Effects Of IL-10Gene Transfectionon Rat Corneal Transplantation

ObjectiveWith adenovirus mediated IL-10transfect corneal as a donor, establish ratpenetrating corneal transplantation model, to study whether it can prolongsurvival time of corneal graft.Methods1、Take Wistar rats corneal in three groups of different nutrient solutionculture3h,4℃. Control group: RPMI-1640medium containing10%FCS; GFPgroup:10%complete medium containing GFP-Adenovirus (transfection titer is108); IL-10group:10%complete medium containing IL-10-GFP-Adenovirus(transfection titer is108). After3h,the three groups of culture medium arereplaced with RPMI-1640medium containing10%FCS. To observe thefluorescent expression of corneal graft use inverted fluorescence microscopeand confocal microscopy after incubation3,24,48and72hours. Westen Blotmethod detection the IL-10protein expression of corneal explants aftertransfection.2、Animals were divided into three groups: control group, GFP group, IL-10group. With Wistar rat as donor, SD rat as receptor to establish ratpenetrating corneal transplantation model, donor corneas were cultured in eachgroup of medium for3h,4℃. After corneal transplantation, we observed thepostoperative corneal allografts and recorded the survival time everyday.Cornea, aqueous humor and eyeball were taken after surgery in12days by RT-PCR method, the ELISA method, histopathology methods to detect thechange of cytokines.Results1、Each group has not see the fluorescent expression in3h,cornealepithelium in GFP group and IL-10group has a small amount of fluorescentprotein expression after incubation24h, fluorescent protein expression to thepeak in48h, to72h fluorescent expression gradually diminishes. Westen Blotshow: IL-10protein has highly expressed in IL-10-GFP-Adenovirus transfectedcorneal.2、The positive control group, GFP group, IL-10group keratoplasty cornealgrafts survival time:10.17±1.94d,12.00±0.89d,16.00±1.67d. IL-10groupcompared with control group, GFP group were significant statisticallydifferences(P＜0.01), control group compared with GFP group had nosignificant difference(P＞0.05).RT-PCR,ELISA show: the expression of IL-2、INF-γ、TGF-β、IL-10、IL-4、TNF-αin IL-10group compared with other groupswere significant statistically differences (P≤0.01). Histopathologicalexamination show: corneal graft in IL-10group had edema, thickening, andless inflammatory cell infiltration.Conclusions1、 GFP-Adenovirus and IL-10-GFP-Adenovirus can successfullytransfected into corneal.2、Corneal graft with the IL-10gene transfection can induce the cornealtransplantation immune tolerance and prolong the survival time of corneal graft.In the corneal transplantation immune rejection, Th1/Th2deviate and Th3increased is one of the mechanisms that induce transplantation tolerance.