| ObjectiveTo investigate the effects of CD25siRNA nanocarrier promoting corneal graft survival in rat models of high-risk penetrating keratoplasty. There are two parts in my study:Study One:To evaluate the safety and efficacy of topical EntransterTM vector and liposome in rat corneal application.Study Two:To explore the effects of CD25siRNA nanocarrier on penetrating corneal graft rejection in rat high-risk corneal transplantation immune rejection.MethodStudy One:80 SD rats were randomly divided into 4 groups (EntransterTM-CD25siRNA group, liposome-CD25siRNA, siRNA group, control group). Right corneal epithelia were completely scraped using a Tooke corneal knife in a rotary motion parallel to the limbus. Thus four reagents were applied topically to rat ocular. 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 alk ali burned SD rats to mimic high-risk rat models.Donor cornea (Wist ar rat) was grafted into the right cornea of recipient (SD rat) on day 14 after alkali burn.Four groups were randomly divided:ontrol grou p (Group A), EntransterTM- control CD25siRNA instilled treatment (Group B), EntransterTM-CD25siRNA instilled treatment (Group C) an d EntransterTM-CD25siRNA twice instilled treatment (Group D, the s econd administration on day 7 post-surgery). the recipient eyes were examined using a slit lamp microscope.then,The mean survival time a nd rejection index(RI) were calculated. The morphology of grafts wer e characterized by HE stainings and TEM.CD25 expression after oper ation was determined by quantitative RT-PCR and immunohistochemi stry (IHC).ResultStudy One:The results showed that no fluorescence was observed in control group and massive green fluorescence was detected in Entr ansterTM group.The green fluorescence in CD25siRNA group was early expression but completely disappeared at 24 hours post-transfection. H E staining,TUNEL assay and CD11b immune detection after liposome a pplication to the cornea revealed inflammation,cell apoptosis and immu ne reaction were significant different from that in other groups.Study Two:The survival curves showed that the mean survival time in rats from group C and D were significantly longer than that in group A and B(p<0.05).No significant differences were found between group A and group B,the same as group C and group D.The grafts in group A and B showed obviously edematous, thickening, collagen fibers arranged disorderly and cell infiltration. IHC results showed that CD25 expressed on the corneal epithelium, stroma and endothelium in all rats,and higher CD25 expression was observed in group A and group B. ultrastructure results showed that the degree of stromal fibroblast apoptosis and necrosis in corneal graft were obviously lower in group C and group D.The expression of CD25 mRNA in the experimental group was significantly lower at all time points.Conclusion1. EntransterTM vector is an effective vector for corneal gene therapy which exhibited low toxicity,no immunogenicity and high transfection efficiency.2. CD25siRNA gene transfer successfully down-regulated CD25 expression, inhibited graft rejection and prolonged the survival time of corneal graft. |
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