The effect of optimized normothermic mechanical perfusion on biliary epithelial cells in liver graft by preserving the donation after cardiac death in rats / 中华肝胆外科杂志

ABSTRACT

Objective To study the effect of bone marrow mesenchymal stem cells (BMMSCs) combined with normothermic mechanical perfusion (NMP) on biliary epithelial cells (BEC) after DCD donor liver transplantation in rats.Methods The third generation of BMMSCs and the BMMSCs modified by Ad/HO-1 (Ad/HO-1/BMMSCs) were cultured,identified and expanded in vitro.To establish a stable NMP system device in vitro.The DCD liver transplantation models were constructed in rats after cardiac ischemia for 30 minutes,220 SD recipient rats were randomly divided into sham operation group (S group,n=44) static cold storage (SCS group,n =44) group,and simple NMP group (P group,n =44),BMMSCs combined with NMP group (BP group,n =44) and BMMSCs modified by Ad/HO-1 combine with NMP group (HBP group,n =44),NMP group,BP group and HBP group were subjected to vitro perfusion for 4h.The group were taken at 0,1,7 and 14 days after transplantation and the relevant indicators were detected,n =6 in each group.The survival rate of the recipient rats,liver function and pathological changes of the bile duct were observed.The expression of cytokeratin 19 (CK19) protein in BEC was detected by immunohistochemistry and Western blot.Apoptotic biliary epithelial cells were detected by TUNEL staining and the expression of apoptosis-related protein caspase-3 was detected by immunohistochemistry.Results The survival time of HBP group was significantly prolonged for (5.6 ±0.8) d in SCS group vs.(18.4 ±2.0) d in NMP group,(20.5 ± 1.5) d in BP group,(82.5 ±3.2) d in HBP group,the differences were statistically significant (all P ＜ O.05).Compared with other groups,the HBP group and the BP group were significantly improved in liver function and biliary pathology,and the expression of CK19 protein in BEC was significantly increased [(0.81 ±0.02) in S group vs.(0.35 ±0.03) in SCS group,(0.47 ±0.02) in NMP group,(0.63 ± 0.02) in BP group,(0.77 ± 0.01) in HBP group on postoperative day (POD) 14],the differences were statistically significant (all P ＜ 0.05).The number of apoptosis and the expression of apoptosis-related protein caspase-3 in HBP group were significantly decreased [(10.0 ± 1.2) in S group vs.(57.3 ±5.5) in SCS group,(40.1 ±4.6) in NMP group,(32.0 ± 2.2) in BP group,(13.7 ± 3.1) in HBP group on POD 14],the difference was statistically significant (all P ＜ 0.05).Compared with the BP group,the protective effect of the HBP group was more obvious,and the difference was statistically significant (P ＜ 0.05).Conclusion By the method of the BMMSCs modified by Ad/HO-1 combined with NMP in vitro preservation of rat,DCD donor liver can significantly improve the effect of BEC on rats and the survival rate after liver transplantation.