced apoptosis between the human LSEC line and human primary LSEC were investigated by TUNEL.

RESULTSThe established human LSEC line maintained a high proliferative ability and has been passaged for more than 80 times in the absence of any growth factors. Immunofluorescence staining showed that the human LSEC line could express classic endothelial cell marks including von Willebrand Factor (vWF), and could take up acetylated low-density lipoproteins (Ac-LDL). The purity of the human LSEC line was confirmed over 95% by flow cytometric analysis. The W-P bodies and the phagocytosis of Dynabeads was demonstrated by transmission electron microscope. And fenestrations could be found cellular surface with scanning electron microscopy. When compared with human primary LSEC, the human LSEC line has an equivalent responsiveness to tumor necrosis factor in up-regulation of ELAM-1 and ICAM-1. The human LSEC line can also release PAI-1, t-PA, u-PA but can not release IL-6 and IL-8 to TNF-alpha. In contrast, human primary LSEC could release IL-6. The human LSEC line showed higher susceptibility to hypoxia-reoxygenation-induced apoptosis, and the percentage of apoptotic cells was as high as (38.4 +/- 6.7)%, while (28.6 +/- 4.5)% and (7.8 +/- 1.2)% respectively in primary LSEC and in human umbilical vein endothelial cells.

CONCLUSIONSThe established human LSEC line maintains the special phenotypes and the major functional characteristics, and especially maintains the high susceptibility to hypoxia-reoxygenation-induced apoptosis. Therefore it is feasible to use this cell line for the study of liver ischemia-reperfusion injury.

Apoptosis ; Cell Line ; Cell Proliferation ; E-Selectin ; metabolism ; Endothelial Cells ; cytology ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Liver ; cytology