| BackgroundCorneal disease is the important cause leading to blindness in our country,and corneal transplantation is the only and effective clinical treatment for corneal blindness.Although eyes ball have the immune privilege and anterior chamber associated immune deviation.the immunological rejection after corneal transplantation is the primary cause of the failure.Therefore,prevention and treatment of rejection is still an important issue in corneal transplantation.Recently,many new cytokines and targeted therapeutic agents have been discovered and proved to play an important role in the immune response.On the one hand,SDF-1,a member of the chemokine family,binds specifically to its receptor CXCR4,which can mediate the maturation and chemotaxis of immune cells by G protein dependent transduction pathway and non G protein dependent transduction pathway.At present,they have been found to promote the rejection in renal transplantation and other transplantation,but there is no report on corneal allograft rejection.On the other hand,Tocilizumab is the interleukin 6 receptor(IL-6R)antagonist,which can inhibite the differentiation of Th17 cells,and promote Treg differentiation,causing graft tolerance.In islet transplantation and other studies have confirmed that tocilizumab can significantly prolong the survival time of the grafts,but there has not been reported in corneal transplantation immune response.Therefore,this study uses SD-Wistar rats penetrating keratoplasty model,on the one hand,choicing SDF-1/CXCR4 cell factor as the research object,observing the expression changes of corneal transplantation at different time points,to explore its potential biological effects and mechanism in the immune response after corneal transplantation;on the other hand,blocking IL-6/STAT3/ROR? t pathway by tocilizumab,compairing survival time of corneal graft,and observing the effect of tocilizumab in diferent level on corneal allograft rejection,providing a new strategy that more effective clinical prevention and treatment of corneal allograft rejection for the future.Part I Role of Stromal Cell-derived Factor-1 and CXC Chemokine Receptor 4 in Rejection Response of Corneal Transplantation in RatsObjectiveTo examine expression of stromal cell-derived factor-1(SDF-1)and CXC chemokine receptor 4(CXCR4)in rat cornea tissue and their role in corneal allograft rejection.MethodsIn this study,SD rats were used as donors and Wistar rats were used as recipients,to establish the model of allogeneic corneal transplantation.They were divided into normal group,autograft group,allograft group and tobradex group.Clinical assessment of the corneal grafts was carried out using Larkin's method;histopathological observation,immunohistochemistry,and real-time quantitative PCR of the corneal grafts were performed on days 5 and 9 after the transplantation.ResultsGraft rejection occurred in none of the rats in autograft group.In rats treated with tobradex,the graft survival time was significantly longer than that in rats with saline treatment(24±0.32 vs 10±0.36 days,P<0.001),and histopathological examination revealed numerous inflammatory cells and neovascularization in the allografts in the latter group.SDF-1 and CXCR4 mRNA expression in the corneal tissue increased significantly in rats receiving allograft transplantation and saline treatment(P<0.001 on day 5 and P<0.01 on day 5),and their expression was obviously lowered in rats with tobradex treatment.Immunohistochemical examination revealed that the expression of SDF-1 and CXCR4,found mainly in the corneal epithelium and stroma,was significantly increased in the allografts in rats with saline treatment.ConclusionSDF-1/CXCR4 may participate in corneal graft rejection in rats early after transplantation possibly through the mechanism that SDF-1 specifically induces CXCR4+ cell maturation and chemotaxis toward the allograft to promote corneal neovascularization.Part? Tocilizumab Promotes Corneal Allograft Survival in RatsObjectiveTo investigate the effect of tocilizumab on rat corneal graft rejection and the underlying mechanisms.MethodsIn this study,SD rats were used as donors and Wistar rats were used as recipients,to establish the model of allogeneic corneal transplantation.They were divided into normal group,autograft group,allograft group and tocilizumab group.Clinical assessment of the corneal grafts was carried out using Larkin's method;histopathological observation,immunohistochemistry,real-time quantitative PCR of the corneal grafts and flow cytometry were performed on days 14 after the transplantation.ResultsThe survival times of allograft and tocilizumab groups were shown to be(10+0.55)and(24 + 1.27)days respectively,showing statistically significant difference between each other(P<0.001).By histopathological examination,we observed that corneal tissues of allograft groups were markedly thickened with severe inflammatory cell infiltration and neovascularization,while the tocilizumab group exhibited less degree of inflammatory cell infiltration in corneal tissue with neither thickening nor angiogenesis.IL-17A,RORy t,IL-6,VEGF mRNA expression levels in corneal tissues of the allograft group were significantly increased,compared with the tocilizumab group(P<0.05).Foxp3 mRNA expression level in corneal tissues of the tocilizumab group were significantly higher than other groups(P<0.001).Immunohistochemical examination revealed that corneal tissue IL-17A and VEGF levels in the allograft group were greatly higher than that of tocilizumab group(P<0.01),while the Foxp3 level in the allograft group was then significantly lower than that of the tocilizumab group(P<0.001).Flow cytometric analysis showed that the blood Th17 cell amount in allograft group was also significantly higher than that of tocilizumab group(P<0.001),while the amount of Treg cells was significantly reduced with comparison to the tocilizumab group(P<0.001).ConclusionTocilizumab could significantly prolong the survival time of rat corneal graft,which might be mediated by changing balance of Th17/Treg cells in the rats and reducing IL-17A,VEGF and IL-6 expression in corneal graft,inhibiting corneal graft rejection,reducing the formation of corneal neovascularization,and consequently prolonging survival time of corneal graft. |
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