| ObjectiveCorneal transplantation is the only effective way for visual rehabilitation caused by irreversible corneal injury or congenital diseases. Immune rejection is the major cause of graft failure. MSCs can exert profound immune suppression 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 further investigate the effect of subconjunctival injection of MSCs with different time on corneal transplantation rejection in rat model and primarily study its mechanisms, to provide new ideas and alternative approaches to clinical treatment of cornea allograft rejection.Methods1. MSCs from bone marrow of Wistar rats were isolated and cultured. Phenotype of the cultured cells was characterized by flow cytometry and inducing differentiation in vitro. The3rd generation of the MSCs was used in the following experiments.2. Corneas of Wistar rats (donors) were transplanted to Lewis rats (recipients). Transplanted rats were divided into four treatment groups randomly: Group A (PBS, the rats received an injection of PBS with equal volume); Group B (MSCs were injected subconjunctivally before transplantation); Group C (MSCs were injected subconjunctivally after transplantation one time); Group D (MSCs were injected subconjunctivally after transplantation two times). After corneal transplantation, grafts were scored for corneal opacity, edema and extent of neovascularization through slit lamp. The graft survival time in different groups were compared.3. The right rat eyeballs were collected for histopathology HE staining and immunohistochemical staining10days after transplantation.4. Corneas were collected for RNA7and10days after transplantation respectively. Total RNA was isolated and reverse transcribed. The transcript levels of Thl cytokines, IFN-y and IL-2, and Th2cytokines, IL-4and IL-10were examined by real-time PCR (RT-PCR).5. Corneas were collected for protein extraction10days after transplantation. Protein levels IL-4and IL-10were measured by ELISA.Results1. MSCs were successfully separated and identified. Red adipose vacuole in endochylema was showed in adipose-like cell and red calcium salts deposition was found in bone-like cells.2. Established rat model of penetrating keratoplasty (PKP).3. MSCs infused preoperation accelerated immune rejection. MSCs infused postoperation with two times led to longer suvival time of corneal allografts.4. In group A, HE staining showed that corneal grafts edema and thickening. There are a lot of neutrophils, lymphocytes, neovascularization and lymphangiogenesis in the corneas. Immunohistochemical staining showed a lot of CD4+T cells in the graft bed and corneal stroma. In group D, corneal grafts were slightly edema and the number of inflammation cells were less in cornea.5. Real-time PCR of corneal graft showed that the level of IFN-ymRNA group D was lower than group A’s and the level of IL-4and IL-10mRNA were higher7days after corneal transplantation. On the10th day, the express of IL-4and IL-10mRNA continued to increase and were significantly higher in group D compared with group A. However, the express of Thl cytokines(IFN-γ、IL-2) were not different from group A and group D.6. The result of ELISA of corneal graft showed that the express of IL-10was significantly higher than normal group and group A.ConclusionsMSCs were injected subconjunctivally can effectively ameliorate immunological rejection and prolong the survival time of corneal graft. The effect may execute through the inhibition of pathogenic T-cell responses and infiltration, modulation of the balance of Thl/Th2, especially the Th2cytokine IL-10. |
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