AIM： To enrich hepatic progenitors using epithelial cell adhesion molecule （EpCAM） as a marker from human fetal liver and investigate the expression of human leukocyte antigen （HLA） and their markers associated w...AIM： To enrich hepatic progenitors using epithelial cell adhesion molecule （EpCAM） as a marker from human fetal liver and investigate the expression of human leukocyte antigen （HLA） and their markers associated with hepatic progenitor cells. METHODS： EpCAM ＋ve cells were isolated using magnetic cell sorting （MACS） from human fetuses （n = 10） at 15-25 wk gestation. Expression of markers for hepatic progenitors such as albumin, alpha-fetoprotein （AFP）, CD29 （integrin ~1）, CD49f （integrin c^6） and CD90 （Thy 1） was studied by using flow cytometry, immunocytochemistry and RT-PCR; HLA class Ⅰ （A, B, C） and class Ⅱ （DR） expression was studied by flow cytometry only. RESULTS： FACS analysis indicated that EpCAM ＋ve cells were positive for CD29, CD49f, CD90, CD34, HLA class I, albumin and AFP but negative for HLA class Ⅱ （DR） and CD45. RT PCR showed that EpCAM ＋ve cells expressed liver epithelial markers （CK18）, biliary specific marker （CK19） and hepatic markers （albumin, AFP）. On immunocytochemical staining, EpCAM ＋ve cells were shown positive signals for CK18 and albumin. CONCLUSION： Our study suggests that these EpCAM ＋ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.展开更多
AIM： Thioacetamide （TAA） has been used in studying liver fibrosis and cirrhosis, however, the mechanisms of TAA-induced apoptosis in liver are still unclear. The hepatic epithelial cell line clone 9 was cultured an...AIM： Thioacetamide （TAA） has been used in studying liver fibrosis and cirrhosis, however, the mechanisms of TAA-induced apoptosis in liver are still unclear. The hepatic epithelial cell line clone 9 was cultured and treated with TAA to investigate the causes of cell death. METHODS： The cell viability of TAA-induced clone 9 cells was determined using MTT assay. Total cellular GSH in TAA-induced clone 9 cells was measured using a slight modification of the Tietze assay. The activity of caspase 3 in TAA-induced clone 9 cells was monitored by the cleavage of DEVD-p-nitroanaline. TUNEL assay and flow cytometry were applied for the determination of DNA fragmentation and the proportion of apoptosis in TAA- induced clone 9 cells, respectively. The alterations of caspase 3, Bad, Bax and Phospho-P53 contents in TAA- induced clone 9 cells were measured by Western blot. RESULTS： The experimental data indicated that TAA caused rat hepatic epithelial cell line clone 9 cell death in a dose-and time-dependent manner; 60% of the cells died （MTT assay） within 24 h after 100 rag/1 TAA was applied. Apoptotic cell percentage （TUNE1 assay） and caspase 3 activities were highest after 100 rag/1 TAA was added for 8 h. The release of GSH and the elevation in caspase content after TAA treatment resulted in clone 9 cell apoptosis via oxidative stress and a caspasedependent mechanism. The phospho-p53, Bax and Bad protein expressions in clone 9 cells were increased after TAA treatment. CONCLUSION： These results reveal that TAA activates p53, increases caspase 3, Bax and Bad protein contents, perhaps causing the release of cytochrome c from mitochondria and the disintegration of membranes, leading to apoptosis of cells.展开更多
Cholangiocytes-the epithelial cells which line the bileducts-are increasingly recognized as importanttransporting epithelia actively involved in the absorptionand secretion of water,ions,and solutes.Thisrecognition is...Cholangiocytes-the epithelial cells which line the bileducts-are increasingly recognized as importanttransporting epithelia actively involved in the absorptionand secretion of water,ions,and solutes.Thisrecognition is due in part to the recent development ofnew experimental models.New biologic concepts haveemerged including the identification and topography ofreceptors and flux proteins on the apical and/orbasolateral membrane which are involved in the molecularmechanisms of ductal bile secretion.Individually isolatedand/or perfused bile duct units from livers of rats andmice serve as new,physiologically relevant in vitromodels to study cholangiocyte transport.Biliary treedimensions and novel insights into anatomic remodeling ofproliferating bile ducts have emerged from three-dimensional reconstruction using CT scanning andsophisticated software.Moreover,new pathologicconcepts have arisen regarding the interaction ofcholangiocytes with pathogens such as Cryptosporidiumparvum.These concepts and associated methodologiesmay provide the framework to develop new therapies forthe cholangiopathies,a group of important hepatobiliarydiseases in which cholangiocytes are the target cell.展开更多
A rat liver epithelial cell line designated LW13 was established using a sequential sedimentation method. The cell line retained many normal properties of liver epithelial cells and showed some structural and function...A rat liver epithelial cell line designated LW13 was established using a sequential sedimentation method. The cell line retained many normal properties of liver epithelial cells and showed some structural and functional features resembling those of liver parenchymal cells. LW13 cells became malignant after the introduction of exogenous transforming EJ Ha ras gene. Tumors produced by inoculation of the transformed cells into baby rats .contained areas of poorly differentiated hepatocellular carcinoma. In situ hybridization analysis confirmed the random rather than specific integration of exogenous ras gene into host chromosomes. Furthermore , an at least tenfold increase in the expression of the endogenous c myc gene was detected among transformed cell lines, suggesting the involvement of the c myc proto oncogene in the in vitro transformation of rat liver epithelial cells by EJ Ha ras oncogene.展开更多
基金Council of Scientific and Industrial Research Network Grant CMM002ICMR Grant (GAP 0215)
文摘AIM： To enrich hepatic progenitors using epithelial cell adhesion molecule （EpCAM） as a marker from human fetal liver and investigate the expression of human leukocyte antigen （HLA） and their markers associated with hepatic progenitor cells. METHODS： EpCAM ＋ve cells were isolated using magnetic cell sorting （MACS） from human fetuses （n = 10） at 15-25 wk gestation. Expression of markers for hepatic progenitors such as albumin, alpha-fetoprotein （AFP）, CD29 （integrin ~1）, CD49f （integrin c^6） and CD90 （Thy 1） was studied by using flow cytometry, immunocytochemistry and RT-PCR; HLA class Ⅰ （A, B, C） and class Ⅱ （DR） expression was studied by flow cytometry only. RESULTS： FACS analysis indicated that EpCAM ＋ve cells were positive for CD29, CD49f, CD90, CD34, HLA class I, albumin and AFP but negative for HLA class Ⅱ （DR） and CD45. RT PCR showed that EpCAM ＋ve cells expressed liver epithelial markers （CK18）, biliary specific marker （CK19） and hepatic markers （albumin, AFP）. On immunocytochemical staining, EpCAM ＋ve cells were shown positive signals for CK18 and albumin. CONCLUSION： Our study suggests that these EpCAM ＋ve cells can be used as hepatic progenitors for cell transplantation with a minimum risk of alloreactivity and these cells may serve as a potential source for enrichment of hepatic progenitor.
基金Supported by the National Science Council, Taiwan, No.92-2317B-259-001
文摘AIM： Thioacetamide （TAA） has been used in studying liver fibrosis and cirrhosis, however, the mechanisms of TAA-induced apoptosis in liver are still unclear. The hepatic epithelial cell line clone 9 was cultured and treated with TAA to investigate the causes of cell death. METHODS： The cell viability of TAA-induced clone 9 cells was determined using MTT assay. Total cellular GSH in TAA-induced clone 9 cells was measured using a slight modification of the Tietze assay. The activity of caspase 3 in TAA-induced clone 9 cells was monitored by the cleavage of DEVD-p-nitroanaline. TUNEL assay and flow cytometry were applied for the determination of DNA fragmentation and the proportion of apoptosis in TAA- induced clone 9 cells, respectively. The alterations of caspase 3, Bad, Bax and Phospho-P53 contents in TAA- induced clone 9 cells were measured by Western blot. RESULTS： The experimental data indicated that TAA caused rat hepatic epithelial cell line clone 9 cell death in a dose-and time-dependent manner; 60% of the cells died （MTT assay） within 24 h after 100 rag/1 TAA was applied. Apoptotic cell percentage （TUNE1 assay） and caspase 3 activities were highest after 100 rag/1 TAA was added for 8 h. The release of GSH and the elevation in caspase content after TAA treatment resulted in clone 9 cell apoptosis via oxidative stress and a caspasedependent mechanism. The phospho-p53, Bax and Bad protein expressions in clone 9 cells were increased after TAA treatment. CONCLUSION： These results reveal that TAA activates p53, increases caspase 3, Bax and Bad protein contents, perhaps causing the release of cytochrome c from mitochondria and the disintegration of membranes, leading to apoptosis of cells.
基金grants DK24031 and DK57993(N.F.LaRusso)from the National Institutes of Healththe Mayo Foundation
文摘Cholangiocytes-the epithelial cells which line the bileducts-are increasingly recognized as importanttransporting epithelia actively involved in the absorptionand secretion of water,ions,and solutes.Thisrecognition is due in part to the recent development ofnew experimental models.New biologic concepts haveemerged including the identification and topography ofreceptors and flux proteins on the apical and/orbasolateral membrane which are involved in the molecularmechanisms of ductal bile secretion.Individually isolatedand/or perfused bile duct units from livers of rats andmice serve as new,physiologically relevant in vitromodels to study cholangiocyte transport.Biliary treedimensions and novel insights into anatomic remodeling ofproliferating bile ducts have emerged from three-dimensional reconstruction using CT scanning andsophisticated software.Moreover,new pathologicconcepts have arisen regarding the interaction ofcholangiocytes with pathogens such as Cryptosporidiumparvum.These concepts and associated methodologiesmay provide the framework to develop new therapies forthe cholangiopathies,a group of important hepatobiliarydiseases in which cholangiocytes are the target cell.
文摘A rat liver epithelial cell line designated LW13 was established using a sequential sedimentation method. The cell line retained many normal properties of liver epithelial cells and showed some structural and functional features resembling those of liver parenchymal cells. LW13 cells became malignant after the introduction of exogenous transforming EJ Ha ras gene. Tumors produced by inoculation of the transformed cells into baby rats .contained areas of poorly differentiated hepatocellular carcinoma. In situ hybridization analysis confirmed the random rather than specific integration of exogenous ras gene into host chromosomes. Furthermore , an at least tenfold increase in the expression of the endogenous c myc gene was detected among transformed cell lines, suggesting the involvement of the c myc proto oncogene in the in vitro transformation of rat liver epithelial cells by EJ Ha ras oncogene.
