The Effect Of Bone Marrow Mesenchymal Stem Cells Modified By Heme Oxygenase-1 Combined With Normathermic Mechanical Perfusion On Biliary Epithelial Cells In Rat Liver Graft After Cardiac Death

Objective:To study the effect of bone marrow mesenchymal stem cells（BMMSCs）modified by heme oxygenase-1（HO-1）combined with normothermic mechanical perfusion（NMP）on biliary epithelial cells（BEC）after DCD donor liver transplantation in rats.Metheds:1.SPF level healthy male SD rats of 2～3 weeks,40～60 g were removed by spinal cord dissection,the femur and tibia were extirpated under aseptic conditions,and cultured to the third generation in an incubator at 37℃and 5%CO₂ using the whole bone marrow adherency screening method.The differentiation ability,growth morphology and surface specific markers of BMMSCs were identified by flow phenotype.2.The third generation BMMSCs were transfected with adenovirus containing HO-1 gene to construct the bone marrow mesenchymal stem cell model transfected with HO-1（Ad-HO-1/BMMSCs）.3.To establish a stable NMP system device in vitro.4.DCD liver transplantation model for 30 minutes of hot ischemia after cardiac death was established in male SD receptor rats aged 6～8 weeks with a body weight of 200～220 g,and the SD recipient rats were random Ly divided into sham operation group（S group）,static cold storage（SCS group）,simple NMP group（P group）,BMMSCs combined with NMP group（BP group）and BMMSCs modified by Ad/HO-1 combine with NMP group（HBP group）,NMP group,BP group and HBP group were subjected to vitro perfusion for 4 h.The groups were taken at 0,1,7 and14 days after transplantation and the relevant indicators were detected.5.The status and survival rate of the recipient rats were observed,and biochemical methods were used to detect liver function in each group of rats,and bile duct pathological changes in each group were analyzed by hematoxylin-eosin staining.Cytokeratin 19（CK19）was detected by Western blotting and immunohistochemical staining.The expression of BEC was determined by Td T-mediated d UTP Nick-End Labeling（TUNEL）staining,and the expression of caspase-3 was detected by immunohistochemistry.Results:1.The morphology,differentiation ability and phenotypic identification of BMMSCs and Ad-HO-1/BMMSCs confirmed that the stem cells used in the experiments were standard BMMSCs and Ad-HO-1/BMMSCs.2.The NMP systemdevices were successfully established in vitro and stably operated.3.Orthotopic liver transplantation model of DCD in male SD rats with warm ischemia after cardiac death for 30 minutes was successfully constructed in vitro.4.The survival rate of rats in HBP group and the BP group was significantly higher than that in the SCS group at 0,1,7 and 14 days after operation.The increase was more significant in the HBP group,with 100%on postoperative day（POD）1,and the14-day survival rate was still significantly higher than that in the SCS group,the NMP group and the BP group[90%（18/20）vs.20%（4/20）,40%（8/20）,45%（9/20）,both P<0.05].Pathological examination of transplanted liver of SD recipient rats in each group on day 7～14 after liver transplantation showed that Ad-HO-1/BMMSCs combined with NMP to protect DCD donor liver could significantly reduce the inflammatory damage of bile duct epithelial cells.Subsequently,our study found that the expression of CK19 protein in the HBP group and BP group was significantly higher than that in the SCS group and NMP group,while the expression of CK19protein in the HBP group from day 7 to day 14 was higher than that in the BP group,and the expression of CK19 protein in the HBP group was not significantly changed compared with that in the S group at each time point.By detecting the apoptotic bile duct epithelial cells,it was found that the number of apoptotic cells in BEC in the SCS group and NMP group was significantly increased compared with that in the HBP group,and the number of apoptotic cells in the BP group was further increased compared with that in the HBP group from day 7 to day 14.These results suggested that the protective effect of the BMMSCs modified by HO-1 gene combined with normothermic mechanical perfusion on BEC was significantly better than that of the SCS group,the NMP group and the BP group.Conclusion:1.The BMMSCs extracted and cultured from the bone marrow of male SD rats are the BMMSCs required for this experiment.The method of transferring the HO-1 gene into BMMSCs to construct Ad-HO-1/BMMSCs is feasible,and provided the cytological basis for subsequent experiments.2.A stable in vitro NMP system and rat orthotopic liver transplantation model were successfully constructed to provide animal model basis for following experiments.3.Preservation of rat DCD donor liver by BMMSCs modified by HO-1 gene combined with NMP can significantly reducethe BEC injury after liver transplantation,thus the prognosis and survival rate after transplantation.