The Role And Mechanism Of CD73 In Endometrial Regenerative Cells Mediated Inhibition Of Mouse Cardiac Allograft Rejection

Background:At present,organ transplantation has become an effective treatment for some patients with end-stage diseases and congenital organ defects.The development and application of immunosuppressive agents have played an indispensable role in organ transplantation.Although the existing immunosuppressive schemes have been continuously optimized in the direction of individualization and combination of drugs,they still bring many immunosuppressant-related complications,including infection,tumor,organ injury and so on.At this time,the finding of Mesenchymal stem cells(MSCs)bring a new choice for the treatment of transplantation rejection.MSCs are multipotent stem cells which can differentiate to multiple lineages including adipocytes,osteoblasts and chondrocytes.Numerous studies have demonstrated that MSCs can suppress the body?s immune function and have a beneficial in the process of organ transplantation.Compared to immunosuppressive drugs,MSCs can be more safety and avoid long-term application.However,there are some limitations such as the invasive harvesting procedure and related complications,limited proliferation capacity and less availability,which hinder the widespread use of MSCs.Endometrial regenerative cells(ERCs)are mesenchymal-like stromal cells which have the same immune regulating properties as MSCs and can be easily isolated from human menstrual blood.Existing studies have also demonstrated that human ERC-based therapy can induce cardiac allograft tolerance.As for the mechanisms of the ERCs,it is not so clear.So,further studies need to be performed to explore the exact mechanisms of ERCs.Interestingly,we find ecto-5'-nucleotidase,known as CD73 which is a rate-limiting enzyme during the generation of extracellular adenosine(ADO),are highly expressed on the surface of ERCs.In addition,CD73.is a potent immunosuppressive factor,for example:the combination of ADO with its four down-stream receptors has been proved to have the function of preventing excessive immune reactions and immune-mediated tissue damage.Thus,the mechanism study of CD73 expression on ERCs mean to the regulation function of ERCs is critical to guide its clinical use.So,this study focuses on the mechanism of CD73 expression on ERCs in cardiac allograft transplantation.Objective:Isolate and purify ERCs,and confirm CD73 expression on the surface of ERCs;Examine the catalytic function of the CD73 expressed on ERCs and explore the effect of CD73 expression on ERCs in regulating dendritic cells(DCs),macrophages and regulatory T cells(Tregs)in vitro.To investigate the mechanism of CD73 expression on ERCs in prolonging the survival time of allogeneic heart graft and inhibiting immune rejection.Methods:This study was made up of three parts.1)Part one:ERCs were extracted from the menstrual blood of healthy female volunteers and cultured in vitro.The cell phenotype of ERCs was identified by flow cytometry analysis.Fluorescence staining was used to confirm the expression of CD73on the surface of ERCs.2)Part two:Pi Color Lock^(TM)Phosphate Detection Reagent was used to detected the catalytic capacity of CD73 expressed on the ERCs.3)Part three:Co-cultures of ERCs with allogeneic splenocytes were used to examine the effects of CD73 expressing ERCs have on regulating DCs,macrophages and Tregs in vitro.The changes in cell population were detected by flow cytometry analysis.Results:Part one:The mesenchymal-like ERCs were successfully extracted from the menstrual blood of the volunteers and achieved amplification in vitro.The identification showed that ERCs had a high positive rate in CD90,CD105 and CD73,but a low positive in CD39.The immunofluorescence also showed that CD73 could express on the surface of ERCs.Part two:CD73 which expressed on ERCs could catalyze AMP to ADO in vitro and the function of CD73 could be blocked by anti-CD73 m Ab.After CD73 on the surface was blocked,its ability to inhibit proliferation of DCs,and to promote differentiation of M2 and Tregs would be suppressed.Part three:The mouse heterotopic cardiac allograft transplantation model was established successfully.The data also showed that the blockage of CD73 expressed on ERCs would suppress the influences of ERCs in prolonging the survival time of the graft,alleviating the pathological changes of the graft,decreasing the infiltration of CD4⁺and CD8⁺cells,promoting proliferation of Tol-DC,M2,and Tregs,reducing the secretion of pro-inflammatory factors IFN-?and TNF-?,increasing the secretion of anti-inflammatory factor IL-10 and promoting the expression of A_2B receptors in heart tissue.Conclusion:Through the three parts of experimental research,we could conclude that:1)The phenotypic stable ERC can be extracted from the menstrual blood of healthy female volunteers,and the surface of ERC can stably express CD732)In vitro,CD73 expressed on the surface of ERCs can catalyze the decomposition of AMP into ADO,and the function of CD73 can be blocked by anti-cd73 monoclonal antibody.Meanwhile,the expression of CD73 is extremely important for ERC to inhibit the maturation of DCs and promote the proliferation of M2 and Tregs.3)CD73 expressing ERCs could inhibit the rejection of allograft in mice and prolong the survival of transplanted heart grafts.Therefore,the successful implementation of this project could provide theoretical and practical basis for the clinical transformation and standardization of ERCs.