| Part1the differential tear proteomic analysis of rat penetrative corneal transplantation model using iTRAQObjectiveTo establish Autologous and homogenous model of corneal transplantation in rat and screen differentially expressed proteins and identify more biomarkers involved in the process of allograft rejection. Then the function and pathway of differentially expressed proteins would be assessed by bioinformatics analysis.Materials and Methods1. Forty two Wistar rats were divided into3groups at random:the autologous corneal transplantation group, the homogeneity penetrability corneal transplantation group (donor:SD rats, recipient:Wistar rats) and the control group.2. Tear samples from the two surgery groups were collected at2nd,7th and14th day respectively while for controls no surgery was done and tears of each individual was collected only once. The volume of each individual was usually7-10μl. Samples collected at same time point in the same group were pooled together so totally7specimens were obtained at last which were stored at-80°C until processed.3. After removal of high abundance proteins by using Removal System (MARS-3) affinity column, proteins from each specimen were quantified and enzymatic.4. The iTRAQ regents113,115,116,117,118,119and121were used to labeled the peptides from the control group tears,2nd day tears in homogeneity PKP group,7th day tears in homogeneity PKP group,14th day tears in homogeneity PKP group,2nd day tears in autologous corneal transplantation group,7th day tears autologous corneal transplantation group and14th day tears autologous corneal transplantation group respectively. Then, the mixture of the peptides was analyzed by two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS). 5. The ProteinPilot software was used for peptide identification and quantification. The fold change cut-off ratio>2or<0.5was selected to designate the proteins of differential expression (P<0.05).6. We observed the differential protein expression in the3groups and the three timing within1group. Bioinformatics techniques including Hierarchical Cluster Analysis, GO analysis and pathway analysis were used for further understanding of the function of the identified differential proteins on allograft corneal rejection.Results1. Compare the clinical evaluation of homogeneity PKP group and autologous corneal transplantation group according to homogeneity penetrability corneal transplantation score. Except that corneal transparency was not statistically significant at2nd day (p=0.123), the indexes including corneal edema, transparency, and neovascularization were all statistically significant at other timing (p<0.05).2. Totally277proteins were identified. The significant proteins identified in homogeneity PKP group at2nd day,7th day and14th day were35,36and31respectively.3. The GO results indicated that the mainly functions of the differentially expressed proteins in our study were neurological disease, free radical scavenging, cell death and survival and cell movement.4. The top enrichments of the pathways of the differentially expressed proteins were LXR/RXR activation, acute phase response signaling, clathrin-mediated endocytosis signaling, actin cytoskeleton signaling.5. Coronin-1A was significantly up-regulated in homogeneity PKP group than that in autologous corneal transplantation group. In homogeneity PKP group, the concentration of coronin-1A peaked on the7th day when rejection began to occur. In contrast, in autologous corneal transplantation group, the concentration of coronin-1A decreased gradually.6. Vitamin-D binding protein (DBP) was significantly up-regulated in homogeneity PKP group than that in autologous corneal transplantation group on the14th day. Conclusion1. The iTRAQ technology provides a good platform to understand the differentially expressed proteins and identify more significant biomarkers involved in process of allograft corneal rejection. It is reliable to obtain proteomic profiles in limited tear samples using iTRAQ for its good reproducibility.2. The bioinformatics analysis can broaden our mind and help us understand the global function and regulation of the differentially expressed proteins. Our study suggests that the nervous system disorders may play a vital role in corneal rejection, and in addition to the apoptosis of endothelial cells, free radical scavenging may be also related to inflammatory cell aggregation, immunologic injury and neovascularization. LXR/RXR activation may influence on graft survival though lipid and glucose metabolism, and the certain gene expression in macrophage. Actin cytoskeleton signaling may have effects on immune cell chemotaxis, antigen recognition, and the formation and activation of immune synapse.3. Coronin-1A may be a biomarker for early allograft rejection. It is close related to T cell migration and the interaction between T cells and APCs.4. Vitamin-D binding protein (DBP) may be close related to macrophage activation and migration. It may be a biomarker for acute corneal rejection.Part2the impact of antirejection drug on tear proteomics in rat modelObjectiveTo investigate the expression of tear differential proteins in rat model with dexamethasone and CsA intervention and to assess the function and pathway of differentially expressed proteins by bioinformatics analysis.Materials and methods1. Forty-two Wistar rats were divided into3groups randomly, dexamethasone group (donor:SD rat, recipient:Wistar rat), CsA group (donor:SD rat, recipient:Wistar rat) and control group. The PKP was just the same as Part1. 2. Tear samples were collected at2nd,7th and14th day respectively as stated in Part1.3. The iTRAQ regents114,115,116,117,118,119and121were used to labele the tear peptides from the control group,2nd day sample in CsA group,7th day sample in CsA group,14th day sample in CsA group,2nd day sample in dexamethasone group,7th day sample in dexamethasone group,14th day sample in dexamethasone group. Other processes and statistical methods were just as stated in Part1.Results1. Totally440proteins were identified by iTRAQ.2. For dexamethasone group, the significant proteins identified at2nd day,7th day and14th day were36,41,9respectively.3. For CsA group, the significant proteins identified at2nd day,7th day and14th day were45,49and43respectively.4. For dexamethasone group, the GO results indicated that the mainly functions of the significant proteins in our study were dermatologic disease, protein synthetizing and neurological disease. The top enrichments of the pathways of the differentially expressed proteins were EIF2signaling, LXR/RXR activation, clathrin-mediated endocytosis signaling, and acute phase response signaling. Compared with the group with no antirejection intervention, the enrichments of LXR/RXR activation, clathrin-mediated endocytosis signaling, and acute phase response signaling were declined greatly.5. For CsA group, the GO results indicated that the mainly functions of the significant proteins in our study were cell death and survival, protein synthetizing, dermatologic disease and immunologic disease. The top enrichments of the pathways of the differentially expressed proteins were LXR/RXR activation, EIF2signaling, clathrin-mediated endocytosis signaling, and acute phase response signaling. Compared with the group with no antirejection intervention, the enrichments of LXR/RXR activation, clathrin-mediated endocytosis signaling, and acute phase response signaling were declined greatly.6. The up-regulated degree of coronin-1A was lower than the group with no antirejection intervention (the group with no antirejection intervention:2d=5.75,7d=8.24,14d=4.29; dexamethasone group:2d=5.86,7d=1.63,14d=1.55; CsA group:2d=3.80,7d=3.40,14d=3.02).7. The up-regulated degree of DBP was lower than the group with no antirejection intervention on the14th day.Conclusion1. EIF2signaling can promote corneal endothelial recover mechanisms so that to prevent apoptosis.2. Acute phase response proteins have closely correlation with allograft rejection and its severity degrees. Thus, tear acute phase response proteins may be a biomarker for prognosis of PKP.3. Antirejection drugs reduce the expression of coronin-1A, which have influence on T cell immigration and its interaction with APCs.4. We may consider DBP as another biomarker for PKP prognosis.Part3the differential tear proteomic analysis of PKP patients using iTRAQObjectiveTo screen differentially expressed tear proteins in PKP patients with and without allograft rejection and to assess the function and pathway of significant proteins by bioinformatics analysis.Materials and method1. The patients undergoing PKP were classified into2groups based on whether allograft rejection occurred:rejection group (n=10) and not rejection group (n=10). Based on the condition of all20cases, routine local and/or systemic anti-rejection medications (corticosteroids and immunosuppression) were employed postoperatively2. For rejection group, tear samples were collected at the time of acute rejection occurred. For not-rejection group, tear samples were obtained at least2weeks after the operation. All sampled were stored at-80℃。3. The iTRAQ regents117and121were used to label the tear peptides from rejection group and not-rejection group. The fold change cut-off ratio>1.3or <0.77was selected to designate the proteins of differential expression (P<0.05). Other processes and statistical methods were just as stated in Part1. 4. Ingenuity pathway analysis microware was used to assess the interaction of differentially expressed proteins.Results1. The10cases with acute rejection included4leukoma,3herpes simplex keratitises,1fungus keratitis,1perforation, and1keratoconus.2. Totally428proteins were identified. The up-regulated differentially expressed proteins were clusterin, proline-rich protein4anda-amylase2B, while the down-regulated differentially expressed proteins were lipocalin-1, extracellular glycoprotein lacritin, serum albumin and transcription factor7-like1.3. The GO results indicated that the mainly functions of the significant proteins in our study were cell-to-cell interaction, cell compromise and organismal injury and abnormalities. The top enrichments of the pathways of the differentially expressed proteins were LXR/RXR activation, primary immunodeficiency signaling and acute phase response signaling.4. IPA results found that ubiquitin conjugation enzyme (UBC) had many interactions with our identified differentially expressed proteins.Conclusion1. High risk factors for allograft rejection include inflammation, neovascularization, lymphangiogenesis, previous rejection, large or eccentric grafts, infection and viral replication. In addition, even for keratoconus, unregular medication can result in corneal rejection.2. Clusterin, proline-rich protein4and a-amylase2B and lipocalin-1may be biomarkers for the occurrence of corneal rejection and the prognosis.3. UBC is the upstream molecular of many significant proteins identified in our study, and it may take part in the process of allograft rejection by regulating downstream proteins. |
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