Changes in the total volume and contour density of oval cells in hepatic lobules of rats with carbon tetrachloride-induced hepatic fibrosis： A stereological study / 临床肝胆病杂志

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.

Kangxianruangan granule­containing serum mediated inhibition of hepatic oval cell differentiation into hepatocellular carcinoma cells via the Wnt­1/ß­catenin signaling pathway.

Hepatocellular carcinoma is a malignancy with poor clinical prognosis. Hepatic oval cells (HOCs) tend to differentiate into cancerous hepatocellular carcinoma cells (HCCs) in the tumor microenvironment. The purpose of the present study was to explore the role of kangxianruangan granule (KXRG)­containing serum in inhibiting the differentiation of HOCs into HCCs via the Wnt­1/ß­catenin signaling pathway. N­methyl­N'­nitro­N­nitrosoguanidine (MNNG) was applied to induce the transformation of the rat HOC cell line WB­F344 into HCCs. The overexpression plasmid, Wnt­1­up, was utilized to increase Wnt­1 expression. Subsequently, high, medium and low concentrations of KXRG were applied to MNNG­treated WB­F344 cells to assess the inhibitory effect of KXRG on cell differentiation. Flow cytometry was conducted to detect the cell cycle distribution, apoptotic rate and expression of cytokeratin­19 (CK­19) protein in cells. An immunofluorescence double staining protocol was used to detect the expression of Wnt­1 and ß­catenin. ELISAs were performed to detect α fetoprotein in the cell supernatants. Reverse transcription­quantitative PCR and western blotting were conducted to detect the mRNA and protein expression levels of Wnt­1, ß­catenin, Cyclin D1, C­myc, matrix metalloproteinase­7 (MMP­7), Axin2 and epithelial cell adhesion molecule (EpCAM) in cells. Compared with the normal group, the apoptotic rate, proportion of S phase cells, concentration of AFP in the cell supernatant, level of CK­19 protein, and mRNA and protein expression levels of Wnt­1, ß­catenin, Cyclin D1, C­myc, MMP­7, Axin2 and EpCAM were all significantly increased in the model group. Addition of KXRG significantly reduced the aforementioned indicators compared with the model group. Moreover, Wnt­1 overexpression further increased the aforementioned indicators compared with the model group, whereas KXRG significantly inhibited these effects. The results indicated that KXRG inhibited the differentiation of HOCs into HCCs via the Wnt­1/ß­catenin signaling pathway, which suggested the potential clinical application of KXRG for the prevention of hepatocellular carcinoma.

Mechanism of Biejiajian Wan in Reversing EMT in Rat Hetapic Oval Cells via Wnt/ β-catenin Pathway / 中国实验方剂学杂志

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.

Matrine promotes hepatic oval cells differentiation into hepatocytes and alleviates liver injury by suppression of Notch signalling pathway.

AIMS: Recent studies have shown that the hyperactive Notch pathway is involved in cirrhosis and hepatocellular carcinoma (HCC) development by regulating differentiation of hepatic oval cells (HOCs) into cancer cells. The aim of this study was to investigate whether matrine can alleviate liver injury and promote HOC differentiation into hepatocytes by suppression of Notch pathway. MAIN METHODS: We evaluated the expression of Notch-1, Jagged-1, and Hes-1 in HCC tissue by immunohistochemistry. Stem cell characteristics of HOCs were evaluated by CCK-8, cell cycle, and apoptosis. The expression of Notch pathway, HOC markers and albumin (ALB) was detected by immunohistochemistry, QRT-PCR and western blotting. The effects of matrine in protecting liver in vivo were investigated in a rat Solt-Farber precancerous model. KEY FINDINGS: We found an abnormal activated Notch pathway in HCC tissue, and the hyperactive Notch pathway was strongly associated with poor liver function in patients with cirrhosis with HCC. Using siNotch-1 to inhibit Notch pathway confirmed that Notch pathway could maintain stem cell characteristics of HOCs. Matrine inhibited stem cell characteristics of HOCs, the expression of Notch pathway and HOC markers but upregulated ALB. Matrine in combined with siNotch-1 RNA decreased the more potently inhibited HOC markers and Notch pathway. In rat Solt-Farber precancerous model, prophylactic application of matrine alleviated liver injury, downregulated Notch pathway and HOC markers, and upregulated ALB in a dose-dependent manner. SIGNIFICANCE: Matrine could promote the differentiation of HOCs into hepatocytes by inhibiting the Notch signalling pathway and alleviate liver injury.

Hepatic stellate cells contribute to liver regeneration through galectins in hepatic stem cell niche.

BACKGROUND: As a critical cellular component in the hepatic stem cell niche, hepatic stellate cells (HSCs) play critical roles in regulating the expansion of hepatic stem cells, liver regeneration, and fibrogenesis. However, the signaling of HSCs, particularly that involved in promoting hepatic stem cell expansion, remains unclear. While the overexpression of galectins has been identified in regenerating liver tissues, their involvement in cell-cell interactions between HSCs and hepatic stem cells remains to be elucidated. METHODS: To generate a liver regeneration rat model and establish a hepatic oval cell microenvironment as a stem cell niche, 2-acetylaminofluorene treatment plus partial hepatectomy was performed. Immunofluorescence staining was conducted to detect the emergence of hepatic stem cells and their niche. Liver parenchymal cells, non-parenchymal cells, and HSCs were isolated for gene and protein expression analysis by qPCR or western blotting. To evaluate the effect of galectins on the colony-forming efficiency of hepatic stem cells, c-Kit-CD29+CD49f+/lowCD45-Ter-119- cells were cultured with recombinant galectin protein, galectin antibody, galectin-producing HSCs, and galectin-knockdown HSCs. RESULTS: Following liver injury, the cytokeratin 19+ ductal cells were robustly induced together with the emergence of OV6+CD44+CD133+EpCAM+ hepatic stem cells. The activated desmin+ HSCs were recruited around the periportal area and markedly enriched in the galectin-positive domain compared to the other non-parenchymal cells. Notably, the HSC fraction isolated from regenerating liver was accompanied by dramatically elevated gene and protein expression of galectins. Hepatic stem cells co-cultured with HSCs significantly enhanced colony-forming efficiency. Conversely, single or double knockdown of galectin-1 and galectin-3 led into a significant function loss, impaired the co-cultured hepatic stem cells to attenuated colony size, inhibited colony frequency, and reduced total cell numbers in colonies. On the other hand, the promotive function of galectins was further confirmed by recombinant galectin protein supplementation and galectins blocking antibodies. CONCLUSIONS: Our findings, for the first time, demonstrated that galectins from activated HSCs contribute to hepatic stem cell expansion during liver regeneration, suggesting that galectins serve as important stem cell niche components.

Knockout of LATS1 induces neoplastic phenotype in hepatic oval cells.

BACKGROUND: Primary liver cancer (PLC) is the second leading cause of cancer-related death worldwide. It has been reported that PLC can be originated from malignant transformed adult hepatic progenitor cells. Mammalian large tumor suppressor kinase 1 (LATS1) is one of the core components of the Hippo pathway and it has been implicated in regulating invasion and metastasis of different cancer cell. However, the underlying connections between hepatic progenitor cells and LATS1 in the pathogenesis of PLC are still elusive. METHODS: LATS1 gene knockout (LATS1-KO) hepatic oval cells (HOCs) were constructed by the CRISPR/Cas9 system. Cell viability was evaluated by the CCK-8 assay. Cell migration was measured by scrape assay. Cell invasion was examined by Transwell assay. Cell apoptosis was evaluated by flow cytometry. The expression of LATS1 and Yes-associated protein (YAP) in HOCs was determined by Q-PCR and Western blot analysis. RESULTS: Here, we found that knockout of LATS1 significantly induced the migration and invasion of WB-F344 cells. Knockdown of YAP suppressed the neoplastic phenotype of LATS1-KO WB-F344 cells. Furthermore, overexpression of YAP promoted the migration and invasion of LATS1-KO WB-F344 cells. CONCLUSIONS: In summary, the current study demonstrated that LATS1 is required for inhibiting the neoplastic phenotype of normal hepatic progenitor cell via downregulating YAP.

Cancer stem cells of hepatocellular carcinoma.

Hepatocellular carcinoma is a malignant tumor arising from hepatocytes. The hepatocellular carcinoma is dictated by a subset of cells with stem cell-like features. These cells are apoptosis-resistant and have particular biomarkers, which serve as seeds in different stages of tumorigenesis including initiation, progression, metastasis, and relapse of hepatocellular carcinoma. Signaling pathways of cancer stem cells are novel targets for the radical intervention of hepatocellular carcinoma.

Embedded Disposable Functionalized Electrochemical Biosensor with a 3D-Printed Flow Cell for Detection of Hepatic Oval Cells (HOCs).

Hepatic oval cells (HOCs) are considered the progeny of the intrahepatic stem cells that are found in a small population in the liver after hepatocyte proliferation is inhibited. Due to their small number, isolation and capture of these cells constitute a challenging task for immunosensor technology. This work describes the development of a 3D-printed continuous flow system and exploits disposable screen-printed electrodes for the rapid detection of HOCs that over-express the OV6 marker on their membrane. Multiwall carbon nanotube (MWCNT) electrodes have a chitosan film that serves as a scaffold for the immobilization of oval cell marker antibodies (anti-OV6-Ab), which enhance the sensitivity of the biomarker and makes the designed sensor specific for oval cells. The developed sensor can be easily embedded into the 3D-printed flow cell to allow cells to be exposed continuously to the functionalized surface. The continuous flow is intended to increase capture of most of the target cells in the specimen. Contact angle measurements were performed to characterize the nature and quality of the modified sensor surface, and electrochemical measurements (cyclic voltammetry (CV) and square wave voltammetry (SWV)) were performed to confirm the efficiency and selectivity of the fabricated sensor to detect HOCs. The proposed method is valuable for capturing rare cells and could provide an effective tool for cancer diagnosis and detection.

Influence of STAT3 on promoting Warburg effect probably by upregula-ting GLUT2 expression in the malignant transformation of WB-F344 rat hepatic oval cells / 中国病理生理杂志

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.

Upregulation of hydroxysteroid sulfotransferase 2B1b promotes hepatic oval cell proliferation by modulating oxysterol-induced LXR activation in a mouse model of liver injury.

Hydroxysteroid sulfotransferase 2B1b (SULT2B1b) sulfates cholesterol and oxysterols. Hepatic oval cells (HOCs), thought to be progenitor cells, can be triggered in chemically injured livers. The present study focused on the role of SULT2B1b in HOC proliferation after liver injury. Our experiments revealed that the expression of SULT2B1b was increased dramatically in a chemical-induced liver injury model, mainly in HOCs. Upon challenge with a hepatotoxic diet containing 0.1 % 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), SULT2B1-/- mice presented alleviated liver injury and less HOC proliferation compared with wild-type (WT) mice, and these findings were verified by serum analysis, histopathology, immunofluorescence staining, RNA-seq, and Western blotting. HOCs derived from SULT2B1-/- mice showed lower proliferative capability than those from WT mice. SULT2B1b overexpression promoted growth of the WB-F344 hepatic oval cell line, whereas SULT2B1b knockdown inhibited growth of these cells. The IL-6/STAT3 signaling pathway also was promoted by SULT2B1b. Liquid chromatography and mass spectrometry indicated that the levels of 22-hydroxycholesterol, 25-hydroxycholesterol, and 24,25-epoxycholesterol were higher in the DDC-injured livers of SULT2B1-/- mice than in livers of WT mice. The above oxysterols are physiological ligands of liver X receptors (LXRs), and SULT2B1b suppressed oxysterol-induced LXR activation. Additional in vivo and in vitro experiments demonstrated that LXR activation could inhibit HOC proliferation and the IL-6/STAT3 signaling pathway, and these effects could be reversed by SULT2B1b. Our data indicate that upregulation of SULT2B1b might promote HOC proliferation and aggravate liver injury via the suppression of oxysterol-induced LXR activation in chemically induced mouse liver injury.

Research advances in signaling pathways involved in liver regeneration mediated by oval cells / 临床肝胆病杂志

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.

In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

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.

Proliferation of Hepatic Oval Cells via Cyclooxygenase-2 and Extracellular Matrix Protein Signaling during Liver Regeneration Following 2-AAF/Partial Hepatectomy in Rats.

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 α-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.

Proliferation of Hepatic Oval Cells via Cyclooxygenase-2 and Extracellular Matrix Protein Signaling during Liver Regeneration Following 2-AAF/Partial Hepatectomy in Rats

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.

Effect of HOCs implantation on protein expression of TGF-?/Smad signaling pathway in liver tissue of hepatic fibrosis rats / 中国病理生理杂志

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.

Derivation, characterization and differentiation in vitro of hepatic oval cells of adult rats / 解放军医学杂志

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.

Hepatic oval cells in rat: immunohistochemistry localization and ultrastructure / 解放军医学杂志

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.