[Study of immuno-tolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells in allogenic transplantation].

OBJECTIVE: To study the immuno-tolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells (BMSCs) in the allogeneic transplantation.

METHODS: Forty female C57BL/6 mice and forty male BALB/C mice were respectively used as donors and recipients in skin allogenic graft model. Forty male BALB/C mice were divided randomly into 4 groups: blank control group, CP group, BMSCs group , CP + BMSCs group, with 10 mice in each group. Before skin graft, high-dose abdominal injection of cyclophosphamide (200 mg/kg, 2 d, q. d.) was performed in recipient mice in CP and CP + BMSCs groups. On the transplantation day, a bonus of 2 x 10(6) BMSCs from the SD rat (SD-BMSCs) were injected through the tail vein in the BMSCs and CP + BMSCs groups. The observation and HE staining of skin grafts were used. The expressions of CD29, CD34, CD45 and CD90 of cells were analyzed by using flow cytometry in order to identify BMSCs. The CD4+, CD25+, Foxp3 and Treg cells of spleen were detected by flow cytometry. Cytokine in peripheral blood of recipient mice were measured by ELISA, including TGF-beta, IL-10 and IFN-gamma. T cells were co-cultured with 60Co-irradiated bone marrow MSCs from different individuals. The proliferative activity of T cells were evaluated with MTT assay.

RESULTS: The skin graft survival time was significantly prolonged in the CP + BMSCs group, as compared with that in the blank control group, the CP group, the BMSCs group, respectively. Cells cultured by whole bone marrow adherent cultivation showed CD29 (99.7%), CD44+ (96.7%), CD34- (1.6%), CD45- (1.3%). Compared with the control group and CP group, the ratio of the CD4+, CD25+, Foxp3+ and Treg cells significantly increased in the SD-BMSCs group and CP + BMSCs group (P < 0.05). Analysis of peripheral blood by ELISA showed significant high level of TGF-beta, IL-10 and low level of IFN-gamma in BMSCs group and CP group,compared with that in control group. When co-cultured with BMSCs from different individuals, T- lymphocytes proliferation decreased apparently in SD-BMSCs group and C57-BMSCs group (P < 0.05), but there was no significant difference between SD-BMSCs group and C57-BMSCs group (P > 0.05).

CONCLUSIONS: The immunotolerance mechanism of the third-party bone marrow-derived mesenchymal stem cells in the allogeneic transplantation might be associated with its effect on the proliferation of Treg cells and increasing expression of TGF-beta and IL-10, decreasing expression of IFN-gamma.