| Objective: To investigate the relationship between activation of Jagged 1/ Notch signaling pathway and epithelial-mesenchymal transition of cholangiocytes after rat liver transplantation.Method: 1) 96 male specific pathogen free Sprague-Dawley rats weighing 250-300 gram were divided into three groups randomly, including liver transplantation group (48 rats), sham operation group (24 rats) and control group (24 rats). The rat liver transplantation models were established by modified Kadama's"two-muff"technique in which the hepatic arteries were ligated without anastomosis. Weight of recipients were higher than donors. The rats in control group received no treatment while those in sham group only received liver dissociation. Venous blood was collected to detect total bilirubin (TBIL),γ-glutamyl transpeptidase (γ-GT) and glutamic-oxaloacetic transaminase (ALT) on 1d, 7d, 14d and 28d postoperation respectively. Immediately, rats were sacrificed and the livers were obtained to receive morphological and immunological assay. The pathological morphology assay was performed by hematoxylin-eosin staining. Expression of molecules of Notch signaling (Jagged 1 and Hes1), mesenchymal cells marks fibroblast specific protein-1(FSP-1), vimentin,α-SMA and biliary epithelial marker (cytokeratin19, CK19) were detected by immunohistochemistry and immunoblotting. 2) In vitro, primary intrahepatic biliary epithelial cells were isolated from SD rats. Cells were identified by immunostaining of CK19 and the viability was evaluated by trypan blue exclusion. Following construction of eukaryon expression vector pEGFP-IRES2-Jagged1, the cells were transfected with Jagged1 or vehicle vector by Lipofectamine TM2000. The untreated group served as control. After successful transfection for 24 hours, the inhibitor of Notch signaling, DAPT (1μmol/L) was added in the Jagged1 group. Followed by incubation for another 72 hours, the expressions of Jagged 1, Hes1, FSP-1, vimentin,α-SMA and CK19 in the cells were detected by western-blot. Cell morphous was observed by light inverted microscope and migration potency was evaluated by Transwell assay.Result: Rat orthotopic liver transplantation models were established successfully and recipients were survival well under normal diets. Obviously cholestasis occurred in rats of transplantation group. TBIL (32.05±3.28μmol/L) andγ-GT (118.3±17.4μmol/L) (P<0.05) dramatically elevated on 14d, however, they descended gradually on 28d posttransplantation. Ductular reactions shown by hematoxylin-eosin staining represented an impairment of periportal region, including bile duct dilation, cholangiocytes hyperplasia and inflammatory cells infiltration. Brown granules shown by immunohistochemistry in cytolemma and cytoplasm of proliferative cholangiocytes declared a strong expression of Jagged 1 and Hes1. Compared with transplantation group, Jagged 1 and Hes1 were weakly expressed in normal bile ducts, periportal region, hepatocytic membrane and hepatic vein endothelial cells in control groups. On the other hand, in transplantation group, mesenchymal cell markers (FSP-1, vimentin, andα-SMA ) were also expressed as brown granules and diffuse distributed in proliferative cholangiocytes, as granules of Jagged 1 and Hes1 in transplantation group. However, in transplantation group the expression of CK19 was negative. We detected the expressions of Jagged 1, Hes1, mesenchymal cell markers (FSP-1, vimentin, andα-SMA) and CK19 at protein level by Western-blot. In control groups, Jagged 1, Hes1 and mesenchymal cell markers were expressed at low levels whereas CK19 was expressed at high level. However, 7 days after liver transplantation, Jagged 1, Hes1, FSP-1, vimentin andα-SMA were strongly expressed and upregulated with time on posttransplantation. Nevertheless, the exprssion of CK19 was obviously downregulated. In vitro, to overexpress Jagged1 we transfected rat intrahepatic biliary epithelial cells (rIBECs) with Jagged1 gene. Along with the strong expression of Jagged1, Hes1 and mesenchymal cell markers (FSP-1, vimentin,α-SMA) we also revealed expression of CK19 at low level. On the contrary, the cells treated with DAPT (the inhibitor of Notch signaling), and the control cells revealed the weak expression of Jagged1, Hes1, FSP-1,vimentin,α-SMA while high expression of CK19. In relative to biological behaviors, the IBECs transfected with Jagged1 revealed clostridial form whereas control rIBECs and rIBECs treated with DAPT revealed cobblestone shaped. Migration assay by Transwell demonstrated that the migration of rIBECs transfected with Jagged1 (21333±520, P<0.05) was significantly enhanced compared with the other three groups (2083±381, 2000±661 and 1833±389).Conclusion: In the early phase (≤4w) of rat orthotopic liver posttransplantation by modified Kadama's"two-muff"technique, ductular reactions represented impairment of bile ducts. During this process, Notch signaling pathway was activated and its activation might be involved in epithelial-mesenchymal transition of intrahepatic biliary epitheliums.
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