| Objectives:Mesenchymal stem cells have high research and application value in the field of transplantation immunity with their low immunogenicity and immunosuppression.However,the effect of using mesenchymal stem cells alone on organ transplantation is not ideal.In clinical practice,mesenchymal stem cells are mostly used together with immunosuppressive agents to treat the immune rejection.However,long-term use of immunosuppressive agents will cause inevitable side effects.Therefore,improving the immunosuppressive effect of mesenchymal stem cells has become an ideal way to solve the immune rejection after organ transplantation.PD-L1 is one of the ligands of PD-1,and plays an important role in negative immunoregulation.Whether mesenchymal stem cells with overexpression of PD-L1 can inhibit graft rejection has not been reported.In this study,By means of lentivirus infection to study the effects of mesenchymal stem cells with overexpression of PD-L1 on the immune rejection of recipient mice.Methods:Primary mesenchymal stem cells were isolated from umbilical cord and cultured,and then identified immunophenotypes and differentiation functions.Through gene recombination method,we constructed lentiviral recombinant vector with green fluorescent protein(GFP).By flow cytometry we detected the expression of the target gene PD-L1 in the human umbilical cord mesenchymal stem cells.By ConA-stimulated lymphocyte proliferation and mixed lymphocyte response experiments,we detected the inhibition of mesenchymal stem cells with overexpression PD-L1 on lymphocyte proliferation.Human umbilical cord mesenchymal stem cells were transplanted under the renal capsule to detect the survival of human umbilical cord under the renal capsule in mice.On the 6th day after human umbilical cord mesenchymal stem cell transplantation,serum,spleen and kidney tissues of the recipient mice were collected and analyzed by ELISA,qRT-PCR,flow cytometry and histology to study the effect of PD-L1 overexpression on the immune rej ection of the recipient mice.Results:We isolated a large number of mesenchymal stem cells easily from human umbilical cord.The cell purity was high,the morphology was uniform,and had the MSCs morphological characteristics.Flow cytometry results showed that the MSCs had high expression of CD73,CD90,CD105,and low expression of CD14,CD34,CD45 and HLA-DR,which were consistent with the marker characteristics of mesenchymal stem cells.The results of induced differentiation in vitro showed that we isolated and cultured mesenchymal stem cells could differentiate into bone,cartilage and adipose cells.After transfection with lentivirus recombinant vector,MSCs can stably express PD-L1 protein.In vitro lymphocyte proliferation and mixed lymphocyte response experiments showed that mesenchymal stem cells with overexpression PD-L1 protein had a stronger inhibitory effect on ConA-stimulated lymphocyte proliferation and mixed lymphocyte response.In vivo living imaging of small animals showed that GFP could last for 1 week under the renal capsule,indicating that we successfully constructed the mesenchymal stem cells renal subcapsular transplantation model,and mesenchymal stem cells could be well survived under the renal capsule of mice.In vivo test results showed that PD-L1-MSCs group compared with MSCs group,the proportion of CD4+T cells have a significant reduction(P<0.05).There was no difference in the proportion of B cells,CD8+T cells and Treg cells between the two groups(P>0.05).In ELISA and qRT-PCR results,the content of IFN-y decreased in PD-L1-MSCs group compared with MSCs group(P<0.01).Conclusions:PD-L1 overexpression enhanced the inhibitory effect of human umbilical cord mesenchymal stem cells on lymphocyte proliferation,and enhanced the immunosuppressive ability of mesenchymal stem cells in transplantation immunity.Our research providing a new idea for the application of mesenchymal cells in transplantation immunity... |
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