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ObjectiveTo quantitatively investigate the changes in the total volume and contour density of hepatic oval cells （HOC） in hepatic lobules of rats with carbon tetrachloride （CCl4）-induced hepatic fibrosis. MethodsA total of 11 healthy male Sprague-Dawley rats were randomly divided into control group with 5 rats and hepatic fibrosis group with 6 rats， and CCl4 and olive oil suspension were injected subcutaneously twice a week， 3 mL/kg each time. After five weeks of hepatic fibrosis modeling， five liver tissue blocks with a size of about 1 mm3 were randomly selected from the liver of each rat to prepare one Epon812 epoxy resin-embedded ultrathin section， and the stereological method and transmission electron microscopy were used for the quantitative analysis of the total volume and contour density of HOC in the hepatic lobules of rats. In addition， four liver tissue blocks with a thickness of 2 mm were randomly selected from the remaining liver of each rat to prepare two paraffin-embedded Masson staining sections， and the degree of liver fibrosis in each rat was qualitatively evaluated according to the Metavir staging criteria for liver fibrosis. The independent-samples t test was used for comparison of continuous data between groups. ResultsThe quantitative stereological analysis showed that the total volume of HOC in hepatic lobules was 15.40±7.63 mm3 in the control group and 146.80±114.00 mm3 in the liver fibrosis group， and compared with the control group， the total volume of HOC in hepatic lobules of rats in the liver fibrosis group was significantly increased by 8.53 times （t=-2.551， P=0.031）； the contour density of HOC in hepatic lobules was 56.20±40.40 in the control group and 566.50±317.00 in the liver fibrosis group， and compared with the control group， the contour density of HOC in hepatic lobules of rats in the liver fibrosis group was significantly increased by 9.08 times （t=-3.539， P=0.006）. Qualitative observation showed that liver fibrosis stage of rats reached stage Ⅱ-Ⅲ according to the Metavir scoring criteria， and massive proliferation of HOC was observed around the proliferation site of hepatic stellate cells in the perisinusoidal space of rats. ConclusionCCl4 induces significant proliferation of HOC in hepatic lobules of rats with liver fibrosis.
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Objective:To study the effect of Biejiajian Wan on the epithelial-mesenchymal transition （EMT） of rat hepatic oval cells induced by transforming growth factor- β1（TGF-β1）， in order to explore its mechanism in reversing EMT. Method:WB-F344 cells were divided into five groups： blank group， TGF-β1 model group （10 μg·L-1TGF-β1）， low-dose group （10 μg·L-1TGF-β1+0.55 g·kg-1Biejiajian Wan）， medium-dose group （10 μg·L-1TGF-β1+1.1 g·kg-1Biejiajian Wan）， high-dose group （10 μg·L-1TGF-β1+2.2 g·kg-1Biejiajian Wan）. Except blank group， TGF-β1 was used to induce WB-F344 cells in all of the remaining groups to construct an EMT model. After the cells were treated with low， medium and high doses of Biejiajian Wan serum， the changes of migration ability of WB-F344 cells were detected by cell scratching test. The expressions of E-cadherin， N-cadherin and Vimentin were detected by Western blot. Real-time PCR was used to detect the changes in the expression of β-catenin mRNA. The expression of β-catenin was detected by cell immunofluorescence assay. Result:Compared with normal WB-F344 cells， the intercellular space of WB-F344 cells became loose from tight， and the morphology changed from cobblestone to fibroblast after TGF-β1 induced WB-F344 cells for 4 days， and the expression of E-cadherin protein decreased， while the expression of N-cadherin protein increased （P<0.01）， indicating that the EMT model of WB-F344 cells was successfully built. Compared with the blank group， the migration ability of WB-F344 cells in TGF-β1 model group was enhanced （P<0.01）， compared with TGF-β1 model group， Biejiajian Wan could significantly inhibit the migration ability of WB-F344 cells； specifically， the low-dose group had no statistical significance， and the medium and high-dose groups had statistical significance （P<0.05）. Western blot results showed that compared with the blank group， the expression of E-cadherin decreased， whereas those of N-cadherin and Vimentin increased in the TGF-β1 model group （P<0.01）， compared with TGF-β1 model group， E-cadherin protein expression was increased in the low， medium and high-dose groups， while the expressions of N-cadherin and Vimentin was decreased； specifically， the low-dose groups had no statistical significance， and the medium and high dose groups had statistical significance （P<0.05，P<0.01）. Real-time PCR results showed that compared with the blank group， the mRNA expression of β-catenin in the TGF-β1 model group was increased （P<0.05）， whereas compared with TGF-β1 model group， the mRNA expression of β-catenin in the low， medium and high-dose groups of Biejiajian Wan was reduced （P<0.01）. The results of cellular immunofluorescence showed that compared with the blank group， the fluorescence expression of β-catenin in the cell nucleus was enhanced in the TGF-β1 model group； and compared with the TGF-β1 model group， the expression of β -catenin in the cell nucleus of the low， medium and high-dose groups of Biejiajian Wan decreased， and the inhibitory effect of Biejiajian Wan on β-catenin in the cell nucleus was positively correlated with its concentration. Conclusion:Biejiajian Wan may reverse the EMT process that TGF-β1 induced WB-F344 cells， and inhibit the migration of WB-F344 cells by inhibiting Wnt/β-catenin signaling pathway.
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AIM:To investigate the influence of signal transducer and activator of transcription 3(STAT3)on Warburg effect in the malignant transformation of WB-F344 rat hepatic oval cells.METHODS:The WB-F344 cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)and hydrogen peroxide(H2O2)to induce the malignant trans-formation.Evaluation of the transformed cells were measured by the soft agar colony formation assay and DNA aneuploidy with flow cytometry.The levels of glucose and lactate in the culture medium of the cells were detected by chromatography. The protein levels of alpha-fetoprotein(AFP),STAT3,p-STAT3 and glucose transporter 2(GLUT2)in the cells were ex-amined by Western blot analysis.The cell proliferation were evaluated by WST-1 assay,viable cell counting,measuring the S-phase fraction(SPF)and proliferation index(PI)using the data from flow cytometry analysis,and detecting proliferating cell nuclear antigen(PCNA)protein expression by Western blot.RESULTS:Compared with the control cells,the forma-tion of colonies in soft agar(P<0.05)and DNA aneuploidy(P<0.01)were elevated in transformed cells,and the ex-pression level of AFP was also augmented(P<0.05).The increases in the level of both glucose consumption(P<0.05) and lactate production(P<0.01)show that Warburg effect was enhanced in transformed cells.Meanwhile, the protein levels of GLUT2(P<0.01)and p-STAT3(P<0.01)in transformed cells were higher than those in the control cells.The cell proliferation parameters including SPF(P<0.01),PI(P<0.01), viable cell number and PCNA expression(P<0.01)in transformed cells were also elevated as compared with the control cells.Interestingly, stattic, an inhibitor of STAT3 activation,resulted in declines in glucose consumption(P<0.05)and lactate production(P<0.01)in the trans-formed cells.In addition,compared with transformed cells,formation of colonies in soft agar(P<0.01),DNA aneuploidy (P<0.01),AFP(P<0.05), GLUT2(P<0.05), and cell proliferation parameters including SPF(P<0.01), PI (P<0.01),viable cell number(P<0.05)and PCNA expression(P<0.05)were also decreased following stattic treat-ment in transformed cells.CONCLUSION:STAT3 promotes Warburg effect and cell proliferation probably by upregula-ting GLUT2 expression in the malignant transformation of hepatic oval cells.
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The apoptosis, necrosis, and senescence of hepatocytes are closely associated with the activation and proliferation of oval cells, and in addition, liver regeneration mediated by oval cells is an important part of regeneration after liver injury. Currently, studies have shown that a variety of cytokines, hormones, and neurotransmitters are involved in this process. This article reviews recent research findings, introduces the research advances in the association between various signaling pathways (TWEAK/Fn14, Hedgehog, and thyroid hormone) and liver regeneration mediated by oval cells, elaborates on the role of each pathway in the regeneration of hepatocytes, and investigates related mechanisms in liver regeneration.
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Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Student t-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.
Subject(s)
Animals , Female , Male , Rats , Cell Proliferation , Cell Differentiation/physiology , Cell Transplantation/methods , Hepatocytes/cytology , Liver Transplantation/methods , Flow Cytometry , Graft Rejection/diagnosis , Hepatectomy , Immunohistochemistry , Liver/anatomy & histology , Liver/surgery , Primary Cell Culture , Rats, Inbred Lew , Real-Time Polymerase Chain Reaction/methods , Survival RateABSTRACT
BACKGROUND/AIMS: In the 2-acetylaminofluorene (2-AAF)/70% partial hepatectomy (PHx) model, the mechanism underlying the differentiation of activated hepatic oval cells (HOCs) into hepatocytes and bile ductile cells is unclear. We investigated the role of cyclooxygenase-2 (COX-2) in HOCs and the relationship between COX-2 and extracellular matrix proteins in cellular proliferation. METHODS: Reverse transcription-polymerase chain reaction, immunohistochemical staining, and Western blotting were used to assess COX-2 expression. The co-localization of COX-2 with Thy1, c-Met, epithelial cell adhesion molecule, and alpha-smooth muscle actin was also examined. Additionally, we investigated whether connective tissue growth factor (CTGF), fibronectin (FN), extracellular signal-regulated kinase 1/2 (P-ERK1/2), and AKT were expressed in HOCs. RESULTS: The expression of COX-2, prostaglandin E2 receptors, and c-Met was upregulated in HOCs. However, HOCs treated with the COX-2 inhibitor NS398 showed decreased COX-2, CTGF, FN, and AKT expression, whereas P-ERK1/2 was unaffected. Additionally, NS398 inhibited HOC proliferation, but not the proliferation of HOCs cultured on FN-coated dishes. Furthermore, the proliferative response of HOCs treated with NS398 was reversed by hepatic growth factor treatment. CONCLUSIONS: These results suggest that HOC proliferation is mediated through COX-2, extracellular FN expression, and AKT activation. Thus, COX-2 plays an important role in HOC proliferation following acute injury.
Subject(s)
Animals , Rats , 2-Acetylaminofluorene , Actins , Antigens, Neoplasm , Bile , Blotting, Western , Cell Adhesion Molecules , Connective Tissue Growth Factor , Cyclooxygenase 2 , Dinoprostone , Epithelial Cells , Extracellular Matrix , Extracellular Matrix Proteins , Fibronectins , Hepatectomy , Hepatocytes , Liver , Liver Regeneration , Muscles , Nitrobenzenes , Phosphotransferases , SulfonamidesABSTRACT
AIM:To investigate the effect of hepatic oval cells (HOCs) on the protein expression of TGF-?/Smad signaling pathway in the liver tissue of hepatic fibrosis rats.METHODS:The SD rat models of liver fibrosis were made by treating with carbon tetrachloride and combined factors.The HOCs was isolated from the model rats.HOCs suspension (0.5 mL at a density of 1 ? 1012cells/L) were transplanted via portal vein into the hepatic fibrosis rats at 8th week and observed continuously for 30 days.Meanwhile,WuLing capsules were used for positive control.The blood samples were collected through trail vein at 8th day,15th day,23th day and 30th day after transplantation of HOCs.The levels of aspartate aminotransferase (AST) and alamine aminotransferase (ALT) in serum were determined by enzyme method.The morphological changes of hepatic tissues were observed under microscope with HE and Musson staining.The protein levels of collagen type I (Col-Ⅰ),extracellular-signal regulated protein kinase (ERK),phosphory-lation extracellular regulatedprotein kinases (p-ERK),TGF-? receptor type Ⅰ (T?RⅠ),TGF-? receptor type Ⅱ (T?RⅡ),mothers against decapentaplegic homolog 2 /3 (Smad 2 /3) and mothers against decapentaplegic homolog (Smad 7) were assessed in liver tissues by Western blotting.RESULTS:In HOCs and WuLing capsules treated groups,the levels of ALT and AST decreased significantly at 15th day,23th day and 30th day after the transplantation of HOC.The damage degree of hepatic fiber hyperplasia of the liver histological structure reduced notably.The expression levels of Col-Ⅰ,ERK,p-ERK,T?RⅠ and T?RⅡ in liver tissues of hepatic fibrosis rats were down-regulated obviously while the expression of Smad 7 increased significantly.CONCLUSION:The implantation of HOCs prevents the progress of liver fibrosis in rats.The mechanism of action is to inhibit the protein expression of p-ERK,T?RⅠ,T?R Ⅱ for TGF-?/Smad signaling pathway of liver tissue.
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Objective To establish a proliferating model of hepatic oval cells (HOCs) with adult Wistar rats, isolate and culture in vitro the HOCs, and to approach the possibility of inducing the HOCs differentiated into hepatocytes. Methods Rats were fed with 0.07% wt/wt ethionine. On day 8, 2/3 partial hepatectomy (2/3 PH) was performed. Isolate, harvest and purify HOCs by type Ⅳ collagenase perfusion with semi in situ 2-step method and Percoll density gradient method. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were added to complete Williams' medium E (WE) to culture the HOCs. HOCs was induced differentiation with HGF, oncostatin m (OSM) and fibroblast growth factor-4 (FGF4) into hepatocytes. Results The concentration of cell after purification was about 1.34?105/ml. Most cells were small, about 1/6~1/3 the size of normal hepatocyte. Ovoid, elliptical or polygonal in shap, the nucleus-cytoplasm ratio was relatively large, and clone-like proliferation appeared 2 weeks later. LSCM revealed positive expression of Thy-1 and C-kit in cytoplasm and membrane of HOCs. ICC showed AFP in cytoplasm of HOCs. Stimulated by inducing system, the shape of HOCs changed gradually. The volume enlarged and cells lost their adherence ability. ICC indicated apparent positive stain of cytoplasm Alb 14 days after differentiated induction, and the positive ratio increased along with the extension of induction duration. Cytochemical tests indicated brown or black sediment with G-6-P staining and red particles with PAS staining, respectively. Conclusion The proliferation model of rats' HOCs was established after ethionine feeding and 2/3 PH. HOCs can be obtained with type IV collagenase perfusion and Percoll density gradient isolation and purification. Clone proliferation can be achieved through culturing HOCs in vitro. Under certain conditions, HOCs can be induced and differentiated into certain typical hepatocyte phenotype.
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Objective To investigate the location and ultrastructure of hepatic stem cells in adult rat. Methods Proliferation of hepatic oval cell of the rat was accomplished. Identification was made with immunohistochemistry with CK18, 19 and CD34 as markers. Ultrastructure of hepatic stem cells was observed with transmission electron microscope. Results The majority of hepatic oval cells were localized in the Hering channel, but some of them were distributed in the hepatic lobules. Electron microscopy revealed three types of oval cells. TypeⅠ cell was small, 7?m in size, with large nucleus but small amounts of cytoplasm and organelles. The cell was recognized as primitive oval cell. Type Ⅱ cell was larger, measuring 8?m in diameter, containing more cytoplasm and organelles. Type Ⅲ cells were larger than both of above cells, and they contained more organelles. Conclusion Hepatic oval cells in adult rat are localized in Hering channel, and they may be stem cells hepatocytes.