| Objective:1. To test whether down-regulation of CD25 expression via siRNA inhibits corneal allograft rejection and to study the dynamics of Treg/Th1 cytokines.2. To investigate whether anti-CD25 and Rapa have the ability to suppress allosensitization via inhibiting DC function and immune-related cytokines as well as their effects on Treg cells.Methods:1. Wistar rat were used as donors and SD rat as receptors to establish the corneal penetrating transplantation model. These models were randomly divided into three groups. They were allograft control group,scamble-siRNA group, and CD25-siRNA group. From 1 week post-transplantation, the recipient eyes were examined every day. On day14 postoperation, HE was performed and the expression of IL-10, TGF-β,IFN-γ, IL-1β, TNF-α, and Foxp3 were detected by immunohistochemistry.On days 3, 7, 14 and 21 post-operation, the frequencies of CD4+ T cells in the peripheral blood were analysised by flow cytometry.2. C57BL/6 mice were used as donors, and Balb/c mice whose corneas were neovascularized by sutures for 2 weeks as receptors to establish thehigh-risk corneal penetrating transplantation model. These operated mice were then randomly divided into four groups. They were allograft control group, anti-CD25 group, rapa group, and combination of both anti-CD25 and Rapa group. On postoperative days 12, HE was performed and the infiltration of DCs was detected by immunohistochemistry. The percentages of CD4+ T, Treg, and DC cells in the DLNs and spleen of each group were determined by flow cytometry. The expression levels of IFN-γ、IL-2 、 IL-1β and TGF-β in the grafts and DLNs of each group were analyzed by Real-Time PCR. Orthotopic grafts were observed by slit-lamp biomicroscopy daily up to 4 weeks.Results:1. Compared with the control and scamble-siRNA group, CD25 siRNA group significantly prolonged graft survival time, with reduced opacity,oedema and neovascularisation(p<0.05). On postoperative days 14,immunohistochemistry showed that higher IL-10, TGF-β expression and less TNF-α, IFN-γ, IL-1β were in the grafts of the CD25 siRNA group.Meanwhile, the expression of Foxp3 showed no significant differences among three groups. On days 3, 7, 14, and 21 postoperation, the percentage of CD4+ T cells in the peripheral blood were gradually increased in the control group. CD4+ T cell populations were lower in the CD25 siRNA group compared with the untreated group(P < 0.05) at all time-points.2. Compared with the control group, three groups with differenttreatment significantly prolonged graft survival time(p<0.05). On postoperative days 12, reduced DC and other inflammatory cells infiltration,as well as lower percentage of DC cells and CD4+ T cells in the draining lymph nodes, were showed in the anti-CD25, Rapa, and combined treatment groups. The percentage of Treg cells in the untreated mice was lower than that of Rapa group, while higher than anti-CD25 group.Moreover, the mice treated with both of anti-CD25 and Rapa has no significantly diffenerce with control in the percentage of Treg cells(P>0.05). The relative mRNA expression of IFN-γ、IL-2、IL-1β in the grafts were significantly reduced in three treatment groups compared with those in the controls on postoperative days 12. Moreover, TGF-β mRNA expression in the anti-CD25 group and the combined treated group were higher than those in the other two groups.Conclusions:1. Downregulation of CD25 expression played a protective role in corneal graft rejection, with longer survival time of grafts.2. This was related with the upregulation expression of IL-10, TGF-βcytokines and downregulation expression of TNF-α, IFN-γ, and IL-1βcytokines.3. Effects of maintaining the low maturation state of DC, reducing migration and expansion of immune cells may also contribute to enhanced allograft survival in corneal transplantation with downregulation of CD25. |
PDF Full Text Request