ABSTRACT
Objective@#To investigate the protective effect of bone marrow mesenchymal stem cells (BMMSC) combined with normothermic mechanical perfusion (NMP) on biliary epithelial cells (BEC) in rats receiving donation after cardiac death (DCD) donor liver transplantation.@*Methods@#The BMMSC were isolated from male Sprague-Dawley (SD) rats aged 2-3 weeks and weighing 40-60 g, and then cultured, identified and expanded to the third generation in vitro. Male SD rats aged 6-8 weeks and weighing 200-220 g were divided into sham-operated group (Sham group), static cold storage (SCS group), simple NMP group (NMP group) and BMMSC combined with NMP group (BMMSC+NMP group) by random number table method with 44 rats in each group. The DCD donor liver transplantation models in rats were reproduced with 30-minute warm ischemic time. While the rats in Sham group merely received perihepatic ligaments-separation, which did not affect their liver blood supply, and then their incisions were sutured after 30 minutes. The DCD donor grafts in SCS group were preserved in the University of Wisconsin (UW) cold storage solution for 4 hours. While the DCD donor grafts in the NMP group and the BMMSC+NMP group were perfused with the DMEM/F12-based culture solution or combined with BMMSC for 4 hours through the established ex vivo NMP system. The orthotopic liver transplantation model was reproduced, and the survival rate of the recipients was observed at 0, 1, 7 and 14 days after liver transplantation. The biochemical liver function of rats in different groups was determined at each time point after operation. The morphological changes in bile ducts of liver grafts were observed by hematoxylin-eosin (HE) staining, and the expression of cytokeratin 19 (CK19) was determined qualitatively by immunohistochemistry and quantitatively by Western Blot after protein extraction from BEC in liver samples.@*Results@#The morphology, differentiation function and phenotypic identification of BMMSC confirmed that the stem cells used in this experiment were standard BMMSC. The survival rates of rats in the NMP group and the BMMSC+NMP group were significantly higher than that in the SCS group at 0, 1, 7 and 14 days after operation. The increase was more significant in the BMMSC+NMP group, with 100% on postoperative day (POD) 0, and the 14-day survival rate was still significantly higher than that in the SCS group and the NMP group [80.0% (16/20) vs. 20.0% (4/20), 70.0% (14/20), both P < 0.05]. As the time after liver transplantation prolonged, the liver function parameters of rats in the SCS group were deteriorated gradually, which reached the peak at 1-7 days after operation. The damage of biliary tissue increased gradually under the microscope, and the injury was most serious on POD 7 in the SCS group, showing a lot of balloon-like changes in hepatocytes, with obvious bile duct dilatation accompanied by large area inflammatory cell infiltration. Immunohistochemistry and Western Blot showed that the expression of CK19 in BEC cytoplasm was decreased gradually in the SCS group, reached the lowest on POD 7, and then gradually increased. The BMMSC+NMP group and the NMP group were significantly better than the SCS group in terms of liver function, pathological injury of biliary tract and CK19 expression in BEC, and the improvement was more significant in the BMMSC+NMP group. These results suggested that the protective effects of BMMSC combined with NMP on BEC was significantly better than that of the SCS and NMP.@*Conclusion@#Preservation of rat DCD donor liver by BMMSC combined with NMP can reduce the BEC injury after liver transplantation significantly, thus improving both the prognosis and the survival rate after transplantation.
ABSTRACT
Objective To investigate the protective effect of bone marrow mesenchymal stem cells (BMMSC) combined with normothermic mechanical perfusion (NMP) on biliary epithelial cells (BEC) in rats receiving donation after cardiac death (DCD) donor liver transplantation. Methods The BMMSC were isolated from male Sprague-Dawley (SD) rats aged 2-3 weeks and weighing 40-60 g, and then cultured, identified and expanded to the third generation in vitro. Male SD rats aged 6-8 weeks and weighing 200-220 g were divided into sham-operated group (Sham group), static cold storage (SCS group), simple NMP group (NMP group) and BMMSC combined with NMP group (BMMSC+NMP group) by random number table method with 44 rats in each group. The DCD donor liver transplantation models in rats were reproduced with 30-minute warm ischemic time. While the rats in Sham group merely received perihepatic ligaments-separation, which did not affect their liver blood supply, and then their incisions were sutured after 30 minutes. The DCD donor grafts in SCS group were preserved in the University of Wisconsin (UW) cold storage solution for 4 hours. While the DCD donor grafts in the NMP group and the BMMSC+NMP group were perfused with the DMEM/F12-based culture solution or combined with BMMSC for 4 hours through the established ex vivo NMP system. The orthotopic liver transplantation model was reproduced, and the survival rate of the recipients was observed at 0, 1, 7 and 14 days after liver transplantation. The biochemical liver function of rats in different groups was determined at each time point after operation. The morphological changes in bile ducts of liver grafts were observed by hematoxylin-eosin (HE) staining, and the expression of cytokeratin 19 (CK19) was determined qualitatively by immunohistochemistry and quantitatively by Western Blot after protein extraction from BEC in liver samples. Results The morphology, differentiation function and phenotypic identification of BMMSC confirmed that the stem cells used in this experiment were standard BMMSC. The survival rates of rats in the NMP group and the BMMSC+NMP group were significantly higher than that in the SCS group at 0, 1, 7 and 14 days after operation. The increase was more significant in the BMMSC+NMP group, with 100% on postoperative day (POD) 0, and the 14-day survival rate was still significantly higher than that in the SCS group and the NMP group [80.0% (16/20) vs. 20.0% (4/20), 70.0% (14/20), both P < 0.05]. As the time after liver transplantation prolonged, the liver function parameters of rats in the SCS group were deteriorated gradually, which reached the peak at 1-7 days after operation. The damage of biliary tissue increased gradually under the microscope, and the injury was most serious on POD 7 in the SCS group, showing a lot of balloon-like changes in hepatocytes, with obvious bile duct dilatation accompanied by large area inflammatory cell infiltration. Immunohistochemistry and Western Blot showed that the expression of CK19 in BEC cytoplasm was decreased gradually in the SCS group, reached the lowest on POD 7, and then gradually increased. The BMMSC+NMP group and the NMP group were significantly better than the SCS group in terms of liver function, pathological injury of biliary tract and CK19 expression in BEC, and the improvement was more significant in the BMMSC+NMP group. These results suggested that the protective effects of BMMSC combined with NMP on BEC was significantly better than that of the SCS and NMP. Conclusion Preservation of rat DCD donor liver by BMMSC combined with NMP can reduce the BEC injury after liver transplantation significantly, thus improving both the prognosis and the survival rate after transplantation.
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Objective To investigate the inhibitory effect of 5-FU on human intrahepatic biliary epithelial cells with TGF-β1-induced mesenchymal transition and potential mechanism.Methods The primary epithelial cells from human intrahepatic bile ducts were cultured,the cells were identified by immunofluorescence microscopy.The cells were randomly divided into 3 groups: normal control group,TGF-β1 group and TGF-β1+5-FU group.The expression of CK-19,E-cadherin,vimentin and α-SMA protein were measured by immunofluorescence microscopy and Western blot assay.Western blot was used to detect the expression levels of TGF-β1.Real-time PCR to detect mRNA expression of TGF-β1,Ⅰand collagen type Ⅲ.Results The primary epithelial cells of intrahepatic bile ducts were successfully cultured.In TGF-β1 group,the protein expression of vimentin,α-SMA and the mRNA expression levels ofⅠand collagen type Ⅲ were significantly higher than that of normal control group(P0.05),TGF-β1 protein and mRNA expressions were significantly lower as compared with that of normal TGF-β1 group(P<0.05).Conclusions 5-FU could inhibit TGF-β1-induced biliary epithelial-mesenchymal transition with down-regulated expression of TGF-β1.
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Objective To investigate the effects of IL-6/STAT3 activation on the proliferation of biliary epithelial cell following liver transplantation in rat.Methods Based on the classic "two-cuff" technique for orthotopic liver transplantation in rat,end-to-end anastomosis between the common hepatic arteries of donor and recipient by the modified "Gao stent" was performed to reconstruct hepatic artery blood flow.Wistar rats were then randomly divided into CP1h group and CP12h group(grafts were preserved in UW solution at 4℃ for 1h and 12h respectively),RPM group(CP12h group treated by rapamycin,RPM)and C group(control).At 1,3,7,14d after operation,liver IL-6 mRNA expression was analyzed by real-time RT-PCR.STAT3 activation was determined using laser confocal scan microscopy(LSM).BEC proliferation was detected by immunohistochemistry.Results In the CP1h group,expression of IL-6 mRNA was upregulated one day after operation(0.41?0.03),and then fell to the C group level subsequently(0.28?0.03,0.23?0.03 and 0.27?0.05 at 3,7 and 14d after operation).STAT3 activation in the BEC(94?8,61?6,39?4 and 34?3)and proliferation of the BEC following liver transplantation(2.1%?0.3%,5.9%?0.5%,2.6%?0.5% and 2.3%?0.5%)showed a similar trend.Compared with the CP1h and C group,the CP12h group demonstrated a significant and persistent up-regulation of IL-6 mRNA(0.60?0.03,0.73?0.02,0.38?0.02 and 0.30?0.04)and STAT3 activation(167?17,247?13,110?9 and 74?8).BEC proliferation in CP12h group were 7.0%?0.5%,27.8%?1.8%,23.1%?1.6% and 17.8%?1.2%,there was a direct correlation between IL-6 mRNA expression and STAT3 activation(r=0.95,P