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

Animals ; Apoptosis ; Cell Line ; Hepatic Stellate Cells ; cytology ; Rats ; Signal Transduction ; Wnt Proteins ; metabolism ; beta Catenin ; metabolism

Cell Line ; Hepatic Stellate Cells ; cytology ; metabolism ; pathology ; Humans ; Oxidative Stress ; Phosphatidylcholines ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo observe the effects of Wnt3a on proliferation and, activation of hepatic stellate cells (HSCs) and their the expression of the transforming growth factor beta (TGFb) and /Smad signaling factors of rat hepatic stellate cells line in vitro using a rat HSC line.

METHODSSynchronized HSC-T6 cells were stimulated with various concentrations of recombinant Wnt3a (50, 100, 200, 250 and 300 ng/mL). Unstimulated cells served as controls. Edu Effects on proliferation were determined by EdU (5-ethynyl-2'-deoxyuridine) incorporation assay and fluorescence microscopy.analysis was used to observe the proliferation of the hepatic stellate cells stimulated by different concentration of recombinant Wnt3a, and the Effects on the protein expression of TGFb/Smad signaling factors was assessed by western blot detection (gray-value analysis) of alpha-smooth muscle actin (a-SMA), a-SMA, TGFb1, Smad3, and and Smad7; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as the normalization control in the hepatic stellate cells was observed by Western blot analysis .The correlation was also observed. The significance of inter-group differences was assessed by one-way ANOVA, and correlations were determined using bivariate statistical modeling.

RESULTSIn general, HSC The proliferation of hepatic stellate cells increased after the addition of in response to Wnt3a stimulation for 24 h, reaching its peak at the maximum proliferation rate was observed with the 200 ng/mL Wnt3a concentration (63.00+/-2.30%), and it increased dramatically compared with those in which was significantly higher than the proliferation rates of the unstimulated control cells, and the cells stimulated with 50, 100 and 150 ng/mLl group (P less than 0.05), but the increase was not significantly different from that in the compared cells stimulated with 250 and 300 ng/mLl group,it had no obvious increase(P more than 0.05).; The Wnt3a stimulation also led to time-dependent increases in the protein expressions of a-SMA, TGFb1, and Smad3 increased with the addition of Wnt3a and the extension of time . For all three, The maximal amount of increased protein expression all reached to the was maximal produced by stimulation when hepatic stellate cells were treated by with 300 ng/mLl Wnt3a for 48 h hours,and the rations of(normalized gray- values:s of a-SMA, 1.0860+/-0.0101; TGFb1, 1.0346+/-0.0118; Smad3, to GAPDH were 1.0860+/-0.0101, 1.0346+/-0.0118, 1.0306+/-0.0122)respectively. However in contrast, the Wnt3a stimulation led to concentration- and time-dependent decreases in Smad7 expression varied inversely, with to them with the minimal ration of it to GAPDH the maximal decrease occurring with 300 ng/mL Wnt3a for 48 h (0.7736+/-0.0139) after being treated by 300 ng/ml Wnt3a for 48h. The comparison was remarkably discrepant, (P less than 0.05).There were positive correlations between a-SMA expression and was found to be positively correlated to TGFb1, Smad3 (r=0.968, P less than 0.05) and; Smad3 (r=0.997, P less than 0.01), but a-SMA and Smad7 had negatively correlated to Smad7 ion(r=0.960, P less than 0.05).

CONCLUSIONWnt3a can increase the stimulates proliferation as well as and activation of rat the hepatic stellate cells HSCs , and upregulate modifies the expression of TGFb/Smad signaling factors, of the hepatic stellate cells, and which may promote the hepatic fibrosis.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Rats ; Signal Transduction ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; metabolism ; Wnt3A Protein ; pharmacology

BACKGROUND/AIMS: Therapies involving bone-marrow-derived mesenchymal stem cells (BM-MSCs) have considerable potential in the management of hepatic disease. BM-MSCs have been investigated in regenerative medicine due to their ability to secrete various growth factors and cytokines that regress hepatic fibrosis and enhance hepatocyte functionality. The aim of this study was to determine the antifibrosis effect of BM-MSCs on activated hepatic stellate cells (HSCs) and the mechanism underlying how BM-MSCs modulate the function of activated HSCs. METHODS: We used HSCs in both direct and indirect co-culture systems with BM-MSCs to evaluate the antifibrosis effect of BM-MSCs. The cell viability and apoptosis were evaluated by a direct co-culture system of activated HSCs with BM-MSCs. The activations of both HSCs alone and HSCs with BM-MSCs in the direct co-culture system were observed by immunocytochemistry for alpha-smooth muscle actin (alpha-SMA). The levels of growth factors and cytokines were evaluated by an indirect co-culture system of activated HSCs with BM-MSCs. RESULTS: The BM-MSCs in the direct co-culture system significantly decreased the production of alpha-SMA and the viability of activated HSCs, whereas they induced the apoptosis of activated HSCs. The BM-MSCs in the indirect co-culture system decreased the production of transforming growth factor-beta1 and interleukin (IL)-6, whereas they increased the production of hepatocyte growth factor and IL-10. These results confirmed that the juxtacrine and paracrine effects of BM-MSCs can inhibit the proliferative, fibrogenic function of activated HSCs and have the potential to reverse the fibrotic process by inhibiting the production of alpha-SMA and inducing the apoptosis of HSCs. CONCLUSIONS: These results have demonstrated that BM-MSCs may exert an antifibrosis effect by modulating the function of activated HSCs.
Apoptosis ; Bone Marrow Cells/*cytology ; Cell Differentiation ; Coculture Techniques ; Hepatic Stellate Cells/*cytology/metabolism ; Hepatocyte Growth Factor/metabolism ; Humans ; Immunophenotyping ; Interleukin-10/metabolism ; Interleukin-6/metabolism ; Liver Cirrhosis ; Mesenchymal Stromal Cells/*cytology/metabolism ; Transforming Growth Factor beta1/metabolism

OBJECTIVETo observe the effect of ligand of peroxisome proliferators-activated receptor gamma (PPAR gamma) 15d-PGJ2 on the proliferation and activation of hepatic stellate cells (HSC) and to study the role played by PPAR gamma during the process of HSC activation.

METHODSBy using RT-PCR and cell culture, we investigated the effects of 5 micro mol/L and 10 micro mol/L 15d-PGJ2 on culture-activated HSC and on PDGF-induced HSC proliferation, production of extracellular matrix and expression of chemokines.

RESULTSThe expression of alpha-SMA was significantly suppressed by 5mumol/L 15d-PGJ2, and the expression of PPAR gamma was significantly higher in the 15d-PGJ2 treated group than in the untreated group (0.64+/-0.03 vs 0.09+/-0.01, t=36.0517, P<0.01); PDGF-induced HSC proliferation was dose-dependently suppressed by 15d-PGJ2; the expressions of PPAR gamma in 5 micro mol/L and also in 10 micro mol/L 15d-PGJ2 plus PDGF pre-treated group increased much more than those in the PDGF-treated group (0.03+/-0.02 vs 0.60+/-0.03, t=42.6616, P<0.01 and 0.03+/-0.02 vs 0.69+/-0.04, t=33.83, P<0.01); the expressions of alpha-SMA, alpha 1 (I)-collagen and MCP-1 were suppressed.

CONCLUSIONActivation of PPAR gamma can modulate pro-fibrotic and pro-inflammatory roles of HSC and the increased expression of PPAR gamma may become a new target for antifibrosis.

Animals ; Cell Differentiation ; Cell Proliferation ; drug effects ; Cells, Cultured ; Hepatic Stellate Cells ; cytology ; metabolism ; Male ; PPAR gamma ; metabolism ; Prostaglandin D2 ; analogs & derivatives ; pharmacology ; Rats ; Rats, Wistar

Mesothelial cells (MCs) cover the surface of visceral organs and the parietal walls of cavities, and they synthesize lubricating fluids to create a slippery surface that facilitates movement between organs without friction. Recent studies have indicated that MCs play active roles in liver development, fibrosis, and regeneration. During liver development, the mesoderm produces MCs that form a single epithelial layer of the mesothelium. MCs exhibit an intermediate phenotype between epithelial cells and mesenchymal cells. Lineage tracing studies have indicated that during liver development, MCs act as mesenchymal progenitor cells that produce hepatic stellate cells, fibroblasts around blood vessels, and smooth muscle cells. Upon liver injury, MCs migrate inward from the liver surface and produce hepatic stellate cells or myofibroblast depending on the etiology, suggesting that MCs are the source of myofibroblasts in capsular fibrosis. Similar to the activation of hepatic stellate cells, transforming growth factor β induces the conversion of MCs into myofibroblasts. Further elucidation of the biological and molecular changes involved in MC activation and fibrogenesis will contribute to the development of novel approaches for the prevention and therapy of liver fibrosis.
Epithelial Cells/*physiology ; Epithelium/metabolism ; Hepatic Stellate Cells/*physiology ; Humans ; Liver/*cytology/injuries/*physiology ; Liver Cirrhosis/etiology/prevention & control ; Liver Regeneration/*physiology ; Mesenchymal Stromal Cells/physiology ; Myofibroblasts/physiology

OBJECTIVETo investigate the dynamic expression of PTEN in fibrogenic liver tissue of rats and its effect on the activation and proliferation of hepatic stellate cells (HSC).

METHODSA rat model of hepatic fibrosis was established by common bile duct ligation (BDL). The expressions of PTEN in the rat liver tissues were detected by immunohistochemical staining, Western blot and real-time PCR assay. The expressions of PTEN in activated HSC in the rat liver tissues were detected by immunofluorescence double labeling confocal laser scanning microscopy. The alpha-SMA in the rat liver tissues was determined by immunohistochemical staining.

RESULTSThe immunohistochemical staining indicated that there was extensive expression of PTEN in the liver tissues of normal rats, it was expressed mainly in the cytoplasm of the HSC. With the aggravation of hepatic fibrosis, the expression of PTEN in the hepatic tissues decreased gradually (P less than 0.01), while the alpha-SMA positive cells in the hepatic tissues increased significantly (P less than 0.01). The expressions of PTEN protein and mRNA in the rat liver tissues at week 1, 2, 3 and 4 after BDL were all lower than those in the sham operation group (P less than 0.01), and the expressions gradually decreased with the development of hepatic fibrosis (P less than 0.01). Immunofluorescence double labeling confocal laser scanning microscopy showed that PTEN were expressed extensively in activated HSC, especially in the cytoplasm, and with the development of hepatic fibrosis, the PTEN-expressing activated HSC accounted for an increasingly smaller percentage of total activated HSC.

CONCLUSIONThe expressions of PTEN mRNA and protein in rat fibrogenic liver tissues were downregulated, and their expressions in HSC in vivo also decreased. The dynamic expressions of PTEN in liver tissues had a significant negative correlation with the activation and proliferation of HSC.

Animals ; Cell Proliferation ; Hepatic Stellate Cells ; cytology ; Liver ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; metabolism ; pathology ; Male ; PTEN Phosphohydrolase ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo analyze differentially expressed proteins of hepatic stellate cells (HSCs) treated with oxymatrine (OMT) liposomes, thus further exploring the molecular mechanism of OMT liposomes for treating liver fibrosis.

METHODSA rat model of CCl4 induced chronic liver fibrosis was established. HSCs were perfusion isolated from modeled SD rats and cultured in vitro . Passage 2 HSCs were divided into the model group (Group A), the OMT-liposome-treated group (Group B), and the liposome-treated control group (Group C). HSCs from normal rats were taken as the normal control group (Group D). The total proteins of HSCs cells were extracted from Group B and D after 7 days of treatment, and separated with isoelectrofocusing two-dimensional electrophoresis (2-DE). A 2-DE system was established to analyze the differences in the protein profile between Group B and Group C. Tow protein dots with most obvious difference were selected to determine the structures and functions of different proteins using peptide mass fingerprinting (PMF).

RESULTS(1) The total number bf proteins decreased after treated with OMT liposomes, with 864 spots before treatment and 756 spots after treatment, and the matching rate was 63%. (2) According to 2-DE results, 10 differential protein spots were found by image analysis of magnifying images in local regions. (3) Two most differently expressed proteins were identified to be ATM (46. 236 kD) and Miz1 (54. 051 kD) by PMF and SWISS-PROT protein database retrieval.

CONCLUSIONAction of OMT liposomes on HSCs of rats with chronic liver fibrosis caused different protein expressions, which might be involved in the signaling pathways of inducing the apoptosis of HSCs.

Alkaloids ; pharmacology ; Animals ; Electrophoresis, Gel, Two-Dimensional ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Liposomes ; Liver Cirrhosis, Experimental ; metabolism ; Male ; Proteome ; metabolism ; Quinolizines ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To determine the effect of heat shock protein 47 (HSP47) on the expression of collagen I induced by transforming growth factor beta(1) (TGF-beta(1)) in hepatic stellate cell-T6 (HSC-T6) cells. METHODS: We used 1 ng/mL and 10 ng/mL recombinant human TGF-beta(1) to stimulate the cultured HSC-T6 cells. Heat shock response (HSR) and antisense oligonucleotides of HSP47 were used to induce and block the expression of HSP47, respectively. The expressions of HSP47 and collagen I were detected by Western blot and the cell viability was observed by MTT assay. RESULTS: Both HSP47 and collagen I were expressed in normal HSC-T6 cells. Collagen I and HSP47 expression could be induced by both 1 ng/mL and 10 ng/mL TGF-beta(1) and collagen I was expressed the most after the treatment with 10 ng/mL TGF-beta(1). Although HSR could not affect the synthesis of collagen I as it induced the HSP47 expression, HSR could promote the expression of collagen I induced by TGF-beta(1). With no effect on the cell viability, antisense oligonucleotides could significantly inhibit HSR-mediated HSP47 expression and TGF-beta(1)-induced collagen I synthesis. CONCLUSION Over-expression of HSP47 enhances TGF-beta(1)-induced expression of collagen I in HSC-T6 cells, and HSP47 may play important roles in the process of hepatic fibrosis.
Cell Line ; Collagen Type I ; metabolism ; HSP47 Heat-Shock Proteins ; metabolism ; Heat-Shock Response ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Transforming Growth Factor beta1 ; pharmacology