| Objective:Through researching the allogeneic skin graft recipient mice with third-party bone marrow mesenchymal stem cells in mice injected, to determine that BMSCs can prolong the skin graft survival of recipient mice and to study the immuno-tolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells (BMSCs) on the allogeneic transplantation.Methods:1 Isolation culture and definition of MSCs in vitro:Femoral bone of SD rat was isolated sterilely, made into single cell suspension of bone marrow. Then adopt whole bone marrow adherent cultivation to culture MSCs in DMEM-F12 medium containing 10% FBS, Every 3 days to remove non-adherent cells. The cells were amplified when reached 90% confluence. Take three generations to adjust the concentration of spare cells. The expressions of CD29,CD34,CD45 and CD90 of cells were analyzed by using flow cytometry in order to identify MSCs.2 Establishment of animal model:Forty C57BL/6 female mice were used for skin graft donors, after anesthesia, making a round skin graft about 1 cm2 in the back. Forty BALB/C male mice were used for skin graft receptors. Cut out the whole thickness of the skin to form a round wound about 1cm2.Establish a stable skin allograft model.3 Injection of cells and drugs. Experimental groups:Forty BALB/C mice were divided randomly into 4 groups:Blank control group:Only carry on skin grafting, not given other treatment; Cy group:high-dose abdominal injection of Cyclophosphamide (200 mg/kg,2 d.q.d.)；SD-MSCs group:On the transplantation day a bonus of 2×106BMSCs of the SD rat (SD-BMSCs) were injected through the tail vein; Cy+SD-MSCs group:high-dose abdominal injection of Cyclophosphamide (200 mg/kg,2 d,q.d.),On the transplantation day a bonus of 2×106 BMSCs of the SD rat (SD-BMSCs) were injected through the tail vein.4 The observation of skin grafts and HE staining:Observe the skin graft of recipient mice in 7 days after transplantation and record the survival time. The skin graft shows 90% Scab as necrosis, if the skin graft completely black, stiff, shedding, it was shown as a rejection and make a pathological grade.take of skin grafts in each group of recipient mice for HE staining.5 Detection of CD4+CD25+Foxp3+Treg cells of spleen:The spleen of recipient mice was isolated in 7 days after transplantation for preparation of single cell suspension, Content of Treg cells were detected by using Fluorescent antibodies (CD4,CD25,Foxp3) after transplantation.6 Detection of Cytokine(TGF-β,IL-10,IFN-γ) in peripheral blood of recipient mice:Cytokine in peripheral blood of recipient mice were measured by ELISA, including TGF-β,IL-10,IFN-γin 7 days after transplantation.7 Detection of Proliferation of T lymphocytes stimulated by different allogeneic MSCs:T cells were co-cultured with 60Co-irradiated bone marrow MSCs from different individuals(C57 mouse or SD rat). The proliferative activity of T cells were evaluated with MTT assayResults:1 Observation and identification of SD-MSCs cultured in vivo: Adherent cell growth can be seen in 48h after primary culture, The morphology of parts of cells become spindle-like, after then cells grow fast, proliferation of cells reach to 90% confluence in 7～11 d, and display swirling shape. Passage cells adhere in 24h,passage the cells in 5～7 d, stable for at least 20 passages; The result of identification of MSCs is:surface marker of mesenchymal stem cell shows CD29+(99.7%),CD44+(96.7%); surface markers of hematopoietic stem cell shows CD34-(1.6%). CD45- (1.3%).2 Results of survival time of skin grafts and HE staining:The survival time of skin grafts is:CP+SD-MSCs group (15.7±1.4) d, Control group(6.1±1.1)d, CP group(12.3±1.5)d. SD-BMSCs group(12.6±1.8) d. The survival time of skin grafts in CP+SD-MSCs group is significantly longer than the other 3 groups (P<0.05):The control group shows complete necrosis of the epidermis and dermis, significant increase in cell number, significant inflammatory response. The CP group and SD-MSCs group shows dermal cells decreased and extensive fibrosis. The CP+MSCs group shows missing part of the epidermal, normal layer of dermis, Hair follicle structure can be seen.3 Results of CD4+CD25+Foxp3+Treg cells of spleen in recipient mice:Compared with the control group and CP group, the ratio of the CD4+CD25+Foxp3+Treg cells significantly increased in the SD-MSCs group and CP+MSCs group(P<0.05).4 Results of Cytokine(TGF-β,IL-10,IFN-γ) in peripheral blood of recipient mice: Analysis of peripheral blood by ELISA showed significant high levels of TGF-β,IL-10 and low level of IFN-y in MSCs group and CP group, compared with control group.5 Results of proliferation of T lymphocytes stimulated by different allogeneic MSCs:When co-cultured with MSCs from different individuals, T-lymphocytes proliferation decreased apparently in SD-MSCs group and C57-MSCs group (P<0.05), but there were no significant different between SD-MSCs group and C57-MSCs group (P>0.05)Conclusion:1 the Third-party bone marrow-derived mesenchymal stem cells can prolong the survival time of allogeneic skin grafts.2 The immuno-tolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells (BMSCs) on the alloeneic transplantation might be associated with its effect on the proliferation of Treg cells.3 The immuno-tolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells (BMSCs) on the alloeneic transplantation might be associated with its effect on increasing expressions of Promoting immuno-tolerance factor, decreasing expressions of promoting immuno-rejection factor.4 There were no significant different in effect on proliferation of T lymphocytes stimulated by different allogeneic MSCs... |
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