| Background and objective The hepatic progenitor cells called hepatoblasts have the ability to differentiate into both mature hepatocytes and cholangiocytes.During intrahepatic bile duct(IHBD)development,hepatoblasts in the vicinity of portal vein mesenchyme(PVM),give rise to cholangiocytes.In contrast,hepatoblasts in the liver parenchyma,distance from the portal vein,differentiated into hepatocytes.In this study,we aim to investigate the role of TGF? signaling pathway in regulation of IHBD development.Methods Part1: Isolation and culture of mouse embryonic fibroblasts(MEFs),P2 MEFs were divided into four groups: control group,recombinant 5 ng/ml TGF?1,recombinant 5 ng/ml TGF?1+10?M SB525334,or 10?M SB525334 for 72 h.Isolation of mouse hepatic progenitor cells(m HPCs)from mouse fetal liver was based on the cell surface antigen delta-like protein 1/preadipocyte factor 1(Dlk/Pref-1)by a fluorescence-activated cell sorter(FACS).Then m HPCs isolated were co-cultured with/withoutmyofibroblasts or fibroblasts by using Transwell.The ?-SMA expression in MEFs after treatment for 72 h and the cell antigen alpha-fetoprotein(AFP),albumin(ALB)and cytokeratin19(CK19)expression in fresh isolated DLK1+cells or co-cultured for 4 days and 6 days were observed with immunocytochemical method.The levels of ??SMA?HGF?TGF?1?Jagged1?PTN?MDK?IGF1 and IGF2 m RNA in myofibroblasts or fibroblasts and the levels of CK19? ALB? AFP?Sox9 m RNA in m HPCs after coculture for 6d were detected by q RT-PCR.Part2: Eight wild-type pregnant mice at E10.5 were divided into two groups.One group were injected i.p.with TGF?RI inhibitor SB525334(5mg/kg)in 2.2% DMSO and 97.8% corn oil from E10.5 to E18.5 every day for 8 days.Control pregnant mice were injected similarly with2.2% DMSO and 97.8% corn oil.The expression of ??SMA?Sox9 and CK19 in liver sections prepared from E14.5,E16.5,E18.5,P0 and P10 were investigated with Immunostaining.The expression of ??SMA,Jagged1,Sox9 and CK19 in SB525334 treatment E18.5 liver sections and controls were detected with Immunostaining.The expression of ??SMA,Jagged1,Sox9 and CK19 in SB525334 treatment liver tissue and controls were evaluated by q RT-PCR.Bile Ducts(BDs)in the hilum PV and BECs in the periphery PV with a distinct surrounding layer of ?-SMA cuboidal cells and a clear lumen around each PV were recorded.These counts were then used tocalculate the BDs/PV or BECs/PV ratio for each animal.Results Part1: The DLK1+ cells isolated by FACS were about 10%~20% of total E14.5 hepatic cells.When co-cultured with MEFs,the division and proliferation were observed in most of DLK1+ cells and grape-like aggregation was formed.Cells began to adhere to growth and began to become spindle-shaped on 4th day.The DLK1+cells isolated freshly by FACS were expressed AFP and low levels of ALB but not expressed CK19.But,these cells expressed CK 19 and weak expression of ALB on 4th day.In addition,the expression of CK19 increased and the expression of ALB almost not detected on 6th day.These results suggested that most of Dlk1+ cells,isolated from E14.5 fetal livers by FACS,proved to be m HPCs.Furthermore,these cells can proliferate quickly and differentiate into cholangiocytes by co-culture with MEFs.By immunocytochemical method,we conformed that TGF? signaling pathway induced fibroblasts transformed into myofibroblasts.After cocultured with myofibroblasts for 6 days,most of m HPCs attached to the wall of cell culture flask and formed bile duct-like branching structures.However,when co-cultured with fibroblasts,some of m HPCs attach to the wall of cell culture flask and yielded large colonies,and not formed bile duct-like branching structures.IHC results showed that m HPCs co-culture with myofibroblasts expressed higher CK19 and lower ALB than co-culturewith fibroblasts.Furthermore,results of q RT-PCR conformed to the IHC,m HPCs co-culture with myofibroblasts expressed higher CK19 and Sox9 than co-culture with fibroblasts(P<0.05),and lower AFP and KI67 than co-culture with fibroblasts(P<0.05).The total number of cells co-culture with fibroblasts was about 2.5 times higher than that co-culture with myofibroblasts(P<0.05).These data indicate that myofibroblasts,compared with fibroblasts,promotes cholangiocyte maturation of hepatoblasts and suppresses proliferation of hepatoblasts in vitro.Part2: IHC showed that ?-SMA positive cells were main located in the PVM,around the blood vessels at E14.5~18.5 and encircled the bile duct after born.Moreover,Jagged1 are expressed in ?-SMA positive portal myofibroblasts(PMFs),and PMFs contribute to the biliary differentiation of adjacent hepatoblasts and bile duct formation.After blocking the TGF? Signaling,the number of bile ducts in hilum PVs and number of CK19 positive BECs in periphery PVs were significantly reduced(P<0.05),suggesting that TGF? signaling plays an important role in the regulating of IHBD development.After blocking the TGF? Signaling,the levels of ?-SMA expression in PVM were decreased compared with control.The q RT-PCR results also showed that ?-SMA transcripts began to decreased after blocking TGF? signaling at E10.5 and were reduced at E18.5 in SB525334 treated livers(P<0.05),indicating PMFs transform form portal mesenchyme cells induced by TGF? signaling during feotal liverdevelopment.Furthermore,Jagged1 expression in PVM was significantly decreased after blocking TGF? signaling.q RT-PCR results also showed that Jagged1 transcripts in SB525334 treated livers decreased after blocking TGF? signaling(P<0.05),implying TGF? signaling regulation of expression of Jagged1 in PVM through transformation of portal mesenchyme cells into PMFs.Interestingly,the expression of Jagged1 in BECs were not significantly decreased,suggesting that TGF? signaling regulation of expression of Jagged1 in PVM but not in BECs.Sox9 is a direct target of Jag1-Notch signaling.SB525334 treatment livers significantly decreased expression of Sox9 around the portal vein compared with controls at E18.5.The q RT-PCR results also showed that Sox9 transcripts in SB525334 treated livers decreased after blocking TGF? signaling.These results su ggested that TGF? Signaling may control IHBD development through Jag1-Notch-Sox9 signaling pathway.Conclusion 1.The Dlk1+ cells,isolation from E14.5 fetal livers by FACS,proved to be m HPCs.Furthermore,these cells can proliferate quickly and differentiation into chlangiocytes by co-culture with MEFs.2.Myofibroblasts,compared with fibroblasts,promotes cholangiocyte maturation of hepatoblasts and suppresses proliferation of hepatoblasts by using Transwell co-culture method.3.TGF? signaling plays an important role in the regulating of IHBDdevelopment in vivo and may control IHBD development through Jag1-Notch-Sox9 signaling pathway. |
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