Transforming Growth Factor-B-Expressing Mesenchymal Stem Cells Induce Local Tolerance In A Rat Liver Transplantation Model Of Acute Rejection

As we all know,end-stage liver disease in China has a high incidence.Liver transplantation is the only effective menthod for the treatment of end-stage liver disease.Immune rejection of transplanted liver is an unavoidable problem and the biggest challenge of liver transplantation?Although the use of immunosuppressive agents can reduce the occurrence of rejection,it also inevitable brought some complications.Recent years,induced spontaneous immune tolerance,with less or without immunosuppressive agents,has become one of the hot spots of immunology research.Naive regulatory T cells(Tregs),which has a strong immunosuppressive effect,have been reported to induce immune tolerance in allogeneic organ transplantation.Trees can be induced from T cells in vivo and in vitro by transforming growth factor(TGF-?).T cells induced Tregs(iTreg)have similar phenotypic and functional properties to nTregs,and can differentiate into Thl and Th17 cells and inhibit the differentiation and development of Th17 cells.The production of iTreg in transplanted liver need two main conditions:1.Sufficient T cells,2.A suitable vector capable of stably expressing the TGF-? gene.This is the most suitable carrier mesenchymal stem cells(MSCs).In this study,we aimed to investigate the therapeutic potential of TGF-?-overexpressing MSCs in inducing a local immunosuppression in liver grafts after transplantation.Part I:Construction of TGF/MSCs system and study of the effect of TGF/MSCs on iTreg generation in vitroObjective:To construction of TGF/MSCs system and study of the effect of TGF/MSCs on iTreg generation in vitro.Methods:Rat MSCs were isolated by direct adherence culture.The cell growth curve was drawn.The cell surface markers of MSCs were detected by flow cytometry.MSCs were transduced with a lentiviral vector expressing the human TGF-?1 gene;The expressions of human an rat TGF-? were detected by ELISA.The immunosuppressive effect of TGF-? transuded MSCs cells(namely TGF/MSC)was mesured by detecting lymphocytes proliferation,IFN-? expresion and the production of iTreg.Results:The MSCs were successfully isolated by direct adherence method and further purifiedby cell passage.The positive rate of surface markers CD44 was 98.12%on the third generation MSCs.At the same time,we successfully constructed the lentivirus carrying human TGF-? gene.Compared to MSC group,TGF/MSCs group had stronger inhibitory effect on the proliferation of lymphocytes and IFN-?expression.Meanwhile,TGF/MSCs can induced more iTreg generation in vitro than MSCs group.Conclusion:MSCs can be isolate and culture by direct adherence method,and with high purity and biological stability.The TGF-? gene can be stably transfected into MSC cells by lentiviral vector.TGF-? gene modified MSCs obtained a stronger immunosuppressive effect in vitro.Part 2:Effect of TGF/MSCs on iTreg generation in vivoObjective:To investigate the effect of TGF-?1 gene modified MSCs on immune rejection induced by allogeneic liver transplantation in rats,and to explore its mechanism of immune tolerance.Methods:A stable orthotopic liver transplantation model was established.MSCs or TGF/MSCs were injected through the portal vein after liver reperfusion.Thesurvival curve was drawn.The levels of TGF-?,IL-1?,IL-6,IL-10,I1-17,Helios and IFN-y in the liver tissue and serum of the recipients were detected by ELISA and RT-PCR.The expression of MSC in the liver was detected by immunofluorescence.The expressions of CD4+CD25+Foxp3+Tregs and iTregs generation were detected by flow cytometry.Results The survival curve showed that the TGF/MSCs group had longer survial than the other groups.Compared with MSC group and LV-TGF group,TGF/MSCs decreased acute rejectioan and attenuated liver damage after liver transplantation.Furthermore,TGF/MSCs can also more effectively inhibited T cell proliferation than LV-TGF group and MSCs group.The expressions of proinflammatory cytokines such as IL-1,IL-6 and IFN were significantly lower in TGF/MSCs group.While the inflammatory suppression factors IL-10 was just the opposite.Compared to MSCs group,there were more MSCs infiltrated into liver graft in TGF/MSCs group.The number of Tregs in TGF/MSCs group was the lowest on the first day after operation,but the Treg increased gradually with the decrease of inflammatory reaction.However,the number of Tregs was higher in TGF/MSCs group after day 5 than other groups.As a distinction between nTreg and iTreg,Hellos+is a marker for nTreg,while iTregs is Helios negative.At day 7 after liver transplantation,the expressions of Helios negative Tregs were increased while TGF/MSCs group have more Helios negative Tregs than LV-TGF group and MSCs group In the TGF/MSCs group,the liver had the most iTreg than blood and spleen.Conclusion TGF/MSCs can induce a local immunosuppressive effect in liver grafts after transplantation through suppressing T cell proliferation and inducing the production of iTreg.The TGF/MSCs-induced iTreg was mainly confined to the transplanted liver.