The Role Of B7-H1 High Expressing Endometrial Regenerative Cells In Inhibition Of Chronic Allograft Vasculopathy

OBJECTIVE: Isolation and identification of ERC from menstrual blood,observation of the expression of its surface programmed death receptor-ligand 1(B7-H1);establishment of aortic allograft mouse model;explore the high expression of B7-H1 in ERC for aortic transplantation Inhibition of arteriosclerosis,a graft vascular lesion in mouse models,and related mechanisms;The role of B7-H1 immunomodulatory molecules on the surface of ERC cells in inhibiting chronic allograft rejection in aortic transplantation.METHODS: 1)Month cup to collect menstrual blood from healthy women of childbearing age,using the standard Ficoll method to isolate mononuclear cells in menstrual blood,and conduct cell culture,and stimulate the upregulation of E7 surface B7-H1 with IFN-? stimulation.2)Male Adult BALB/c(H-2d)donor mice and C57BL/6(H-2b)recipient mice and male BALB/c(H-2d)donors collect 3-4 mm descending abdominal aortic segments An animal model of mouse aortic transplantation was established at the same location as the transplanted male C57BL/6(H-2b)receptor,and the technical success was defined as the absence of paralysis survival during the first 3 days after transplantation.3)Postoperative male C57BL/6(H-2b)abdominal aortic allograft mice were randomly divided into four groups(n=6,each group): Group 1;untreated group(untreated);Group 2;B7-H1 antibody pretreated ERC group;group 3,ERC treatment group;group 4,IFN-? pretreated ERC treatment group.All four mice had free diet and water.On the second postoperative day,well-grown fourth-generation ERC cells were transplanted.After tail vein injection of ERC(1×106/0.2 ml/body),anti-B7-H1 antibody pretreated ERC group was incubated with B7-H1 monoclonal antibody 0.5 ?g/106 cells for 30 min and injected via tail vein on the surface of ERC cells B7-H1 is closed before transplantation.Aortic allografts were collected on the 40 th day after transplantation.Transplanted aortic tissue sections(4?m)were stained with hematoxylin and eosin(HE)and pathological changes in the severity of chronic rejection of the grafted vessels were examined microscopically.The intimal thickness of the graft was measured by Image J software.The spleens of the mice in each group were collected,and the spleens were assayed for anti-CD3,CD4,CD8,CD11 c,CD25,CD68,CD86,CD206,Foxp3,Ig M,Ig G and MHC II,and mixed lymphocyte reaction(MLR)by flow cytometry.Analyze the changes in the above indicators.P<0.05 was considered statistically significant.RESULTS: 1)Endometrial regenerative cells(ERC)isolated from menstrual blood are MSC-like cells,and the ERC surface is capable of expressing an immunoregulatory molecule B7-H1 that plays an important role in inducing immune tolerance.At the same time,as the concentration of IFN-? stimulating factor is gradually increased,the expression of B7-H1 on the surface of ERC is also correspondingly increased,and is dose-dependent.2)Anastomosis of 20 aortic grafts was performed using this cuff-fitting technique.The implants were implanted on average 23 minutes(range,18-27 minutes).No graft vascular obstruction was collected when specimens were collected.3)In vivo experiments showed that tail vein injection of ERC has a significant protective effect on chronic rejection of aortic allograft in mice.Compared with untreated group,the number of CD3+CD4+T and CD3+CD8+ T cells in the spleen of ERC group was significantly reduced(P< 0.05),donor-reactive Ig M and Ig G antibodies were relatively decreased(P<0.05),the number of CD4+CD25+Foxp3+ Tregs cells was significantly increased(P<0.05),and the number of CD11c+MHC-II+ DCs cells was significantly decreased(P<0.05).Compared with ERC group,anti-B7-H1 antibody pretreatment ERC treatment group increased the number of splenic CD3+CD4+ T,CD3+CD8+ T cells,donor-reactive Ig M and Ig G antibodies(P<0.05),The number of CD4+CD25+ Foxp3+ Tregs cells was relatively decreased(P<0.05),and the number of CD11c+MHC-II+ DCs cells was relatively increased(P<0.05).Therefore,when the anti-B7-H1 antibody pretreated ERC,the ERC-induced aortic graft model was significantly reduced.The protective effect of chronic rejection in mice;Compared with the ERC group,the stimulator IFN-? increased the expression of B7-H1 on the surface of ERC,and the number of CD3+CD4+ T and CD3+CD8+ T cells in the spleen of ERC group further decreased(P<0.05).Donor-reactive Ig M and Ig G antibodies further decreased(P<0.05),The number of CD4+CD25+Foxp3+ Tregs cells increased further(P<0.05),and the number of CD11c+MHC-II+ DCs cells decreased significantly(P<0.05).Conclusion: IFN-? can significantly promote the high expression of B7-H1 on the surface of ERC cells in a dose-dependent manner.ERC can reduce the intimal hyperplasia of graft arteries and significantly improve the pathological manifestations of chronic rejection.ERC can inhibit the number of T cells,total macrophages and Ig M and Ig G levels;promote the number and function of Tol-DC,Tregs,M2 ratio,play an immunomodulatory role.B7-H1 plays a crucial role in the suppression of chronic rejection by ERC.