Experimental Study Of The Relationship Between ICOS And Corneal Allograft Rejection

Background:Cornea transplantation immunological rejection is a T-cell mediated delayed hypersensitivity and a complete process with practice and regulation of many immunology cells and molecules.The activation of T-cell needs the participation of two signals,the MHC antigen peptide signal and the co-stimulatory signal.If lack of the co-stimulatory signal supplied by the co-stimulator,T-cell can not be activated and continue stay in a inability statement.In different phase ofT-cell activation,many co-stimulators educe important regulation effect.Among this inducible co-stimulator, ICOS is an important co-stimulator find out recently.The discovery of initial research find out that ICOS is thought to be the new family member of CD28 family.ICOS mediated co-stimulate passageway educe important effect in multiple organ transplantation immunological rejection, immunological rejection can be effectively regulated by this passageway and we can explore useful strategy and mean to prevent the immunological rejection activation from this.Homogeneity corneal transplantation is the most effective method to treat cornea blind in nowadays.But the immunological rejection after operation is still the main cause of the operation failure.Elevate the successive rate of cornea transplantation is an problem need to be urgently solved in clinical practice.Therefore,continue research the cornea transplantation acute immunology rejection mechanism and positively explore satisfactory anti-rejection method from many routes got great meanings.Partâ… Research of the role of ICOS co-stimulatory passageway in the cornea transplantation Immunological rejectionObjective:Research the relationship between ICOS co-stimulator passageway and cornea transplantation acute immunological rejection with homogeneity penetrability corneal transplantation rat model.Methods:Divide healthy Wistar and SD rats into transplantation group and normal control group.Transplantation group conclude 10 donor Wistar rats and 20 acceptor SD rats,build the homogeneity penetrability cornea transplantation rat model;control group consist of 5 healthy normal SD rats(10 eyes).Observe cornea implant with slit lamp microscope everyday after operation and score three indexes,corneal opacity,edema and new vessals.Get the cornea implant on 7 and 14 days after operation and carry out pathological observation.Test the implant ICOS mRNA expression with RT-PCR and ICOS albumen level in implant lymphocyte with immunohistochemical method;while at the same time,test the peripheral blood CD3⁺ICOS⁺T/CD3⁺T expression with flow cytometry and contrast with normal rats.Results:(1) In homogeneity penetrability corneal transplantation rat model, immunology rejection does not happen on the 7th day.On the 14th day,all the acceptors had acute immunology rejection.(2) Cornea implant become edema and thick after transplantation,fibers in stroma are confused arranged,lots of histomonocytes and lymphocytes infiltrate around the incision and suture line. Inflammation cells also can be seen in the center of implant and become more obvious on the 14th day after operation than on the 7th day.Inflammation cell mainly reside in the corneal epithelium and superficial stroma.(3) ICOS positive cells got buffy dying cellular membrane and cytoplasm.ICOS positive lymphocytes are not seen on various membranes of the normal rat cornea;on the 7th day and 14th day after operation,ICOS positive lymphocytes are found on the implant epithelium and stroma.The score of positive cell:7th day after operation(2.000±0.667) 14th days after operation(3.700±0.483).The statistic result show:ICOS protein positive cell score is higher on the 14th after operation than on the 7th day and there is significant difference(t=-6.530,P=0.000).(4) The expression of ICOS mRNA can not be detected on normal rat cornea;the ratio of brightness of electrophoresis strip of the ICOS PCR product in cornea implant on the 7th day after operation is(1.168±0.190) and(1.525±0.139) on the 14th day.There is significant difference between the two ratios(t=-4.812,P=0.000).(5) Peripheral blood CD3⁺ICOS⁺T/CD3⁺T expression on normal rat is(7.354±1.114)%,(31.521±1.995)%on the 7th day after operation and(45.602±2.034)%on the 14th day after operation.Statistic result show homoscedasticity(P=0.156).Compare with normal rat,peripheral blood CD3+ICOS+T/CD3+T expression on the 7th and 14th day both are evaluated (F=2067.263,P=0.000),the elevation on the 14th day is more significant than the 7th day.There is significant difference(P=0.000).Conclusions:(1) In homogeneity penetrability corneal transplantation rat model, immunology rejection does not happen on the 7^th day.On the 14^th day,all the acceptors had acute immunology rejection.(2) There is no ICOS positive lymphocyte in normal rat cornea,ICOS mRNA expression is not detected either.(3) ICOS protein and mRNA expression can be detected on implant tissue after transplantation,and the expression on the 14^th day after operation is higher than the 7^th day.(4) Compare with the peripheral blood CD3⁺ICOS⁺T/CD3⁺T expression in normal rat,CD3⁺ICOS⁺T/CD3⁺T expression in peripheral blood are elevated on the 7th and 14th days after transplantation,and the elevation on the 14th day is even more significant.These results suggest that co-stimulator ICOS has a close relationship with comeal transplantation immunological rejection.Partâ…¡Experimental study of transfer to corneal tissues of rats mediated by recombinant adenovirus carried the EGFP geneObjective:Investigate the transfection efficacy and safety of recombinant adenovirus vector with the report gene(EGFP) to rat corneas through different ways and seek for the best method of rAdV vector-mediated corneal gene transfer in situ. Methods:80 SD rats were divided into 4 groups randomly.Inject 15μl of Ad-EGFP (2.3×1011v.p./ml)subconjunctivally in group A and15μ1 of sodium chloride in Group B.Rats in Group C and D were injected with 15μl Ad-EGFP(2.3×1011v.p./ml) and sodium chloride into anterior chamber separately.Observe the cornea and other anterior segment tissues in vivo with slit lamp microscope everyday.Enucleate the eyeballs at 1st d,3rd d,5th d,7th day randomly after injection,and its paraffin sections by HE staining were used for observing the pathological changes.Expression of the transferred gene was monitored by confocal microscopy.The corneal samples were fixed and evaluated by transmission electron microscope examination.Results:(1) Corneas of the 4 groups rats remained transparent with no edema, aqueous liquid keep clear and the lens are transparent during the whole experiment period.(2) No apparent inflammation and neovascularization were observed in the cornea and other tissues of the anterior segment of the eye in all groups.(3) Ad-EGFP injection group(group A,C)showed positive staining on 1st d,3rd d,5th d,7th.The fluorescence intensity expression of GFP was increased with time and peaked on the 5th d,followed by a decline on 7th d and 14th d.In group A,positive staining was occured in comeal epithelium and stroma but not in endothelium.In group C,the positive staining was only occured in comeal endothelium,not in epithelium and stroma.Eyes injected with sodium chloride(groupB,D)showed no positive staining at all experiment period.(4) There was no apparent cell damage and significant toxicity in all groups with the transmission electron microscope.Virus particles were observed in comeal stroma(group A) after one day,which was black, high electron density particles group.In group C,Virus particles were observed in corneal endothelium.Conclusions:(1) These results demonstrate that reported genes can be introduced to keratocytes in situ by adenoviral vectors through different way.(2) Report gene expression could only occuer in corneal epithelium and stroma after inject Ad-EGFP into bulbar conjunctiva.(3) Report gene expression could only occuerd in corneal endothelial cell after injecting Ad-EGFP into anterior chamber.(4) Efficient and almost non-toxic transfer of the reported gene into comeal tissues can be successfully achieved by adenoviral. Partâ…¢The effect of blockade ICOS co-stimulator pathway with re-combinate adenovirus vector RNA intervention technique to cornea transplantation immunologyObjective:Observe the inhibitory action of Ad-silCOS-EGFP to the cornea transplantation acute immunological rejection with the co-stimulator passageway blockade.Methods:Build homogeneity penetrability corneal transplantation rat model with Wistar rat(donor) and SD(acceptor) rat.Divide the rats into four group with 20 rats in each group:penetrability corneal transplantation group as the control group(group A);Ad-silCOS-EGFP injection group(group B),inject Ad-silCOS-EGFP(titre 3.7×1011v.p./ml) 15μl in bulbar conjunctiva immediately after operation;Ad-EGFP injection group(group C),inject Ad-EGFP(titre2.3×1011v.p./ml) 15μl in bulbar conjunctiva immediately after operation;saline injection group(group D),inject 0.9%NaCl injection 15μl in bulbar conjunctiva immediately after operation.Observe the cornea implant situation with slit lamp microscope everyday after operation, observe the change of rejection index RI and record the implant survival time.Select 10 rats in each group on the 5th day after operation,test one part of the cornea with immunohistochemistry and RT-PCR technique;and the other part is made into frozen action and observe with confocal microscopy.Record the corneal implant survival time of the rest rats in each group.Results:(1) The average survival time of cornea implant in each group:group A(9.800±0.632)days;group B(14.700±1.418)days;group C(10.000±1.054)days; group D(10.100±0.876)days.Statistic result show homoscedasticity(P=0.306).There is significant difference between the average survival time of cornea implant in each group(F=52.383,P=0.000).Result of multiple comparison:there is significant difference between group B and the other groups(all the P value＜0.05);there is no significant difference between group A,group C and group D.The average survival time of corneal implant in group B(Ad-siICOS-EGFP injection group) is the longest. (2) Green fluorescence expression can be found in cornea implant in group B(inject Ad-siICOS-EGFP in bulbar conjunctiva group) and group C(inject Ad- EGFP in bulbar conjunctiva group),fluorescence can be found on the cornea epithelium and all the stroma.There is no green fluorescence expression in cornea implant of group A and group D.(3) ICOS positive cells got buffy dying cellular membrane and cytoplasm.ICOS positive lymphocytes can be seen in each group's corneal implant, mainly concentrate on the epithelium and stroma of corneal implant.ICOS positive lymphocyte in group B(Ad-siICOS-EGFP injection group) is significantly less than the group A,group C and group D;The score of ICOS protein immunohistochemistry dying positive cell in each group's implant:group A(1.9004±0.568);group B(1.100±0.316 );group C(1.8004±0.632);group D(1.900±0.568).Statistic result show homoscedasticity(P=0.368).there is significant difference between the scores of each group(F=5.214,P=0.004).The result of multiple comparsion:there is significant difference between group B and the other groups(all the Pvalue＜0.05);No difference between group A,group C and group D in statistic.The score of ICOS protein immunohistochemistry dying positive cell in group B is the lowest.(4) The ratio of electrophoresis strip brightness of the ICOS PCR product in cornea implant:group A(1.004±0.164);group B(0.621±0.096);group C(0.9294±0.134);group D (0.9184±0.120).Statistic result show homoscedasticity(P=0.446).There is significant difference(F=16.717,P=0.000).Multiple comparison result:there is significant difference between group B and each of the other groups(all P value＜0.05);no significant difference is existed between group A,group C and group D. The ratio of electrophoresis strip brightness of the ICOS PCR product of implant tissue in Group B(Ad-siICOS-EGFP injection group) is the lowest. Conclusions:(1) Ad-siICOS-EGFP can effectively inhibit the lymphocyte ICOSmRNA and ICOS protein expression in rat cornea implant therefore blockade the ICOS co-stimulator passage.(2) Inject Ad-siICOS-EGFP in bulbar conjunctiva can inhibit the acute immunological rejection in rat cornea transplantation therefore extend the survival time of cornea implant.