Effects Of CD25SiRNA Gene Therapy On Penetrating Keratoplasty

Background:CD25 is the a chain of IL-2R, which participates in the IL-2R-mediated signals.Previous studied suggested that IL-2/IL-2R/CD25 mediated signals could induce corneal graft rejection and were the main cause of corneal graft failure. The CD25mAb application could efficiently inhibit the corneal graft rejection and prolonged the mean survival time of grafts.Our group previous study confirmed that Lentiviral mediated CD25siRNA could prolong the survival time of grafts.However,the nanoparticle vector-CD25siRNA has not been used to corneal transplantation and its outcome,side effects and mechanisms are unknown.All the experiments were divided into two parts.Objective:Study one:To evaluate the safety and efficacy of topical EntransterTM-CD25siRNA vector and liposome-CD25siRNA in rat corneal application,and to choose the optimal time of therapy.Study two:To investigate the effects of EntransterTM-CD25siRNA gene transfer on high-risk rat corneal graft rejection.Methods:Study one:80 SD rats were randomly divided into 4 groups (EntransterTM-CD25siRNA group, liposome-CD25siRNA, siRNA group, control group). Eyes were examined with a slit lamp about corneal redness, edema,and inflammation at 12 hours,1,3 and 7 days post-transfection respectively.Fluorescence detection,TUNEL assay,HE,clinical assessment were used to evaluate the safety and efficacy of CD25siRNA gene transfer in normal SD rat corneas.Study two:Orthotopic corneal transplants were performed in Wistar-SD rats. Corneal recipients were divided into control group, EntransterTM-control CD25siRNA group and EntransterTM-CD25siRNA group. After surgery, the recipient eyes were examined daily for 21 days, and HE,immunohistochemistry,western blotting and qRT-PCR detection were performed on postoperative days 3,7,14 and 21. The expression of IL-10,TGF-β,INF-γ,IL-1β,TNF-α and FOXP3 were detected by immunohistochemistry.Results:Study one:The results showed that no fluorescence was observed in control group and massive green fluorescence was detected in EntransterTM group.The green fluorescence in CD25siRNA group was early expression but completely disappeared at 24 hours post-transfection. HE staining,TUNEL assay and CD11b immune detection after Liposome application to the cornea revealed inflammation,cell apoptosis and immune reaction were significant different from that in other groups.Study two:CD25siRNA treatment significantly prolonged graft survival time,with better graft structure compared to other two groups.Meanwhile,lower CD25mRNA and CD25 protein expression were detected in CD25siRNA group than that in other two groups(p<0.05).Meanwhile, immunohistochemistry showed that less TNF-α, INF-γ and IL-1β and higher IL-10 and TGF-β cells were seen in the corneas from the Entranster group.Conclusions:Study one:EntransterTM vector is an effective vector for corneal gene therapy which exhibited low toxicity,no immunogenicity and high transfection efficiency.Study two:CD25siRNA gene therapy played a protective role in corneal graft rejection,with longer survival time of grafts and better graft morphology This was related with the up-regulation of IL-10 and TGF-β cytokine expression and down-regulation of TNF-α, INF-γ and IL-1β cytokine expression.