Investigation Of The Combinative Effect Of Mesenchymal Stem Cells With CsA In Corneal Allograft Rejection

ObjectiveCorneal allograft immunological rejection is 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. Based on the previous study of the immunomodulate effect of MSCs on corneal allograft rejection, we futher investigate the effect of MSCs with different doses of 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.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 twelve treatment groups randomly: Group A (PBS); Group B (MSCs infusion pretransplanted via the tail vein) Group C (MSCs posttransplanted); Group D (MSCs infusion when rejection happened); Group E (MSCs posttransplanted with daily intramuscular lmg/kgCsA injection); Group F (daily intramuscular lmg/kgCsA injection); Group G (MSCs with2mg/kg CsA); Group H (2mg/kg CsA, daily); Group I (MSCs with5mg/kg CsA); Group J (5mg/kg CsA, daily); Group K (MSCs with10mg/kg CsA); Group L (10mg/kg CsA, daily).After PKP, grafts were scored for corneal transparency, edema and extent of neovascularization.3. Mononuclear cells (MNCs) were obtained from the spleen and the lymph nodes of Group A, C, G and H, 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 Thl and Th2cytokine production were measured in supernatants derived from72hours cultures of MNCs stimulated by ConA under various conditions, using a commercially available ELISA kit.5. We collected spleen and the lymph nodes of Group A and C rat model, which were untreated or treated with MSCs. Cells were counted and examined by flow cytometry to evaluate the expression of CD4, CD25and Foxp3.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. MSCs infused postoperation led to longer suvival time of corneal allografts.4. CsA inhabits corneal allograft rejection in a dose-depended way.5. MSCs and CsA have combined effect. MSCs combined with1mg/kg CsA accelerated graft rejection compared with MSCs or CsA (1mg/kg) alone, while2mg/kg CsA together with MSCs exhibited a synergistic suppressive effect. When the dose of CsA was increased to5mg/kg, the synergistic effect was lost.6. MSCs with/without CsA can significantly inhibite proliferation of T cells in vitro and prevented T cells response on in vivo injection(P<0.05).7. MSCs with/without CsA treatment significantly reduced Thl and elevated IL-4cytokine secretions in PKP rats.8. MSCs treatment significantly increased the proportion of CD4+CD25+Foxp3+Tregs.ConclusionsMSCs can effectively ameliorate immunological rejection, through the inhibition of pathogenic T-cell responses, modulation of the balance of Th1/Th2and increasing the proportion of regulatory T cells. The combined effect of MSCs and CsA was highly dependent on CsA dose.