Molecular Mechanism Of Heme Oxygenase-1 Modified BMMSCs Combined With Normothermic Machine Perfusion To Improve The Prognosis Of DCD Liver Transplantation

Objective:To investigate the molecular mechanism of heme oxygenase-1(HO-1)modified bone marrow mesenchymal stem cells(BMMSCs)combined with normothermic machine perfusion(NMP)preservation of donation after cardiac death(DCD)liver in improving the prognosis of transplant recipients the study.Methods:1.BMMSCs were were isolated from rat bone marrow by adherent method,and identify BMMSCs by cell morphology,differentiation potential,and biomarkers;2.BMMSCs were modified with the adenovirus for HO-1 gene and verified HO-1gene expression of HO-1/BMMSCs;3.Establish a rat DCD model,obtain DCD donor liver,and establish a stable NMP system for rats;4.Use the NMP system to infuse the DCD liver with HO-1/BMMSCs,and complete the orthotopic liver transplantation in rats;5.For the study of liver ischemia reperfusion injury(IRI),rats of the same strain were used for rat liver transplantation.The experiment was divided into 5 groups according to the different treatment methods of the liver:Sham operation(Sham)group,static cold storage(SCS)group,NMP group,BMMSCs combined with NMP(BMP)group,HO-1/BMMSCs combined with NMP(BMP)group.Liver enzyme and cytokine levels,liver histology,and expression of Toll-like receptor 4(TLR4)pathway-related molecules were evaluated;6.For the study of acute rejection,different strains of rats were used to complete the acute rejection model of liver transplantation.The experiment was divided into 6 groups according to the treatment of the liver:Sham group,SCS group,NMP group,BMP group,BMP group,NMP group combined postoperative application of tacrolimus(FK506)group.The liver,spleen,and serum were collected at 7 and 14 days post-transplantation.The severity of acute rejection was evaluated based on the liver histopathology.Gene chip was used to detect differential gene expression,and flow cytometry was used to detect the NKT cells.Results:1.The BMMSCs obtained by the adherence method,which confirmed that the BMMSCs prepared by us conform to international standards.HO-1/BMMSCs were successfully obtained by adenovirus transfection;2.NMP can colonize BMMSCs in the donor liver during in vitro preservation,and the colonization of HO-1/BMMSCs in the liver grafts was better than BMMSCs after liver transplantation;3.The research results of IRI in the same strain of rat transplantation model was that,after liver transplantation,the SCS group showed significantly increased transaminase levels,liver tissue damage,and shorter survival time(2.5 d).The HMP group showed lower transaminase levels,prolonged survival time(>60 d),and decreased serum and liver proinflammatory cytokine levels(P<0.05).Compared with the recipients in the NMP and SCS groups,the expression of HMGB1 of mononuclear cells and liver tissues in the HMP and BMP groups was significantly reduced(P<0.05),and the expression of TLR4 signaling pathway marker proteins was significantly reduced(P<0.05),and the indicators in the HMP group were significantly lower than those in the BMP group(P<0.05);4.The results of the study in the acute rejection liver transplantation model were:when the donor liver contained HO-1/BMMSCs,the acute rejection was obviously controlled,and the survival time was significantly prolonged.The application of HO-1/BMMSCs reduces the number of NKT cells in the liver following transplantation,increases the expression of the NKT cell co-inhibitory receptors,BTLA and CD160,and reduces the level of NKT cell expression of IFN-γ.Thus,NK cell and CD8~+T cell activation was inhibited,which reduced acute of rejection of the transplanted liver.Conclusions:1.NMP provides a new method for the application of BMMSCs,and HO-1 gene modification enhances the function of BMMSCs;2.HO-1/BMMSCs combined with NMP reduces the IRI of DCD liver grafts,and the mechanism is related to the regulation of the inflammatory response pathway mediated by TLR4/NF-кb;3.HO-1/BMMSCs perfused by the NMP system reduces the number of NKT cells in the liver grafts and up-regulates the expression of NKT cell co-inhibitory receptors,which results in inhibiting signal transmission to NKT cells and reducing NKT cell IFN-γlevels.