| Objective: To establish the model of abdominal cardiac transplantation in rats from SD to Wistar rats,and infused mesenchymal stem cells (Mscs) to receptor via peripheral intravenous, to observe whether effective amplification receptor CD4~+CD25~+foxp3+ regulatory T cells (Treg), and through the experiment of MSCs were cecultured with allogenenic CD4+T lymphocytes,to reveal its mechanism.Methods: MSCs were cocultured with allogenenic CD4+T lymphocytes in different proportion,the groups of the experiment: Group A(CD4+T), Group B (MSCs), Group C1 (MSCs /CD4+T 1:1), Group C2 (MSCs/CD4+T 1:10), Group C3 (MSCs/CD4+T 1:100). To observe cells form with Inverted microscope, to detect whether effective amplification receptor CD4~+CD25~+foxp3+regulatory T cells and the expression of Foxp3 protein in the Treg cells. To detect the changing of IL-2, IL-10, TGF-β1 in the supernate with ELISA Donor(SD rats) and receptor(Wistar rats) of 15 were randomly divided into three groups, GroupⅠ(control group): The heart from SD rat was transplanted into the abdomen of Wistar rat and receptor without any treatment. GroupⅡ(PBS group): receptor was injected PBS 0.2 ml via the neck vein before preoperative 5th day. GroupⅢ( group MSCs): receptor was injected mesenchymal stem cells (MSCs) 1×106 via the neck vein before preoperative 5th day. Testing Items: cardiac allograft survival time, pathological changes, receptor CD4~+CD25~+foxp3+Treg cell change, the expression of Foxp3 protein in the Treg cells and serum TGF-β1, IL-10 ,IL-2change.Results: The vitro part: (1)the results of CD4~+CD25~+Treg/CD4+T and the expression of Foxp3 protein on the Treg cells in Supernatant fluid: Group A:6.44±0.55%, 70.64±1.69%, Group B: 0±0%, 0±0%, Group C1: 65.98±3.11%, 84.10±3.03%, Group C2: 51.92±1.86%, 78.23±3.26%, Group C3: 30.52±3.40%, 77.14±1.98%. The CD4~+CD25~+Treg/CD4+T of Group C1,C2,C3 in the Supernatant fluid was significantly higher compared with Group A, P<0.05. The expression of Foxp3 protein in the CD4~+CD25~+Treg cells in Supernatant fluid had no significant difference between Group C1,C2,C3 and Group A, P>0.05. (2) the results of receptor TGF-β1 in supernatant fluid: Group A: 110.77±6.55 pg/mL, Group B: 396.34±6.48 pg/mL, Group C1:688.38±9.92 pg/mL, Group C2:705.07±6.78 pg/mL, Group C3:783.58±19.74 pg/mL. the TGF-β1 levels of Group C1, C2, C3 was significantly higher compared with Group A, B, P<0.05. There had no significant difference among Group C1, C2, C3. (3) the results of receptor IL-10 in supernatant fluid: Group A: 6.68±0.56 pg/mL, Group B: 0±0 pg/mL, Group C1:50.32±1.55 pg/mL, Group C2:52.62±0.80 pg/mL, Group C3组:69.60±1.55 pg/mL. the IL-10 levels of Group C1, C2, C3 was significantly higher compared with Group A, B, P<0.05. There had no significant difference among Group C1, C2, C3. (4) the results of receptor IL-2 in supernatant fluid: Group A: 11.98±0.38 pg/mL, Group B: 0±0 pg/mL, Group C1:4.43±0.19 pg/mL, Group C2:3.89±0.60 pg/mL, Group C3组:3.84±0.63 pg/mL. The IL-2 levels of Group C1, C2, C3 was significantly lower compared with Group A, B, P<0.05. There had no significant difference among Group C1, C2, C3.The body part :(1)Heart mean survival time (MST): GroupⅠ: 6.0±0.71d, GroupⅡ: 5.8±0.84d, GroupⅢ: 17.0±0.71d. The MST of GroupⅢwas significantly prolonger than GroupⅠ,Ⅱ, P<0.05. (2) the results of receptor CD4~+CD25~+Treg/CD4+T and the expression of Foxp3 protein in the Treg cells in peripheral blood: GroupⅠ: 2.18±0.36%, 72.84±1.91%, GroupⅡ: 2.46±0.44%, 72.48±2.37%, GroupⅢ: 8.72±0.68%, 73.24±2.78%, The peripheral blood CD4~+CD25~+Treg/CD4+T of GroupⅢwas significantly higher compared with Group A,B, P<0.05. The expression of Foxp3 protein in the CD4~+CD25~+Treg cells in peripheral blood had no significant difference between GroupⅢand GroupⅠ,Ⅱ, P>0.05. (3) the results of receptor TGF-β1 in peripheral blood: GroupⅡ: 165.73±8.43pg/mL; GroupⅡ: 178.75±9.02pg/mL; GroupⅢ: 341.76±13.82pg/mL; the TGF-β1 levels of GroupⅢwas significantly higher compared with GroupⅠ,Ⅱ, P<0.05. (4) the results of receptor IL-10 in peripheral blood: GroupⅠ: 16.20±0.61pg/mL; GroupⅡ: 18.81±0.42pg/mL; GroupⅢ: 47.38±1.38 pg/mL. the TGF-β1 levels of GroupⅢwas significantly higher compared with GroupⅠ,ⅡP<0.05. It had no significant difference between GroupⅠand GroupⅡ, P>0.05. (5) the results of receptor IL-2 in peripheral blood: GroupⅠ: 90.34±3.43pg/mL; GroupⅡ: 85.90±1.67pg/mL; GroupⅢ: 44.85±3.39 pg/mL. the IL-2 levels of GroupⅢwas significantly lower compared with GroupⅠ,ⅡP<0.05. It had no significant difference between GroupⅠand GroupⅡ, P>0.05.Conclusions 1, MSCs were cultured with CD4 + T, it can significantly improve the CD4+ CD25+ Treg ratio, and dose-related with the MSCs, but the expression of Foxp3 protein had no significant role in promoting . 2, Peripheral venous transplantation of marrow mesenchymal stem cells (MSCs) can make the CD4~+CD25~+foxp3+Treg cells increased significantly and extend heart survival time. 3, Interaction with Treg cells and MSCs, by down IL-2, increased IL-10, TGF-β1 expression of immunosuppressive factors such as to achieve the immune suppression. Keywords: mesenchymal stem cells, CD4~+CD25~+Treg, Foxp3, Cardiac... |
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