Animals ; Apoptosis ; physiology ; Bile Duct Diseases ; pathology ; Bile Ducts ; cytology ; pathology ; Cell Proliferation ; Epithelial Cells ; metabolism ; pathology ; Humans

BACKGROUNDThe possible role of substance P (SP) during wound healing has been the primary research focus in recent years, but its effect on the healing process after bile duct injury is little understood. This study aimed to investigate the effects of SP on growth of fibroblast-like cells derived from rabbit bile duct.

METHODSFibroblast-like cells derived from rabbit bile duct were identified and divided randomly into control and experimental groups. SP-treated cells at different concentrations of 10(-9)-10(-5) mol/L and control group were incubated, respectively, for 48 hours. After incubating, the effects of SP on cell proliferation were assessed by cell counts and MTT test. Apoptosis rate (AR) of cells was measured by flow cytometry.

RESULTSCultured rabbit bile duct cells were fibroblast-like in morphology, and these cells were stained positively for vimentin and negatively for desmin. After SP was added to nonconfluent cells for 48 hours, cell numbers were significantly increased in experimental groups than in controls (P < 0.05). The maximum stimulation of cell proliferation was achieved at SP of 10(-5) mol/L. Bile duct fibroblast-like cells in the SP group showed a higher proliferating activity and lower AR than those in the control group or in the SP + Spantide group (P < 0.05). Spantide partly inhibited the effects of SP on fibroblast-like cells. Examination under transmission electron microscopy revealed rough endoplasmic reticulum and prominent Golgi complexes after SP treatment.

CONCLUSIONSSP has a growth regulatory property on cultivated bile duct fibroblast-like cells in vitro, suggesting that SP may involve in wound healing after bile duct injury by promoting wound fibroblast proliferation and inhibiting apoptosis and participate in pathological scar formation.

Animals ; Bile Ducts ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Fibroblasts ; cytology ; drug effects ; Male ; Rabbits ; Substance P ; pharmacology

Cholangiocytes (epithelial cells lining the intra- and extrahepatic bile ducts) and hepatocytes are two major components of liver epithelia. Although cholangiocytes are less numerous than hepatocytes, they are involved in both bile secretion and diverse cellular processes such as cell-cycle phenomena, cell signaling, and interactions with other cells, matrix components, foreign organisms, and xenobiotics. Cholangiocytes are also targets in several human diseases including cholangiocarcinoma, primary sclerosing cholangitis, autoimmune cholangitis, and vanishing bile-duct syndrome. The rapid advances in experimental biology technologies are greatly expanding interest in and knowledge of the physiology and pathophysiology of cholangiocytes. This review focuses on the progress of in vivo and in vitro experimental models in elucidating the physiologic functions of cholangiocytes and the pathophysiology of various cholangiopathies. The following aspects are reviewed: isolation of cholangiocytes from the liver and their heterogeneity, various culture systems, establishment of cholangiocyte cell lines, isolation and usage of intrahepatic bile-duct units, three-dimensional modeling of the bile duct, experimental models for inducing cholangiocyte proliferation, and various cholangiopathies such as cholangiocarcinoma, primary sclerosing cholangitis, and autoimmune cholangitis.
Animals ; Bile Duct Diseases/etiology ; Bile Ducts/*cytology/physiology/*physiopathology ; Disease Models, Animal ; Epithelial Cells/metabolism/physiology ; Humans ; Imaging, Three-Dimensional ; Mice ; Models, Animal

OBJECTIVETo investigate the transformative potential of hepatic progenitor cells to differentiate into liver stem cells using a normal adult mouse system and to determine the effects of HBx protein in these liver stem cells' differentiation into hepatic cells.

METHODSHepatic progenitor cells were obtained from mice by means of an optimized two-step digestion and perfusion method followed by joint differential centrifugation and density gradient centrifugation. Transformation of the hepatic progenitor cells into liver stem cells was observed by immunofluorescent detection of CD 133, EPCAM, CD49f and CK19. Differentiation of the resultant liver stem cells into hepatic cells and bile duct epithelial cells was observed after DMSO addition by Periodic Acid-Schiff (PAS) staining followed by cell immunofluorescence and flow cytometry. To determine the effects of HBx on these liver stem cells' ability to differentiate into hepatic cells, cell transfection was used followed by observation of morphology and proliferation capacity.

RESULTSCell viability of the isolated hepatic progenitor cells was 78.67+/-4.04%. Stimulation with EGF and collagen led to growth of some of the paving-stone shaped cells attached to the hepatic progenitor cells which had gathered into spherical clumps, as is the nature of stem cells. The liver stem cells showed high expression of CD133, CD49f and CK19, and low expression of EPCAM. Under the effect of DMSO, the liver stem cells differentiated into hepatocytes and bile duct epithelial cells. After HBx transfecfion, the liver stem cells maintained the characteristic shape of stem cells and showed enhanced proliferation.

CONCLUSIONEPCAM-positive adult hepatic progenitor cells can transform into liver stem cells.The HBx protein may play an important role in maintaining the stability of liver stem cells in the adult mouse.

Animals ; Antigens, Neoplasm ; metabolism ; Bile Ducts ; cytology ; Cell Adhesion Molecules ; metabolism ; Cell Differentiation ; Epithelial Cell Adhesion Molecule ; Epithelial Cells ; cytology ; Flow Cytometry ; Hepatocytes ; cytology ; Liver ; cytology ; Mice ; Stem Cells ; cytology

OBJECTIVETo study the clinicopathologic features of combined hepatocellular-cholangiocarcinoma (cholangiolocellular type, CLC type) with stem cell features and its relationship to hepatic progenitor cells (HPCs).

METHODSClinical and histologic features of 26 cases of combined hepatocellular-cholangiocarcinoma (CLC type) were reviewed. Histochemistry was performed to confirm the type of mucin and immunohistochemical study was carried out for hepatocytic markers (Hep Par-1 and AFP) and biliary/HPCs markers (CK7, CK9, EMA, EpCAM, NCAM, CKIT).

RESULTSThe age of patients ranged from 51 to 82 years (mean 64 years). All 26 cases contained CLC and hepatocellular carcinoma components. CLC area was composed of mixtures of small monotonous glands with abundant fibrous stroma and lymphocytic infiltrate. Tumor cells were cuboidal, smaller in size than normal hepatocytes, with basophilic cytoplasm and round nuclei. All cases, especially at the tumor boundary, showed HCC-like trabecular areas characterized by mildly atypical tumor cells with abundant eosinophilic cytoplasm and little stroma. Out of 26 cases, 21 showed definite glandular formation with mucin production, representing intrahepatic cholangiocarcinoma areas. The three distinct areas showed transitional zones merging with each other. The surrounding liver tissue showed cirrhosis and chronic hepatitis with varying degrees of fibrosis and periportal ductular reaction. Immunohistochemistry showed that biliary/HPC markers (CK7, CK9, EMA, EpCAM, NCAM and CKIT) were strongly positive in CLC area in almost all cases, similar to the staining pattern of ductular reaction. In HCC-like areas, CK7 and CK19 were positive in all cases and the expression rates of EMA, EpCAM, NCAM, CKIT, AFP, Hep Par-1 were 80.8% (21/26), 88.5% (23/26), 84.6% (22/26), 88.5% (23/26), 46.2% (12/26) and 53.8% (14/26) respectively, similar to the staining pattern of intermediate hepatocytes. In ICC areas, CK7, CK9, EMA and EpCAM were positive in all cases without the expression of NCAM and CKIT.

CONCLUSIONThe clinicopathologic findings and immunohistochemical results in this study highly suggest a hepatic progenitor cell origin of combined hepatocellular-cholangiocarcinoma (CLC type).

Bile Duct Neoplasms ; pathology ; Biomarkers ; metabolism ; Carcinoma, Hepatocellular ; pathology ; Cholangiocarcinoma ; pathology ; Hepatocytes ; cytology ; Humans ; Immunohistochemistry ; Liver Cirrhosis ; pathology ; Liver Neoplasms ; pathology ; Mucins ; metabolism ; Stem Cells ; cytology

Animals ; Bile Ducts ; cytology ; Cell Transdifferentiation ; Hepatocytes ; cytology ; Liver ; embryology ; metabolism ; Mice ; Mice, Inbred C57BL ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis

The purpose of this study was to explore the role of epithelial-mesenchymal transition in the pathogenesis of hepatolithiasis. Thirty-one patients with primary hepatolithiasis were enrolled in this study. Expressions of E-cadherin, alpha-catenin, alpha-SMA, vimentin, S100A4, TGF-beta1 and P-smad2/3 in hepatolithiasis bile duct epithelial cells were examined by immunohistochemistry staining. The results showed that the expressions of the epithelial markers E-cadherin and alpha-catenin were frequently lost in hepatolithiasis (32.3% and 25.9% of cases, respectively), while the mesenchymal markers vimentin, alpha-SMA and S100A4 were found to be present in hepatolithiasis (35.5%, 29.0%, and 32.3% of cases, respectively). The increased mesenchymal marker expression was correlated with decreased epithelial marker expression. The expressions of TGF-beta1 and P-smad2/3 in hepatolithiasis were correlated with the expression of S100A4. These data indicate that TGF-beta1-mediated epithelial-mesenchymal transition might be involved in the formation of hepatolithiasis.
Adult ; *Bile Ducts/cytology/metabolism/pathology ; Biological Markers/*metabolism ; Cell Differentiation/*physiology ; Epithelial Cells/cytology/*physiology ; Epithelium/physiology ; Female ; *Gallstones/metabolism/pathology ; Humans ; Liver Diseases/metabolism/*pathology ; Male ; Mesoderm/cytology/*physiology ; Middle Aged

OBJECTIVETo investigate the mechanism of hepatocytes transdifferentiation to bile duct epithelial cells (BECs) and intervention of Huangqi decoction (HQD) on hepatic fibrosis formation in rats with secondary cholestasis.

METHODSSeventy-five SD male rats were made into cholestatic hepatic fibrosis model animals by bile duct ligation, and randomized into the control group (n = 50) and the HQD group (n = 15). Starting from one week after modeling, they were administered orally with saline and HQD respectively for four weeks. Besides, a sham-operated group was set up with 10 rats operated by choledochus segregating only and administered after then with saline. Rats were killed in batches at different time points, i.e. each five from the control group and sham-operated group at the end of the 1st week, five from the control group for each time at the end of the 2nd, 3rd and 4th week, and all the remaining rats at the end of the 5th week. Their liver tissues were taken for histological change examination, content of hydroxyproline (Hyp) determination; protein expression of BECs marker cytokeratin 7 (CK7) and the hepatocyte specific antigen HepPar detection by Western blot, and CK7-Hep Par co-localization by laser confocal microscopy. Then IPP software was used to analyze Sirius red stained positive areas of CK7 and Hep Par, as well as the average IOD of CK7/Hep Par co-localization.

RESULTSHepatocytes in hepatic tissues (Hep Par positive cell) in the model rats decreased gradually along was time went by after modeling (Sham > M1w > M2w > M3w > M4w > M5w), which was in parallel with the increase of BECs (CK7 positive cells), degree of fibrosis, Hyp content and CK7 protein expression. Increasing of co-localized positive cells of CK7/Hep Par began at 1 week and reached the peak 3 weeks after modeling, then it decreased gradually. The Hep Par protein expression was negatively correlated with that of CK7; the Hep Par positive cell expression was negatively correlated with CK7 positive cell expression and collagen deposition; while the CK7 positive cell expression was positively correlated with the collagen deposition in the liver tissue. Compared with the model control group, the mortality, CK7/Hep Par co-localized positive cells, fibrosis degree, Hyp content and CK7 protein expression were lesser obviously (P < 0.01), while Hep Par positive cell and protein expressions were higher significantly in the HQD group.

CONCLUSIONSHepatocytes transdifferentiation to BECs might be a key pathological element for secondary cholestatic hepatic fibrosis formation; the restraining action of HQD is possibly a major action mechanism of HQD for effectively intervening and treating secondary cholestasis hepatic fibrosis.

Animals ; Astragalus Plant ; Bile Ducts ; cytology ; Cell Transdifferentiation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; Hepatocytes ; cytology ; drug effects ; Liver ; Liver Cirrhosis, Biliary ; drug therapy ; pathology ; Male ; Phytotherapy ; Rats ; Rats, Sprague-Dawley

BACKGROUND/AIMS: Biliary drainage is performed in many patients with cholangiocarcinoma (CCA) to relieve obstructive jaundice. For those who have undergone biliary drainage, bile cytology can be easily performed since the access is already achieved. This study aims to determine the clinical usefulness of bile cytology for the diagnosis of CCA and to evaluate factors affecting its diagnostic yield. METHODS: A total of 766 consecutive patients with CCA underwent bile cytology via endoscopic nasobiliary drainage or percutaneous transhepatic biliary drainage from January 2000 to June 2012. Data were collected by retrospectively reviewing the medical records. We evaluated the diagnostic yield of bile cytology with/without other sampling methods including brush cytology and endobiliary forcep biopsy, and the optimal number of repeated bile sampling. Several factors affecting diagnostic yield were then analyzed. RESULTS: The sensitivity of bile cytology, endobiliary forceps biopsy, and a combination of both sampling methods were 24.7% (189/766), 74.4% (259/348), and 77.9% (271/348), respectively. The cumulative positive rate of bile sampling increased from 40.7% (77/189) at first sampling to 93.1% (176/189) at third sampling. On multivariate analysis, factors associated with positive bile cytology were perihilar tumor location, intraductal growing tumor type, tumor extent > or =20 mm, poorly differentiated grade tumor, and three or more samplings. CONCLUSIONS: Although bile cytology itself has a low sensitivity in diagnosing CCA, it has an additive role when combined with endobiliary forceps biopsy. Due to the relative ease and low cost, bile cytology can be considered a reasonable complementary diagnostic tool for diagnosing CCA.
Aged ; Bile/*cytology ; Bile Duct Neoplasms/*diagnosis/pathology/radiography ; CA-19-9 Antigen/metabolism ; Cholangiocarcinoma/*diagnosis/pathology/radiography ; Drainage ; Female ; Humans ; Male ; Middle Aged ; Multivariate Analysis ; Neoplasm Staging ; Retrospective Studies

Fifty three bile specimens from 42 patients were reviewed to assess the diagnostic role of the bile cytology and to define more reliable cytologic indicators of malignancy. Forty three bile specimens came from 34 patients with malignant biliary strictures and 10 bile specimens were from eight patients with benign conditions. There were no false positives. The diagnostic specificity of bile cytology was 100% while diagnostic sensitivity was 55.8%. Overall diagnostic accuracy was 64.2%. We identified four key criteria as cytologic indicators of malignancy among 20 variables by using multiple regression analysis: loss of honeycomb arrangement, hyperchromatism, increased N/C ratio, and coarse chromatin. When bile specimens with three or more of these four criteria are thought to represent malignancy, the sensitivity of diagnosis of malignancy was 65.2%, specificity was 90% and diagnostic accuracy was 69.8%.
Adult ; Aged ; Bile/cytology* ; Bile Duct Neoplasms/diagnosis* ; Cell Nucleus/pathology ; Cholestasis/diagnosis* ; Chromatin/pathology ; Diagnosis, Differential ; Female ; Human ; Male ; Middle Age ; Pancreatic Neoplasms/diagnosis* ; Regression Analysis ; Sensitivity and Specificity