The Research Of The Immunomodulatory Effect Of Mesenchymal Stem Cells Toward The B Cells In The Rejection Of Acute Graft-Versus-Host Disease And Free Skin Allograft

Object Because of its immunomodulatory effect,mesenchymal stem cells(MSC)were applied in transplantation to reduce the dose of immunosuppressor.In the case of acute graft-versus-host disease(aGVHD),the immunomodulatory effect of MSCs on B cells has not been well demonstrated.We test activation markers and co-stimulatory molecules of B cells and cytokine,in order to study the role of MSC in the case of aGVHD and the role on B cells.After MSC infusion in vivo,few MSCs can reach the secondary lymphoid organs,which results in the immune regulation of MSCs in vivo is not as good as in vitro.In order to increase the targeting of MSCs to secondary lymphoid organs,MSCs were overexpressed CCR7,and whether this kind of MSCs could alleviate the rejection of rat allogeneic skin grafts and how this kind of MSC react towards B cells in the rejection.Theoretical support for clinical reduction of transplant immune rejection was provided.Method Part I:Bone marrow MSC from C57 mouses' femur and tibia marrow cavity was isolated using whole bone marrow adherence method,and the cell surface markers and multi-inducible differentiation were identified.Part II:The mouse aGVHD model was established.The experimental group's infusion was combined with 1×106 MSCs.The meau survival time of the mice was observed.The alloreactive cytotoxic assay of the recipient spleen cells on P815 cells was taken on the fifth day after operation.At 12h,24h,D3 and D5,receptor spleen cells were used to detect changes in lymphocyte subsets and their activation markers(CD69)and costimulatory molecules(CD86).B cell stimulating factor IL-4 and Bregs regulatory factor IL were detected.-10 mRNA content.Part ?:B220-spleen mononuclear cells were obtained by immunomagnetic bead sorting system,and mouse aGVHD model was established by using this kind of cells.The experimental group was combined with co-infusion of 1×106 MSCs to observe the average survival time of mice.The expression of activation markers(CD69)and costimulatory molecules(CD86)of lymphocyte subsets were detected by the 24 h receptor spleen cells.Part IV:packaging lentivirus expressing rat CCR7,measuring the lentivirus titer of packaging;using this lentivirus to transfect C3H10T1/2 cells,detecting the immunophenotype,proliferation and multi-directional differentiation of MSC-CCR7 Biological characteristics such as ability.Part V:stablished of rat allogeneic back free skin graft model,divided into three groups,blank control group,MSC-CCR7 infusion group and MSC-GFP infusion group,all the groups got infusion within 24 hours before transplantation.The group only infused 0.5ml PSB,the other two groups infused 2×106 corresponding cells;opened the bandage 7 days after surgery,observed the survival time of the skin,and performed skin biopsy in D7,D10,D14;lymphocytes and the spleen mononuclear cells in D3,D7,D10 and D14 for T cell activation(CD3+CD69),Treg subset,B cells activation(CD45R+CD69)and co-stimulation molecular expression(CD86,CD40)were examined by FCM.Serum was taken for cytokine(IFN-?,IL-2,IL-6,IL-17,IL-4,IL-10,TNF-?)examined by FCM.Results Part I:Whole bone marrow adherence method can isolate mouse bone marrow mesenchymal stem cells.Part ?:aGVHD+MSC group had longer survival time than aGVHD group;aGVHD+MSC group spleen mononuclear cells were less specific cytotoxic to P815 cells than aGVHD group;MSCS infusion inhibited the expression of CD69 and CD86 on donor-derived B cell surface,mRNA levels of IL-4 in B cells were significantly reduced,and IL-10 levels were elevated.Part III:B cells were almost depleted after sorting;the survival time of B220"aGVHD group and B220-aGVHD+MSC group were significantly longer than those of aGVHD group and aGVHD+MSC group,and the ratio of CD69 in CD3 T lymphocytes was lower expressed in B220-aGVHD group and B220-aGVHD+MSC group.Part IV:Using liposome transfection lentiviral packaging+PEG6000 concentration,high titer virus can be obtained(CCR7 virus titer is 5.6×109/ml,GFP virus titer is 6.0×1010/ml);MSC-CCR7 cells transfected by lentivirus are CCR7 positive;surface markers of MSC,MSC-CCR7,MSC-GFP consist with MSC standards.And the three kinds of MSCs were all capable of multi-direction differentiation.;there was no statistical difference in growth rate among the three cells.Part V:The survival time of allografts in MSC-CCR7 group was longer than that in MSC-GFP group and blank control group(P<0.05);the initial signs of acute rejection were later than that in the other two groups;the pathological manifestations of MSC-CCR7 group were the least at the same sampling time point;the expression of activated T and B cells,B costimulatory molecule CD86 and CD40 in MSC-CCR7 group was lower than that in other two groups,Treg contained.The quantity of IL-4 in MSC-CCR7 infusion group was higher than that in other two groups;IFN-y,IL-2,IL-4,IL-6 and TNF-a increased at first and decreased then,while IL-17 and IL-10 increased continuously;IL-4 in MSC-GFP infusion group and blank control group decreased and then increased,while IFN-y,IL-2,IL-6,IL-17 and TNF-a increased at first and decreased,and IL-10 continued to increase.Conclusion ?Bone marrow mesenchymal stem cells(BMSCs)can be obtained by whole bone marrow adherence+continuous passage method;?co-infusion of MSC can prolong the MST of aGVHD mice and reduce the spesific cytotoxity of splenic mononuclear cells to donor cells:?co-infusion of MSC can reduce the activation ratio of T cells,the level of costimulatory molecules secreted by B cells,and the level of B cell stimulator IL-4 and Breg inducible factor IL-10 were increased to inhibit the development of aGVHD;?Deleate B cells further prolonged the MST of aGVHD mice;?Clearance of B cells and infusion of MSC could reduce the proportion of T cells activated in the 24 hour;?Lentivirus transfection+drug screening successfully obtained stem cells with high expression of CCR7 gene and still in line with mesenchymal characteristics.?Mesenchymal stem cells overexpressing CCR7 can prolong the survival time of rat skin allograft and alleviate the rejection reaction of rat skin allograft.?MSC-CCR7 infusion can allevate the rejection reaction of light allograft skin transplantation by increasing the proportion of Treg cells,increasing the secretion of anti-inflammatory cytokines and reduce the secretion of pro-inflammatory cytokines,reducing and postpone T cell activation,B cell activation and antigen presentation.