Study On The Lentiviral Mediated CD25siRNA In Rat High-risk Corneal Transplantation Immune Rejection

ObjectiveTo determine whether Lentiviral mediated CD25siRNA（LV-CD25siRNA）can transfer to the corneal tissues of rats in rat high-risk cornealtransplantation immune rejection.There are two parts in my study:Study One: Study on the effectiveness and toxicity of Lentiviralvector-mediated enhanced green fluorescent protein transfection in ratcorneaStudy Two: Study of the Lentiviral mediated CD25siRNA in RatHigh-risk Corneal Transplantation Immune Rejection.MethodStudy One:23SD rats were divided into five groups,including MOI=5eyedrop group(group A), MOI=5subconjunctival injection group(groupB),MOI=10eyedrop group(group C),MOI=10subconjunctival injectiongroup(group D),MOI=10in vitro transfection group(group E).9SD rats ingroup A,B,C and D.5SD rats in group E.Group A and B used the same9SD rats，group C and D too. Each group was treated respectively withcorresponding concentration of LV-EGFP.To observe the fluorescenceintensity of corneas by inverted fluorescence microscope and cornealcellular morphology by HE stain and convert fluorescence intensity tooptical density(OD).Study Two: Alkali burn (NaOH) method is used to prepare SD rats with high-risk corneal transplantation, and then corneal transplantation is performed from Lewis rats to SD rats. After that intervened by4different methods: A LV-CD25siRNA eye drops, B CyclosporinA, C Negative control viruses (Lentiviral containing negative controlsiRNA), D Saline solution control group. Corneal tissues of every group after transplantation and HE staining is detected. The decreasingcondition of CD25and VEGF-A in the corneal tissues after3,7,14days uses Reverse transcription-quantitative Polymerase Chain Reaction and Western blot.ResultStudy One: Group A OD=0.1803±0.04395,group B OD=0.1061±0.04341,group C OD=0.2369±0.01565,group D OD=0.2002±0.03072,groupE OD=0.2434±0.01730.Compare with the same MOI groups (compareA with B, compare C with D), fluorescence spreaded more uniformly and expressed more strongly between eyedrop groups and subconjunctiva l injection groups(P<0.05). Compare with the same transfection way groups (compare A with C, compare B with D).High MOI groups can express more EGFP than low MOI groups(P<0.05).Group E compared withgroup C, the two groups expressed the same EGFP(P>0.05). Analysingfrom HE stain,corneal cells were normal.Apoptotic cells were not foundin each group.7days later, corneal endothelial cells were still growingwell with no endothelial cell shrinkage, deformation or missing in vitro transfection group.Study Two: Observing under lit lamp:10days after operation, it isvisible in A group photo graft central mild edema, opacity and no CNV graft,in B group and D group showed corneal opacity completely edema andmounts of CNV in clusters of corneal graft, in C group corneal mild edema,opacity and at6point to9point showed a large number of CNV into the graft.Each group means survival time of corneal graft (A12.17±0.34d, B9.5±0.35d, C6.67±0.26d, D6.33±0.57d). Through the SNK-q test compareA group with other three groups compared, graft survival time issignificantly different (P<0.05,).Corneal pathological examination:14days after operation, corneal HEstained can be seen, each group corneal grafts are thickened, mainly thecorneal stromal layer thickened, A group (CD25group) epithelial cells arearranged regularly, closely, no significant edema, corneal stromainflammatory cell infiltration and CNV less, of corneal endothelium cells neat and no missing. B, C, D group show epithelial layer cells loss andloosely arranged, the matrix can be seen a large number of inflammatory cellinfiltration and CNV, fine with endothelial cells. This result was consistentwith that we observed under slit lamp.Corneal CD25and the expression of VEGF-A mRNA:Compare Agroup (LV-CD25siRNA group) with B group (CsA group), at the3d,7d and14d three time points, A group’s (LV-CD25siRNA group) mRNA of theCD25gene is significantly lower than that in B group (CsA group), thedifference is statistically significant (p<0.05). But view from the mRNAexpression of VEGF, A group (LV-CD25siRNA group) and B group (CsAgroup), the difference is statistically significant (p<0.05). But the expressionview from the mRNA expression of VEGF, A group (LV-CD25siRNA group)and B group (CsA group), the difference is not statistical significant at3d,7dand14d (P>0.05).Western blot results:Expression of VEGF-A in normal cornea is few,after corneal transplantation the expression of VEGF-A in each group issignificantly increased, in A group (0.362±0.09),C group (0.994±0.19) andD group (1.07±0.15)compare with the two control group, the expression ofVEGF-A protein(P<0.05) reduced, B group (0.384±0.13) compare with Agroup, the expression of VEGF-A protein is not statistically significant(P>0.05).14days each group Western blot results. Conclusion1. Lentiviral vector-mediated enhanced green fluorescent protein cantransfect rat cornea effectively with low MOI.Transfecting rat cornea bytaking eye drops were more effective than the way by subconjunctivalinjecting. Increasing MOI can increase effectiveness of transfection.Ratcornea continuous transfection were safe with low MOI.2. this research using lentiviral vector mediated CD25siRNA transfersrat cornea after the high-risk corneal transplantation operation, not onlyprolongs the occurring time of corneal transplantation immune rejection,but also suppresses the development of corneal neovascularization in adegree, and this inhibitory effects may not be done by the inhibition ofVEGF-A, may be the occurrence of costimulatory effect of multiplecytokines.