| BackgroundCorneal allograft is one of the most successful solid organ transplantation, however rejection after the transplantation is the major cause of the failure.Studies have shown that:corneal allograft rejection is a multifactor participated, extremely intricated, and highly controled process.After the transplantation, as the presenting of the antigen, CD4+T cell activate, leading to the differentiation of Thl and secretion of IL-2、IFN-y and TNF-a who can mediated destructive DTH,they play a leading role in corneal allograft rejection.Kuffova believes that at the early stage of the transplantation immune reactions manifest as inflammation.Holland also observed that in the early days of the corneal rejection exist nonspecific inflammation:T cells and macrophages gathered around the incision and sutures, corneal graft are thickened.Holland emphasized that the rejection of the cellular immune response is different from the isograft of cellular immunity, nonspecific inflammation around the early postoperative incision and sutures is an important factor of the growth of new blood vessels which were induced by donor damaged endothelial cell.In inflammatory plant bed, the dendritic cells come from the donor transitions to the host’s neck lymph nodes to activate T cells directly induce rejection.The pathological changes of the acute rejection in cornea allograft have two main characteristics:the corneal stromal layer infiltrated with inflammatory cells and neovascularization.Corneal stroma inflammatory infiltration was based on monocytes, and lymphocytes、plasmacyte、neutrophils and so on.The inflammation may led to the disappearing of the advantage as an immune amnesty regional in cornea, rejection occurred early and severe, even immune inhibitors can not regulate.Although the initial rejection was caused by the acquired immune system of T-lymphocytes in the host, new evidence suggests that the innate immune system also plays an important role.Toll-like receptors was a bridge which link innate immunity and acquired immune, and Toll-like receptor 2 (TLR2) was the one most widely expressed、most largely identified of pathogenic microorganisms and types of molecules in human cloning TLRs family.It was distributed in the APC、 inflammatory cells、a variety of tissue cells and tumor cells, and played a innate immunity role in the infection of bacteria、fungi、viruses and other pathogens, and connect the acquired immune through its intracellular signal transduction. Compared with other TLR, TLR2 had more features:other TLR needs to form complex with TLR2 so as to work.At present, the study found that TLR2 at least have two intracellular signal transduction pathways.According to the differece of adaptor protein, it can be divided into MyD88 protein dependent and nondependent ways,and main for the former.Studies have found that TLR2 monoclonal antibody can relieve acute rejection after cardiac graft and prolong the survival time by blocking TLR2-Myd88-NF-KB and TLR2-Myd88-IFR1 pathway.Other scholars thought MyD88 defecting mice in the renal transplantation may lead to donor specific immune tolerance in order to avoid acute or chronic rejection.Study confirmed that using in combination of CD40L antibody can reduce the infiltration of inflammatory cells after transplantation both in MyD88 defect donor and receptor.These studies suggest that:TLR2 may participated in the allograft immune rejection after organ transplantation through the MyD88 transmiting signals.Our previous study have also confirmed that TLR2 may participate in the rat cornea transplant rejection,the expression of TLR2mRNA was detected specially increased 9 days postoperatively, which was in conformity with the histopathological result, immunofluorescence result showed that TLR2 express specificity increased 9 days after corneal transplantation along with the membrane transfer. Now we use TLR2 monoclonal antibody blocking the expression of TLR2 to observe the survival situation of the graft,observe the rejection of graft 9-15 days after the transplant and HE dyeing, and do immunohistochemical and real-time fluorescent quantitative PCR detectionin 9 days postoperation to detect the expression and distribution of TLR2 and its downstream signaling molecule MyD88, so as to confirm the important role of TLR2 and its downstream signaling molecule MyD88 in corneal transplantation rejection.Thoroughly understanding of TLR2/MyD88 signaling pathways can not only help us clear the role of TLR2 in immune rejection and its molecular basis, but also explore the immune response mechanism of some diseases such as microbial infection、tumor、allergy and autoimmune diseases, providing a new pointcut for the treatment of the disease and development of vaccine in the future, open up a broader way for clinical treatment. This paper fromed the perspective of animal experiments with rat corneal transplantation, and based on the research of using TLR2 monoclonal antibody after corneal transplantation on rats, in order to further confirm the key role of TLR2 and its downstream signaling molecules MyD88 in corneal transplantation rejection, thus providing theoretical support for clinical prophylaxis and treatment of corneal allograft rejection.Part I An experiment research about effect of TLR2 monoclonal antibody on the survival condition of rat corneal allograft after transplantationObjectiveTo observe the survival condition of TLR2 monoclonal antibody to rat corneal graft after penetrating keratoplasty (PKP).MethodsThere were three groups:normal group、group A and group mAb, Penetrating keratoplasty (PKP) was performed in 2 groups of rats for allograft corneal transplantation (group A), and allograft corneal transplantation with TLR2 monoclonal antibody treated (group mAb).Group mAb was treated with 15ug TLR2 monoclonal antibody, while group A with equal dose of saline.They were injected on day 0、2、4、6 and 8 from the bulbar conjunctiva respectively altogether for 5 times.The opacity and neovascularization of cornea were examined by a slit-lamp microscope and scored based on the rejection index, make the normal cornea in reference.Two experimenter counted for average and observed the average time of the corneal allograft rejection for each rat.Make each group of eyeballs paraffin embeded on day 9 and 15 after the transplantation for HE staining to observe the change of corneal tissue structure in each layer.ResultsAs time goes by, edema、opacities and neovascularization occurred after the operation in all the operational groups, in particular for group A. The rejection time of group mAb was (15.67±0.753) d, significantly delayed comparing with group A (9.67±0.876)d, there was obvious difference between them (p=0.001). HE staining showed that severe corneal edema、disordered stroma structure、a lots of inflammatory cells infiltration and new blood vessel lumen in stroma were seen in group allograft while mild inflammatory response was found in group mAb.ConclusionTLR2 monoclonal antibody can significantly prolong the survival time in rat corneal graft after transplantation and reduce the inflammatory response after transplantation.PartⅡ Explore the role of TLR2/MyD88 signaling system following rat corneal graft rejectionObjectiveTo explore the role of TLR2/MyD88 signaling system after penetrating keratoplasty(PKP) in rats.MethodsThere were 4 groups:normal group、group I、group A and group mAb. PKP was performed in 3 groups of rat for corneal isograft,corneal allograft,and allograft treated with TLR2 monoclonal antibody.Group isograft and allograft were treated with saline while group mAb with equal doses of 0.5g·L-1 TLR2 monoclonal antibody, They were injected on day O、2、4、6 and 8 after the operation from the bulbar conjunctiva respectively,altogether for 5 times.The opacity and neovascularization of cornea were examined by a slit-lamp microscope and scored based on the rejection index, make the normal cornea in reference.Two experimenter counted for average.Make each group of eyeballs paraffin embeded on day 9 after the transplantation for HE and IHC staining. The detection of the expression of TLR2mRNA and MyD88mRNA were using qPCR.ResultsOver time, edema、opacities and neovascularization of the corneal allograft occurred after the operation in all the groups, in particular for group A.HE staining results showed that normal cornea tissue composed by five layers, each layer structure was clear, collagens were rows loose and neat in stroma; On day 9 after the operation,severe corneal edema、disordered stroma structure、a lots of inflammatory cells infiltration and new blood vessel lumen in stroma were seen in group A while mild inflammatory response and clear structure was found in group I and mAb.IHC result displayed that TLR2 and MyD88 are weakly expressed in normal group、 group isograft and group mAb in corneal epithelium, and they were increased in group A both in epithelium and stroma,particularly stroma.We can see the relative expression of TLR2 mRNA and MyD88 mRNA in normalgroup was 1.01±0.213、 1.00±0.07,group I 3.40±1.007、2.42±0.479、group A 23.79±1.974,4.11±0.572 and group mAb 12.87±2.001、2.32±0.758.TLR2 mRNA and MyD88 mRNA expressed much higher in group A compared with group I and normal control, in addition, the expression in group mAb was much less than group A,and they had a identical trend. Perform a statistical analysis in the expression of TLR2mRNA and MyD88mRNA, and make a test for homogeneity of variance, they were both have a constant variances with valuel.081 and 0.145 respectively. Again use the LSD method between each other, we got the data that the expression of MyD88mRNA in three surgical intervention group were significantly increased compared with normal group (P= 0.012,0.016,0.012), and obviously lower in group mAb compared with group A(P= 0.003), the expression of TLR2mRNA has no statistically significant difference between the normal group and group I(P= 0.098), while the normal group、group mAb and group A statistically significant difference, and the trend of its expression was consistent with MyD88.ConclusionTLR2 monoclonal antibody inhibits TLR2 and its downstream signal MyD88’s expression; TLR2/MyD88 signaling system involved in corneal transplantation immune rejection, and effect the outcome of corneal graft. |
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