AIM: To elucidate the interaction between nonparenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). METHODS: We examined the localiza...AIM: To elucidate the interaction between nonparenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). METHODS: We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2acetylaminofluorene (2-AAF)/PH rat model. RESULTS: By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic lobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells (HSCs), laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver lobule structures began to recover.CONCLUSION: Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions.展开更多
AIM:To evaluate the effect of intrahepatic transplantation of hepatic oval cells(HOC)on fulminant hepatic failure(FHF)in rats.METHODS:HOC obtained from rats were labeled with green fluocescent protein(GFP)or 5,6carbox...AIM:To evaluate the effect of intrahepatic transplantation of hepatic oval cells(HOC)on fulminant hepatic failure(FHF)in rats.METHODS:HOC obtained from rats were labeled with green fluocescent protein(GFP)or 5,6carboxyfluorescein diacetate succinmidyl ester(CFDASE).Cell fluorescence was observed under fluorescent microscope at 6,24,48 and 72 h after labeling.CFDASE labeled HOC(5×10 6 cells each rat)were injected into livers of rats with FHF induced by D-galactosamine.Serum albumin(ALB),alanine aminotransferase(ALT),aspartate aminotransferase(AST)and total bilirubin(TBil)levels were measured at different time points.Liver function of rats was examined on days 3,7,14 and 21 after HOC transplantation.RESULTS:The positive rate of GFP and CFDA-SE labeled HOC was 10%and 90%,respectively,with no significant change in cell viabilities.The survival rate was higher in HOC transplantation group than in control group,especially 48(9/15 vs 6/15)and 72 h(9/15 vs 4/15)after HOC transplantation.The serum ALT,AST and TBil levels were decreased while the serum Alb level was increased after HOC transplantation.Fluorescence became faded and diffused in liver tissues,suggesting that proliferation and differentiation occur in transplanted HOC.CONCLUSION:CFDA-SE is superior to GFP in labeling HOC,although fluorescence intensity is decreased progressively with cell division.HOC transplantation can improve the liver function and increase the survival rate of recipients.展开更多
OBJECTIVE: To study the relationship between oval cells and primary hepatocarcinoma and theexpression of c-kit and proliferating cell nuclear antigen (PCNA) in oval cells of rats with hepatocellularcarcinoma.METHODS: ...OBJECTIVE: To study the relationship between oval cells and primary hepatocarcinoma and theexpression of c-kit and proliferating cell nuclear antigen (PCNA) in oval cells of rats with hepatocellularcarcinoma.METHODS: A hundred and twenty clean SD rats were divided into three groups: normal group,cancer-induction group and intervention group. The normal group was fed with standard forage while therest two groups were fed with 3’-methyl-2-methylamino-azobenzene (DAB) to induce carcinoma for 14weeks and then fed with standard forage and water. Uscharidin was injected abdominally to theintervention group from the first week to the 14th week. All rats were killed and biopsy specimens weretaken from the left and right liver lobes for immunohistochemical staining of c-kit and PCNA on the 2nd,4th, 6th, 8th, 10th, 12th, 14th, 16th, 18th, 20th, 22nd, and 24th week.RESULTS: From the 2nd to 14th week after liver infection, c-kit positive cells, mainly oval cells werefound in the portal area in the carcinoma-induction group and dotted positive pigmentations in liverlobules. In the 22nd week, a large number of cancerous nodes occurred and nuclei heteromorphi-m wasapparent; the number of positive cell decreased but positive cells could be sparsely observed in cancerousnodes. In the 2nd week of the carcinoma-induction process, PCNA positive cells were oval cells in theportal area. In the 4th week, a lot of hepatic cells were positively stained, especially in the central veinarea. In the 6th week, PCNA positive cells could be seen in the lobules of the liver. In the 8th week, thenumber of PCNA cells decreased comparatively. From the 10th to 14th week, oval cells in the portal areawere still over-expressed. From the 16th to 24th week, a large number of cancerous nodes occurred andPCNA was over-expressed in some of them. In necrotic cancerous nodes, the para-cancerous PCNApositive cells were sparsely distributed and their number was less than that of PCNA positive cells ofcancerous tissues.CONCLUSIONS: Hepatic stem cells originating from the terminal biliary plexus of the portal area areinvolved in the development of hepatocarcinoma because c-kit positive cells expressed in cancerousnodes, accompany the whole process of the development. In the middle inflammatory period ofcarcinoma-induction, the expression of PCNA in hepatic cells peaked, but the index decreased in the lateinflammatory period and in the proliferated fibrosis stage. The expression of PCNA is a tortuous process,going up, down, then up again from normal tissues to cancerous tissues. Combined with pathologicalfindings, PCNA can be considered as a warning index for carcinomatous cells.展开更多
BACKGROUND: This study was designed to assess theroles of oval cells and c-myc mRNA in the process of hepa-tocarcinogenesis and to clarify the function of carcinogenec-myc in the development of hepatocellular carcinom...BACKGROUND: This study was designed to assess theroles of oval cells and c-myc mRNA in the process of hepa-tocarcinogenesis and to clarify the function of carcinogenec-myc in the development of hepatocellular carcinoma( HCC) and the mechanism of inhibitory function of uscha-ridin on HCC in mouse hepatocarcinogenesis.METHODS: A total of 120 clean SD mice were divided intonormal group, cancer induction group, and interventiongroup. The normal group was fed with standard foragewhile the rest two groups were given p-dimethylaminoazo-benzene (DAB) to induce cancer. Thirteen weeks after in-duction of cancer, the two groups were fed with standardforage and water. Once the pattern was set up, the inter-vention group was given uscharidin injection into the ab-dominal cavity from the first week to the 14th week. Onthe 2nd, 4th, 6th, 8th, 10th, 12th, 14th, 16th, 18th, 20th,22nd, and 24th week, all mice were killed and biopsiedfrom the liver lobe for pathological analysis. At the sametime, the number of tumor nodes was counted and the ex-pression of c-myc mRNA was tested by RT-PCR.RESULTS: Since the 2nd week after cancer induction, pro-liferated oval cells could be seen in the portal area. Initially,the oval cells appeared in the cortical layer of the portalarea, then proliferated gradually and immigrated into theliver parenchyma. In the period of fibrosis after liver proli-feration, proliferated heaps of oval cells were noted in bothportal and peripheral areas. In the period of carcinomatouschange, oval cells could be seen both outside and inside ofcancer nodes, but most of them were distributed outside.The c-myc gene was expressed negatively in the liver tissueof mice. The quantity of the expression began to increaseat the time of infection of the liver and tended to increasewith the degree of hepatic injury. In the period of cancera-tion, the expression level of c-myc mRNA increased gra-dually. The intervention of uscharidin could not inhibit butdelay the increase of the expression of c-myc mRNA.CONCLUSION: Oval cells are closely related to hepatocar-cinoma cells, which play an important role in the occur-rence and development of hepatocarcinogenesis. Uschari-din can inhibit the occurrence of hepatocarcinogenesis orlocal spreading at the early stage of cancer induction byDAB, but it cannot inhibit the expression of c-myc.展开更多
AIM:To investigate the ultrastructure of oval cells in children with chronic hepatitis B,with special emphasis on their location in areas of collagen fibroplasia. METHODS:Morphological investigations were conducted on...AIM:To investigate the ultrastructure of oval cells in children with chronic hepatitis B,with special emphasis on their location in areas of collagen fibroplasia. METHODS:Morphological investigations were conducted on biopsy material obtained from 40 children,aged 3-16 years with chronic hepatitis B. The stage of fibrosis was assessed histologically using the arbitrary semiquantitative numerical scoring system proposed by Ishak et al. The material for ultrastructural investigation was fixed in glutaraldehyde and paraformaldehyde and processed for transmission-electron microscopic analysis. RESULTS:Ultrastructural examination of biopsy specimens obtained from children with chronic hepatitis B showed the presence of two types of oval cells,the hepatic progenitor cells and intermediate hepatic-like cells. These cells were present in the parenchyma and were seen most commonly in areas of intense periportal fibrosis (at least stage 2 according to Ishak et al) and in the vicinity of the limiting plate of the lobule. The activated nonparenchymal hepatic cells,i.e. transformed hepatic stellate cells and Kupffer cells were seen in close proximity to the intermediate hepatic-like cells. CONCLUSION:We found a distinct relationship between the prevalence of oval cells (hepatic progenitor cells and intermediate hepatocyte-like cells) and fibrosis stage in pediatric patients with chronic hepatitis B.展开更多
AIM:To investigate the effect of the neuropeptides bombesin(BBS)and neurotensin(NT)on oval cell proliferation in partially hepatectomized rats not pretreated with a known hepatocyte inhibitor.METHODS:Seventy male Wist...AIM:To investigate the effect of the neuropeptides bombesin(BBS)and neurotensin(NT)on oval cell proliferation in partially hepatectomized rats not pretreated with a known hepatocyte inhibitor.METHODS:Seventy male Wistar rats were randomly divided into five groups:Ⅰ=controls,Ⅱ=sham operated,Ⅲ=partial hepatectomy 70%(PHx),Ⅳ=PHx+ BBS(30μg/kg per day),Ⅴ=PHx+NT(300μg/kg per day).Forty eight hours after liver resection,portal en-dotoxin levels and hepatic glutathione redox state were determined.α-fetoprotein(AFP)mRNA(in situ hybridisation),cytokeratin-19 and Ki67 antigen expression (immunohistochemistry)and apoptosis(TUNEL)were evaluated on liver tissue samples.Cells with morphological features of oval cells that were cytokeratin-19 (+)and AFP mRNA(+)were scored in morphometric analysis and their proliferation was recorded.In addition,the proliferation and apoptotic rates of hepatocytes were determined.RESULTS:In the control and sham operated groups,oval cells were significantly less compared to groups Ⅲ,ⅣandⅤ(P<0.001).The neuropeptides BBS and NT significantly increased the proliferation of oval cells compared to groupⅢ(P<0.001).In addition,BBS and NT induced a significant increase of hepatocyte proliferation(P<0.001),whereas it decreased their apoptotic activity(P<0.001)compared to groupⅢ.BBS and NT significantly decreased portal endotoxemia (P<0.001)and increased the hepatic GSH:GSSG ratio (P<0.05 and P<0.001,respectively)compared to groupⅢ.CONCLUSION:BBS and NT stimulated oval cell proliferation in a model of liver regeneration,without use of concomitant suppression of hepatocyte proliferation as oval cell activation stimuli,and improved the hepatocyte regenerative response.This peptides-induced combined stimulation of oval cell and hepatocyte proliferation might serve as a possible treatment modality for several liver diseases.展开更多
Objective To acquire oval cells (progenitor stem cells) from adult rat liver of different models including diabetic rats. Methods Thirty Sprague-Dawley (SD) rats were divided into 5 groups randomly: control, 2-acetyla...Objective To acquire oval cells (progenitor stem cells) from adult rat liver of different models including diabetic rats. Methods Thirty Sprague-Dawley (SD) rats were divided into 5 groups randomly: control, 2-acetylaminofluorene (2-AAF), 2-AAF+partial hepatectomy (PH), 2-AAF+carbon tetrachloride (CCl4), and diabetic groups. As two-step collagenase perfusion protocol of Seglen, oval cells were isolated by Percoll density gradient centrifugation. Thy1.1 positive cells were sorted by flow cytometry, and then cultured in Dulbecco's minimum Eagle's medium (DMEM). Immunofluorescence staining was applied to labelling Thy1.1. Results Different rates of Thy1.1 positive oval cells were found in different rat model groups: 0.5% in 2-AAF, 0.3% in 2-AAF+PH, 0.2% in 2-AAF+CCl4 , 0.1% in diabetic, and 0.0% in control. Isolated cells adhered to plate with fusiform or polygon as epithelial cells. Conclusion Progenitor stem cells exist in injured liver tissue including those from diabetic rats.展开更多
Summary:Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia.Several models have been established to activate the oval cells by incorporating a varie...Summary:Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia.Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens,alone or combined with surgical treatment.Those models are obviously not suitable for the study on human hepatic oval cells.It is necessary to establish a new and efficient model to study the human hepatic oval cells.In this study,the hepatocyte growth factor(HGF)and epidermal growth factor(EGF)were used to induce differentiation of mouse embryonic stem(ES)cells into hepatic oval cells.We first confirmed that hepatic oval cells derived from ES cells,which are bipotential,do exist during the course of mouse ES cells’differentiation into hepatic parenchymal cells.RT-PCR and transmission electron microscopy were applied in this study.The ratio of Sca-1+/CD34+cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8,whereas that in the control group remained at a low level.The differentiation ratio of Sca-1+/CD34+cells in the induction group was significantly higher than that in the control group.About 92.48%of the sorted Sca-1+/CD34+cells on the day 8 were A6 positive.Highly purified A6+/Sca-1+/CD34+hepatic oval cells derived from ES cells could be obtained by FACS.The differentiation ratio of hepatic oval cells in the induction group(up to 4.46%)was significantly higher than that in the control group.The number of hepatic oval cells could be increased significantly by HGF and EGF.The study also examined the ultrastructures of ES-derived hepatic oval cells’membrane surface by atomic force microscopy.The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.展开更多
AIM: To investigate the potential of bone marrow mononuclear cells (BM-MCs) in the regeneration of hepatic lesions induced by Schistosoma mansoni (S.mansoni) chronic infection. METHODS: Female mice chronically infecte...AIM: To investigate the potential of bone marrow mononuclear cells (BM-MCs) in the regeneration of hepatic lesions induced by Schistosoma mansoni (S.mansoni) chronic infection. METHODS: Female mice chronically infected with S.mansoni were treated with BM-MCs obtained from male green fluorescent protein (GFP) transgenic mice by intravenous or intralobular injections. Control mice received injections of saline in similar conditions. Enzyme-linked immunosorbent assay (ELISA) assay fortransforming growth factor-beta (TGF-β), polymerase chain reaction (PCR) for GFP DNA, immunofluorescence and morphometric studies were performed. RESULTS: Transplanted GFP+ cells migrated to granuloma areas and reduced the percentage of liver fibrosis. The presence of donor-derived cells was confirmed by Fluorescence in situ hybridization (FISH) analysis for detection of cells bearing Y chromosome and by PCR analysis for detection of GFP DNA. The levels of TGF-β, a cytokine associated with fibrosis deposition, in liver fragments of mice submitted to therapy were reduced. The number of oval cells in liver sections of S.mansoni-infected mice increased 3-4 fold after transplantation. A partial recovery in albumin expression, which is decreased upon infection with S.mansoni, was found in livers of infected mice after cellular therapy. CONCLUSION: In conclusion, transplanted BMCs migrate to and reduce the damage of chronic fibrotic liver lesions caused by S.mansoni.展开更多
基金Supported by A grant from National Natural Science Foundation of China
文摘AIM: To elucidate the interaction between nonparenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). METHODS: We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2acetylaminofluorene (2-AAF)/PH rat model. RESULTS: By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic lobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells (HSCs), laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver lobule structures began to recover.CONCLUSION: Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions.
基金Supported by Tianjin Science Committee,Grant No.05SYSYJC02600Tianjin Health Bureau,Grant No.05KYR01
文摘AIM:To evaluate the effect of intrahepatic transplantation of hepatic oval cells(HOC)on fulminant hepatic failure(FHF)in rats.METHODS:HOC obtained from rats were labeled with green fluocescent protein(GFP)or 5,6carboxyfluorescein diacetate succinmidyl ester(CFDASE).Cell fluorescence was observed under fluorescent microscope at 6,24,48 and 72 h after labeling.CFDASE labeled HOC(5×10 6 cells each rat)were injected into livers of rats with FHF induced by D-galactosamine.Serum albumin(ALB),alanine aminotransferase(ALT),aspartate aminotransferase(AST)and total bilirubin(TBil)levels were measured at different time points.Liver function of rats was examined on days 3,7,14 and 21 after HOC transplantation.RESULTS:The positive rate of GFP and CFDA-SE labeled HOC was 10%and 90%,respectively,with no significant change in cell viabilities.The survival rate was higher in HOC transplantation group than in control group,especially 48(9/15 vs 6/15)and 72 h(9/15 vs 4/15)after HOC transplantation.The serum ALT,AST and TBil levels were decreased while the serum Alb level was increased after HOC transplantation.Fluorescence became faded and diffused in liver tissues,suggesting that proliferation and differentiation occur in transplanted HOC.CONCLUSION:CFDA-SE is superior to GFP in labeling HOC,although fluorescence intensity is decreased progressively with cell division.HOC transplantation can improve the liver function and increase the survival rate of recipients.
基金This work was supported by the grants from Science Foundation of Guangdong Province (No. 00593 and 01059. 2001).
文摘OBJECTIVE: To study the relationship between oval cells and primary hepatocarcinoma and theexpression of c-kit and proliferating cell nuclear antigen (PCNA) in oval cells of rats with hepatocellularcarcinoma.METHODS: A hundred and twenty clean SD rats were divided into three groups: normal group,cancer-induction group and intervention group. The normal group was fed with standard forage while therest two groups were fed with 3’-methyl-2-methylamino-azobenzene (DAB) to induce carcinoma for 14weeks and then fed with standard forage and water. Uscharidin was injected abdominally to theintervention group from the first week to the 14th week. All rats were killed and biopsy specimens weretaken from the left and right liver lobes for immunohistochemical staining of c-kit and PCNA on the 2nd,4th, 6th, 8th, 10th, 12th, 14th, 16th, 18th, 20th, 22nd, and 24th week.RESULTS: From the 2nd to 14th week after liver infection, c-kit positive cells, mainly oval cells werefound in the portal area in the carcinoma-induction group and dotted positive pigmentations in liverlobules. In the 22nd week, a large number of cancerous nodes occurred and nuclei heteromorphi-m wasapparent; the number of positive cell decreased but positive cells could be sparsely observed in cancerousnodes. In the 2nd week of the carcinoma-induction process, PCNA positive cells were oval cells in theportal area. In the 4th week, a lot of hepatic cells were positively stained, especially in the central veinarea. In the 6th week, PCNA positive cells could be seen in the lobules of the liver. In the 8th week, thenumber of PCNA cells decreased comparatively. From the 10th to 14th week, oval cells in the portal areawere still over-expressed. From the 16th to 24th week, a large number of cancerous nodes occurred andPCNA was over-expressed in some of them. In necrotic cancerous nodes, the para-cancerous PCNApositive cells were sparsely distributed and their number was less than that of PCNA positive cells ofcancerous tissues.CONCLUSIONS: Hepatic stem cells originating from the terminal biliary plexus of the portal area areinvolved in the development of hepatocarcinoma because c-kit positive cells expressed in cancerousnodes, accompany the whole process of the development. In the middle inflammatory period ofcarcinoma-induction, the expression of PCNA in hepatic cells peaked, but the index decreased in the lateinflammatory period and in the proliferated fibrosis stage. The expression of PCNA is a tortuous process,going up, down, then up again from normal tissues to cancerous tissues. Combined with pathologicalfindings, PCNA can be considered as a warning index for carcinomatous cells.
基金This work was supported by two grants from the Science Fundation ofGuangdong Province, China (No. 010593 No. 020097).
文摘BACKGROUND: This study was designed to assess theroles of oval cells and c-myc mRNA in the process of hepa-tocarcinogenesis and to clarify the function of carcinogenec-myc in the development of hepatocellular carcinoma( HCC) and the mechanism of inhibitory function of uscha-ridin on HCC in mouse hepatocarcinogenesis.METHODS: A total of 120 clean SD mice were divided intonormal group, cancer induction group, and interventiongroup. The normal group was fed with standard foragewhile the rest two groups were given p-dimethylaminoazo-benzene (DAB) to induce cancer. Thirteen weeks after in-duction of cancer, the two groups were fed with standardforage and water. Once the pattern was set up, the inter-vention group was given uscharidin injection into the ab-dominal cavity from the first week to the 14th week. Onthe 2nd, 4th, 6th, 8th, 10th, 12th, 14th, 16th, 18th, 20th,22nd, and 24th week, all mice were killed and biopsiedfrom the liver lobe for pathological analysis. At the sametime, the number of tumor nodes was counted and the ex-pression of c-myc mRNA was tested by RT-PCR.RESULTS: Since the 2nd week after cancer induction, pro-liferated oval cells could be seen in the portal area. Initially,the oval cells appeared in the cortical layer of the portalarea, then proliferated gradually and immigrated into theliver parenchyma. In the period of fibrosis after liver proli-feration, proliferated heaps of oval cells were noted in bothportal and peripheral areas. In the period of carcinomatouschange, oval cells could be seen both outside and inside ofcancer nodes, but most of them were distributed outside.The c-myc gene was expressed negatively in the liver tissueof mice. The quantity of the expression began to increaseat the time of infection of the liver and tended to increasewith the degree of hepatic injury. In the period of cancera-tion, the expression level of c-myc mRNA increased gra-dually. The intervention of uscharidin could not inhibit butdelay the increase of the expression of c-myc mRNA.CONCLUSION: Oval cells are closely related to hepatocar-cinoma cells, which play an important role in the occur-rence and development of hepatocarcinogenesis. Uschari-din can inhibit the occurrence of hepatocarcinogenesis orlocal spreading at the early stage of cancer induction byDAB, but it cannot inhibit the expression of c-myc.
文摘AIM:To investigate the ultrastructure of oval cells in children with chronic hepatitis B,with special emphasis on their location in areas of collagen fibroplasia. METHODS:Morphological investigations were conducted on biopsy material obtained from 40 children,aged 3-16 years with chronic hepatitis B. The stage of fibrosis was assessed histologically using the arbitrary semiquantitative numerical scoring system proposed by Ishak et al. The material for ultrastructural investigation was fixed in glutaraldehyde and paraformaldehyde and processed for transmission-electron microscopic analysis. RESULTS:Ultrastructural examination of biopsy specimens obtained from children with chronic hepatitis B showed the presence of two types of oval cells,the hepatic progenitor cells and intermediate hepatic-like cells. These cells were present in the parenchyma and were seen most commonly in areas of intense periportal fibrosis (at least stage 2 according to Ishak et al) and in the vicinity of the limiting plate of the lobule. The activated nonparenchymal hepatic cells,i.e. transformed hepatic stellate cells and Kupffer cells were seen in close proximity to the intermediate hepatic-like cells. CONCLUSION:We found a distinct relationship between the prevalence of oval cells (hepatic progenitor cells and intermediate hepatocyte-like cells) and fibrosis stage in pediatric patients with chronic hepatitis B.
文摘AIM:To investigate the effect of the neuropeptides bombesin(BBS)and neurotensin(NT)on oval cell proliferation in partially hepatectomized rats not pretreated with a known hepatocyte inhibitor.METHODS:Seventy male Wistar rats were randomly divided into five groups:Ⅰ=controls,Ⅱ=sham operated,Ⅲ=partial hepatectomy 70%(PHx),Ⅳ=PHx+ BBS(30μg/kg per day),Ⅴ=PHx+NT(300μg/kg per day).Forty eight hours after liver resection,portal en-dotoxin levels and hepatic glutathione redox state were determined.α-fetoprotein(AFP)mRNA(in situ hybridisation),cytokeratin-19 and Ki67 antigen expression (immunohistochemistry)and apoptosis(TUNEL)were evaluated on liver tissue samples.Cells with morphological features of oval cells that were cytokeratin-19 (+)and AFP mRNA(+)were scored in morphometric analysis and their proliferation was recorded.In addition,the proliferation and apoptotic rates of hepatocytes were determined.RESULTS:In the control and sham operated groups,oval cells were significantly less compared to groups Ⅲ,ⅣandⅤ(P<0.001).The neuropeptides BBS and NT significantly increased the proliferation of oval cells compared to groupⅢ(P<0.001).In addition,BBS and NT induced a significant increase of hepatocyte proliferation(P<0.001),whereas it decreased their apoptotic activity(P<0.001)compared to groupⅢ.BBS and NT significantly decreased portal endotoxemia (P<0.001)and increased the hepatic GSH:GSSG ratio (P<0.05 and P<0.001,respectively)compared to groupⅢ.CONCLUSION:BBS and NT stimulated oval cell proliferation in a model of liver regeneration,without use of concomitant suppression of hepatocyte proliferation as oval cell activation stimuli,and improved the hepatocyte regenerative response.This peptides-induced combined stimulation of oval cell and hepatocyte proliferation might serve as a possible treatment modality for several liver diseases.
基金Supported by Shanghai Municipal Education Commission Key Project (07ZZ35)
文摘Objective To acquire oval cells (progenitor stem cells) from adult rat liver of different models including diabetic rats. Methods Thirty Sprague-Dawley (SD) rats were divided into 5 groups randomly: control, 2-acetylaminofluorene (2-AAF), 2-AAF+partial hepatectomy (PH), 2-AAF+carbon tetrachloride (CCl4), and diabetic groups. As two-step collagenase perfusion protocol of Seglen, oval cells were isolated by Percoll density gradient centrifugation. Thy1.1 positive cells were sorted by flow cytometry, and then cultured in Dulbecco's minimum Eagle's medium (DMEM). Immunofluorescence staining was applied to labelling Thy1.1. Results Different rates of Thy1.1 positive oval cells were found in different rat model groups: 0.5% in 2-AAF, 0.3% in 2-AAF+PH, 0.2% in 2-AAF+CCl4 , 0.1% in diabetic, and 0.0% in control. Isolated cells adhered to plate with fusiform or polygon as epithelial cells. Conclusion Progenitor stem cells exist in injured liver tissue including those from diabetic rats.
基金supported by grants from the Special Funds for Major State Basic Research of China(No.2001CB510101)the National Natural Science Foundation of China(No.30230350 and 60278014)the Natural Science Foundation of Guangdong Province(No.36704)
文摘Summary:Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia.Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens,alone or combined with surgical treatment.Those models are obviously not suitable for the study on human hepatic oval cells.It is necessary to establish a new and efficient model to study the human hepatic oval cells.In this study,the hepatocyte growth factor(HGF)and epidermal growth factor(EGF)were used to induce differentiation of mouse embryonic stem(ES)cells into hepatic oval cells.We first confirmed that hepatic oval cells derived from ES cells,which are bipotential,do exist during the course of mouse ES cells’differentiation into hepatic parenchymal cells.RT-PCR and transmission electron microscopy were applied in this study.The ratio of Sca-1+/CD34+cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8,whereas that in the control group remained at a low level.The differentiation ratio of Sca-1+/CD34+cells in the induction group was significantly higher than that in the control group.About 92.48%of the sorted Sca-1+/CD34+cells on the day 8 were A6 positive.Highly purified A6+/Sca-1+/CD34+hepatic oval cells derived from ES cells could be obtained by FACS.The differentiation ratio of hepatic oval cells in the induction group(up to 4.46%)was significantly higher than that in the control group.The number of hepatic oval cells could be increased significantly by HGF and EGF.The study also examined the ultrastructures of ES-derived hepatic oval cells’membrane surface by atomic force microscopy.The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.
基金Instituto do Milênio de Bioengenharia Tecidual (IMBT, CNPq/MCT)Rede Nordeste de Biotecnologia (RENORBIO, FINEP/MCT)+1 种基金Fundao de Amparo à Pesquisa do Estado da Bahia (FAPESB)Fundao Oswaldo Cruz (FIOCRUZ)
文摘AIM: To investigate the potential of bone marrow mononuclear cells (BM-MCs) in the regeneration of hepatic lesions induced by Schistosoma mansoni (S.mansoni) chronic infection. METHODS: Female mice chronically infected with S.mansoni were treated with BM-MCs obtained from male green fluorescent protein (GFP) transgenic mice by intravenous or intralobular injections. Control mice received injections of saline in similar conditions. Enzyme-linked immunosorbent assay (ELISA) assay fortransforming growth factor-beta (TGF-β), polymerase chain reaction (PCR) for GFP DNA, immunofluorescence and morphometric studies were performed. RESULTS: Transplanted GFP+ cells migrated to granuloma areas and reduced the percentage of liver fibrosis. The presence of donor-derived cells was confirmed by Fluorescence in situ hybridization (FISH) analysis for detection of cells bearing Y chromosome and by PCR analysis for detection of GFP DNA. The levels of TGF-β, a cytokine associated with fibrosis deposition, in liver fragments of mice submitted to therapy were reduced. The number of oval cells in liver sections of S.mansoni-infected mice increased 3-4 fold after transplantation. A partial recovery in albumin expression, which is decreased upon infection with S.mansoni, was found in livers of infected mice after cellular therapy. CONCLUSION: In conclusion, transplanted BMCs migrate to and reduce the damage of chronic fibrotic liver lesions caused by S.mansoni.
