| BackgroundCorneal blindness is one common clinical ophthalmocace, which could lead to the loss of visual acuity and affect the visual quality of patients with corneal blindness seriously. Corneal transplantation is one of the most effective treatment of irreversible corneal blindness.In recent years, with the continuous study of the immune tolerance mechanism of corneal transplantation, the rapid development of microtechnique, the perfect preservation technology of donor corneal tissue, which makes the success rate of keratoplasty surgery rise more than 90%.Corneal transplantation is the highest rate of successful operation among all organ and tissue transplantation, but the success rate of the operation is descent with the prolong of time after operation, especially corneal blindness patients with corneal neovascularization, alkali chemical burns, microbial infection, the history of many keratoplasty, and so on. Such high-risk corneal transplantation factors could lead to the increased rejection rate of corneal transplantation.Although the success rate of corneal tissue and organ transplantation is the highest, corneal graft rejection is still one main cause of corneal graft failure. Although the mechanism of corneal graft rejection has been studied a lot, it is still not very clear, how to prevent corneal allograft rejection and improve the survival rate of corneal graft still need study furtherly.The mechanism of corneal graft rejection is complex, involving many factors. The host antigen presenting cells (antigen presention cells, APCs) could reach corneal limbus to capture corneal allogeneic antigen by blood pipeline network. The costimulation factors could active T lymph cells with the assistance of antigen presentation cells, donor antigen could induce donor antigen specific effects by transitional drainage to the mandibular lymph nodes and neck lymph nodes, which is also corneal allograft rejection reaction.Dendritic cells (DC) from corneal edge may be actived by the donor antigen to induce indirect immune rejection to form graft versus host disease. DC derived from comeal graft may also be actived by the host antigen to form direct immune rejection, that is also graft versus host disease. However corneal graft rejection is mainly composed of host versus graft rejection reaction. APC mainly includes DC and macrophages. DC is the strongest antigen-presenting function of cells, therefore regulatory DC plays an important role in the prevention and treatment of corneal allograft rejection.It was shown that toll receptors were widely expressed in ocular surface tissue and DC, which participate in various ocular surface inflammation reaction. The Toll like receptor 2 (Toll-like receptor 2, TLR2) involved in the process of the prevention of corneal graft rejection reaction by glucocorticoid and the expression of NF-kappa B nuclear transcription factor decreased during the preventive effect of immunosuppressant cyclosporin A on corneal graft rejection reaction.It demonstrated that the MyD88 (myeloid differentiation factor 88) was the linking protein of TLR2 and NF-kappa B signal transduction, which played an important role in the inflammation of ocular surface. It was showed that MyD88 gene knockout could promote the recovery of renal function after kidney transplantation to induce immune tolerance in mice.Therefore, it is very important to study how to prevent corneal allograft rejection by blocking the adaptor protein molecular MyD88 and NF-κB nuclear transcription factor in the MyD88 dependent NF-κB signaling pathway, which was very important significance to induce corneal transplantation immune tolerance. Then, according to the basis of the previous studies and the progress of latest study, we could speculate that MyD88 dependent NF-kappa B pathway has an important role in the prevention of corneal allograft rejection.So MyD88 and NF-kappa B were the key target factor, which were intervened respectively to inhibit their expression in rat local ocular surface, and the rejection occurrence of corneal graft after corneal transplantation were observed. This was helpful for us to furtherly understand the mechanism of corneal allograft rejection, which was favor for the prevention and treatment of corneal graft rejection, to improve the success rate of corneal transplantation operation to form stable recovery of corneal blindness and better postoperative visual acuity.Part one. Effect of NF-kappa B inhibitor on rat corneal allograft immune rejectionObjectiveTo investigate the effect of NF-κB inhibitor in the MyD88-dependent NF-κB signal pathway on the prevention of penetrating corneal graft rejection.MethodsThe establishment of 40 rats penetrating corneal transplantation models were randomly divided into four groups:Isograft group、Allograft group、Dexamethasone group and PDTC group. On the first postoperative day, Allograft group and Isograft group were treated with tobramycin eyedrops, Dexamethasone group received TobraDex eyedrops, NF-kappa B inhibitors group were given tobramycin eyedrops and 10mg/ml PDTC eyedrops, one drop each time, four times every day. The postoperative observation and RI (rejection index) and survival analysis of corneal graft were analysized, the HE staining of corneal graft and TNF-alpha were analysized after postoperative 15 days. The mRNA and protein expression of TLR2, MyD88 and NF-kappa B P65 were analysized by RT-PCR and Western blotting.ResultsOn the postoperative 5,7,9,11,13 and 15 days, the rejection index in the allograft group was higher than the other three groups with statistically difference (P<0.05). The median survival time of corneal graft in the PDTC group(MST,23.00days, n=10), Allograft group(MST,14.00days, n=10) and the dexamethasone group (MST, 24.40days, n=10), the MST in the four groups were various with statistical difference (χ2=66.491, P<0.05). The expression of TNF-a in the Dexamethasone group and the PDTC group was less than that of allograft group. On the postoperative 15 day, the mRNA and protein expression of TLR2, MyD88 and NF-kappa B P65 in the dexamethasone group and PDTC group were significantly less than the allograft group with statistical difference (P<0.05).ConclusionDexamethasone has an inhibitory effect on corneal allograft rejection through the MyD88 dependent NF-kappa B signal pathway. PDTC may inhibit rats corneal allograft rejection by inhibiting the NF-kappa B activity, MyD88 dependent NF-kappa B signaling pathway may be an important regulatory pathway on the prevention of corneal allograft rejection.Part two. Effect of MyD88 inhibitor on rat corneal graft immune rejectionObjectiveTo investigate the effect of MyD88 inhibitor on the prevention of rat corneal allograft immune rejection.MethodsThe establishment of rats penetrating corneal transplantation models were randomly divided into three groups (n=6):Allograft group、Dexamethasone group and MyD88 inhibitor group. On the first postoperative day, Allograft group treated with tobramycin eyedrops, Dexamethasone group received TobraDex eyedrops, MyD88 inhibitors group were given tobramycin eyedrops and 2mg/ml ST2825 eyedrops, one drop each time, four times every day. The corneal graft rejection were observed by slit lamp microscope.The score of RI and survival analysis(mean survival time, MST) were analysized in three groups.ResultsOn the postoperative 5,7,9,11,13 and 15 days, the scores of RI among the three groups, such as the Allograft group, the Dexamethasone group and the MyD88 inhibitor group, were statistically significant difference(F=161.486, P<0.05). On the postoperative 5,7,9,11,13 and 15 days, the scores of RI in the Dexamethasone group and the ST2825 group were lower than that of the allograft group with statistical difference(P<0.05). The RI was no statistical difference in the ST2825 group compared with the Dexamethasone group(P=0.458).With the elapse of time, the rejection index of corneal graft in each group increased. The median survival time of corneal graft in Allograft group (MST, 13.00days, n=6), ST2825 group (MST,23.00days, n=6) and dexamethasone group (MST,24.00days,n=6). Overall median survival time among the three groups was with statistically significant difference(χ2=24.110, P<0.05). The median survival time of corneal graft in ST2825 group and dexamethasone group was higher than in the allograft group with statistical difference(χ2=12.230, P<0.05). The median survival time of corneal graft in the dexamethasone group was higher than that in PDTC group with no statistical difference (χ2=2.360, P=0.124).ConclusionMyD88 inhibitor 2mg/ml ST2825 eyedrops have no obvious ocular toxicity in the short term by slit lamp microscope, it has a certain inhibitory effect on rat corneal allograft immune rejection, which may have some relationship with the inhibitory expression and activity of MyD88 in ocular tissue.Part three. Effect of NF-kappa B in rat dendritic cells on corneal allograft rejectionObjectiveTo investigate the effect of DC transfected by Lv-siRNA-Rel-A on rat corneal allograft rejection.Methods(1). DC derived from rat myeloid cells was cultured in RPMI-1640 medium with the application of 5ng/ml GM-CSF(granulocyte macrophage colony-stimulating factor) and 5ng/ml IL-4 to induce DC differentiation. The DC surface costimulatory molecules of CD86, MHC Ⅱ and OX62 were assayed by flow cytometric analysis. The stimulation effect of allogeneic T cells by DC was detected by MLR (mixed lymphocyte reaction).(2). The gene silencing effect of Rel-A-siRNA in dendritic cells by Western blotting analysis. SiRNA sequence was applied efficiently to inhibit the expression of Rel-A by Lv-siRNA-Rel-A vector construction. The suppression effect of Rel-A expression in rats myeloid derived DC by Lv-siRNA-Rel-A vector transfection was verified by Western blotting analysis. The inhibitory effect of siRNA-Rel-A on the expression of Rel-A in DC was assayed by western blotting. The phenotype of DC with CD80 and CD86 and MHC Ⅱ after transfection was analyzed by flow cytometry. The ability of DC to stimulate the proliferation of allogeneic T cells was detected by MLR. Transfected DC cells were randomly divided into four experimental groups:A. control group (without any interference factors); B. transfection reagent control group (transfected with empty RNAi-Mate and without the Rel-A-siRNA); C. siRNA negative control group (universal negative transfection and nonhomologous gene sequence control); D. Rel-A-siRNA group.(3).2×106 Lv-shRNA-Rel-A DCs were cultured in RPMI-1640 medium loaded with the donor antigen for 7 days, which were injected into rats penetrating corneal transplantation models and were divided into six groups:Isograft group, Allograft group, reagent transfection group, DC group, DC with empty vector group, DC treatment group. The RI scores of corneal graft rejection were observed by slit lamp microscope. The mRNA and protein expression of TLR2, MyD88, NF-kappa B p65 were detected by Western blotting and RT-PCR analysis.Results(1). RPMI-1640 medium containing 5ng/ml GM-CSF and 5ng/ml IL-4 could obtain immature DC derived from rat myeloid, the expression level of CD86 was low, the ability of DC to stimulate the proliferation of allogeneic T cells was low in RPMI-1640 medium. The costimulatory molecules expression of MHC Ⅱ, CD86 and OX62 among the three groups were with statistical difference (F=969.213, P<0.05). The MHC II expression of the DC surface was higher than that of CD86 (P=0.039) and lower than that of OX62 (P<0.05) with statistical difference. The expression of CD86 was lower than OX62 with statistical difference(P<0.05). Expression positive rate of MHC Ⅱ, CD86 and OX62 increased gradually with the prolongation of culture time. It demonstrated that the phenotype of DC continued to mature, the purification of DC was more and more, and its antigen presenting ability strengthened constantly.(2). The results of Mixed lymphocyte reaction (MLR). The proliferative ability of allogeneic T lymphocyte stimulated by DC in the three groups (2.5×103 、 5×103、 1×104 DC group) was statistical difference (F=23.886, P<0.05). The optical absorption A value in the 2.5×103 DC group was less than in the 5×103 DC group (P=0.001) and the 1×104 DC group (P<0.05) with statistical significance, optical absorption A value was lower in 5×103 DC group than the 1×104 DC group with statistical significant(P=0.008). The proliferative ability of allogeneic T cells stimulated by DC (A value) enhanced significantly which were cultured for 12 days.(3). Flow cytometry analysis of DC phenotype transfected with Lv-shRNA-Rel-A. It was showed that the expression of CD86 and CD80 in RNA interference group (Lv-siRNA-Rel-A transfection DC group) decreased compared with the empty vector DC group(P<0.05). The expression of CD80 and CD86 was significantly lower than that in the control group with significant difference (P< 0.05), which indicate that siRNA-Rel-A could reduce the DC surface costimulatory molecules expression of CD80 and CD86. The expression of MHC II on DC surface did not decrease obviously (P=0.08).(4). The results of mixed lymphocyte reaction (MLR) on Lv-shRNA-Rel-A DC. The proliferative ability of allogeneic T lymphocyte cells stimulated by DC was various among four groups with statistical difference (F=23.890, P<0.05). It included blank control group (group A), transfection reagent control group (group B), siRNA negative control group (group C), and Rel-A-siRNA group (group D).The proliferative ability of allogeneic T lymphocyte cells stimulated by DC in LPS-DC group was weaker than the LPS-DC group with statistical difference (P< 0.05). It showed that DC transfected with Lv-shRNA-Rel-A could reduce the stimulation of T cells proliferation.(5). The protein expression of Rel-A in dendritic cells. The blank control group (group A), reagent transfection group (group B), empty vector group (group C), siRNA-Rel-A group (group D), the expression of Rel-A protein in four groups was various. The expression of Rel-A protein in siRNA-Rel-A group decreased more than empty vector group and blank control group(P<0.05).(6). The RI score of corneal graft rejection. Repeated measures analysis of variance was used among the five groups (Isograft group, Allograft group, DC group, Empty vector group, and DC treatment group). Sphericity test statistics(Mauchly’s Test of Sphericity), W=0.967,χ2=0.642, P=0.725, which accepted the globular hypothesis. On the postoperative 5 day,10 day,15 day, the scores of RI in the five groups were various with statistically significant difference (F=78.560, P<0.05).The scores of RI was low in treatment group than in the allograft group with statistical difference(P<0.05). The treatment group was compared with the Isograft group without statistically significant differences (P=0.037). With the prolong of time, the rejection index of corneal graft in each group increased.(7). The protein expression of Rel-A in rats cornea. On 15 days after surgery, the relatived protein expression of Rel-A among the six experimental groups was various with statistical difference(P<0.05). Lv-siRNA-Rel-A-DC was injected into rat corneal transplantation model in the DC treatment group. The protein expression of Rel-A decreased in the corneal tissue and the activity of NF-kappa B also decreased in the treatment group.(8). The mRNA expression of TLR2 and MyD88 in each group. Certain amount mRNA expression of TLR2 was observed in normal cornea. On days 15 after operation, the expression of TLR2 mRNA showed significant differences among Normal group, Isograft group, Allograft group, DC group, Empty vector group and DC treatment group(F=27.807, P<0.001). The mRNA expression of TLR2 in DC group was significantly lower than that of Allograft group (P=0.029). The mRNA expression of TLR2 in the treatment group was not significantly lower than that of Allograft group (P=0.079).Certain amount mRNA expression of MyD88 was observed in normal cornea. On days 15 after operation, the expression of MyD88 mRNA showed significant differences among Normal group, Isograft group, Allograft group, DC group, Empty vector group and DC treatment group(F=85.844,P<0.001). The mRNA expression of MyD88 in DC group was significantly lower than that of Allograft group (P=0.035). The mRNA expression of MyD88 in the treatment group was significantly lower than that of Allograft group (P=0.044).ConclusionLv-siRNA-Rel-A transfected DC could inhibit the expression of Rel-A gene in DC and the expression of CD80 and CD86 molecules on the DC surface, and to maintain immature DC. Lv-siRNA-Rel-A transfected DC could inhibit the proliferative reaction of allogeneic T cell. The Lv-siRNA-Rel-A transfected DC loaded with donor antigen was injected into rat corneal transplantation model in vivo, which could inhibit corneal allograft rejection probably by MyD88-dependent NF-kappa B pathway. The MyD88-dependent NF-kappa B pathway may play an important role to maintain rats corneal transplantation immune tolerance. |
PDF Full Text Request