| ObjectiveCorneal transplantation is one of the most successful organ or tissue transplantations, but immunological rejection reaction is still the major cause of human corneal allograft failure. MSCs can exert profound immunosuppression both in vitro and in vivo by inhibiting the proliferation and function of a number of immune cell types. In the present study, we plan to investigate the effect of MSCs with/without cyclosporin A (CsA) on corneal transplantation rejection in rat model and clarify its mechanism, to provide new ideas and alternative approaches to clinical treatment of cornea allograft rejection reaction.Methods1. The phenotype of cultured cells was characterized by flow cytometry and inducing differentiation in vitro.2. Corneas of Wistar rats (donors) were transplanted to Lewis rats (recipients). Transplanted rats were divided into six treatment groups:group A (PBS), Group B (daily intramuscular CsA injection), Group C (MSCs infusion via the tail vein, pretransplanted), Group D (MSCs, posttransplanted) and group E/F (MSCs, pretransplanted/posttransplanted with CsA). After PKP, grafts were scored for corneal transparency, edema and extent of neovascularization. In group A and group D, the experimental eyes were processed for histology.3. Mononuclear cells (MNCs) were obtained from the spleen and the lymph nodes which were incubated in the presence of ConA for72hours. T-cell proliferation was studied thereafter by a standard3H-thymidine incorporation assay.4. The levels of Th1and Th2cytokine production were measured in supernatants derived from72hours cultures of MNCs stimulated by ConA under various conditions, using a commercially available ELISA kit.5. At the end of the experiment, we collected peripheral blood from IRBP-immunized rats that were treated or untreated with MSCs. Cells were then counted and examined by flow cytometry to evaluate the expression of CD4and CD25. 6. Total RNA was isolated from spleen and lymph nodes and Real-time polymerase chain reaction (PCR) was performed to measure FoxP3Gene expression levels.Results1. MSCs were successfully separated and identified. Red adipose vacuole in endochylema was showed in adipose-like cell and blank calcium salts deposition was found in bone-like cells.2. Established rat model of penetrating keratoplasty (PKP).3. The survival time of the corneal grafts in group B, group D, group E and group F was significantly prolonged as compared with the control group(P<0.05). The survival time of the corneal grafts in group B, E and F is longer than that in group D. However, there was no significant difference among group B, group E and group F (P>0.05). The histopathological findings showed that the inflammation cells, neovascularity in group D were significantly fewer than that in group A.4. MSCs significantly inhibited proliferation of pathogenic T cells in vitro and prevented T cells response on in vivo injection (P<0.05).5. MSCs treatment significantly reduced Thl and elevated IL-4cytokine secretions in PKP rats.6. MSCs treatment significantly increased the proportion of Tregs, and real-time quantitative PCR results showed that the expression of FOXP3-mRNA in MSCs-treated group were significantly higher.ConclusionsTaken together, these results suggest that MSCs can effectively prevent and ameliorate immunological rejection, through the inhibition of pathogenic T-cell responses, modulation of the balance of Thl/Th2and increasing the proportion of regulatory T cells, which confirms the therapeutic plasticity of MSCs on immunological diseases due to their capacity for modulating systemic autoimmunity. |
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