The Mechanism Of IL-34 In Rejection Of Rat Corneal Transplantation And An Integrated Deep Sequencing Analysis Of MicroRNAs In Transplanted Corneas

Part1 The Mechanism of IL-34 in Rejection of Rat Corneal TransplantationObjective To investigate the expression of IL-34 and its mechanism in immune rejection after corneal transplantation in rats.Methods SD rats were used as donors and Wistar rats as recipients to establish corneal transplantation experimental models.60 Wistar rats were randomly divided into 3 groups according to the random number table method.Group B was treated by corneal autograft group,and corneal allograft was performed in group C and D.Another 10 cases were served as normal control group(group A).After B,C group was treated with Tarivid eye drops postoperation,D group was treated with TobraDex postoperation.The survival rate of corneal grafts and survival analysis were evaluated according to the rejection criteria of Larkin et al.The corneal grafts were taken from the rest of the rats at fourteenth days after the operation.Histopathological,immunohistochemical and RT-PCR examinations were performed.Results Survival analysis showed that corneal allograft did not occur in group A and B,and mean survival time(MST)was 26 ± 0.97d in group D,which was much higher than that in group C,and the average survival time of cornea was 10±1.55 d(P<0.001).HE staining showed that there were a lot of inflammatory cell infiltration and neovascularization in the corneal tissue of group C,and there were only a few inflammatory cells and neovascularization in group D.Immunohistochemistry showed that IL-34 protein expression in C group(0.0894±0.0056)was significantly higher than that of group A(0.0377±0.0023),B group(0.0684±0.0044)and group D(0.0445±0.0045)(F=145.21,P<0.01),and mainly concentrated in the epithelial and stromal layer.RT-PCR showed that the expression levels of IL-34 and IL-1β,IL-17A,TNF-α mRNA in corneal tissue of group C were significantly higher than those in group A,group B and group D(P<0.05).Conclusion IL-34 is involved in the rejection after corneal transplantation in rats,and the delay of rejection can be delayed by inhibiting the expression of IL-34 and related signaling pathways.Part 2 An Integrated Deep Sequencing Analysis of MicroRNAs in Transplanted CorneasObjective To analyze the differential expression and regulation network of microRNA after corneal transplantation in rats by high-throughput sequencing technology and bioinformatics technology,so as to provide theoretical and experimental basis for exploring new therapeutic targets of corneal allograft rejection.Methods High throughput sequencing technology was used to screen the expression of miRNA in normal control group(group A),autokeratoplasty(B group)and keratoplasty group(C group).Target genes prediction software was used to predict miRNA target genes(targetScan,Miranda,miRDB and CLIP).Bioinformatics analysis of GO,KEGG,PPI and MetaCore was carried out by using CO predicted target genes,and RT-PCR was used to verify the key miRNA.Results In the comparison group of B group and A group,there were 22 obvious upregulated and 4 down regulated miRNA.17 of the comparison group of C group and B group significantly increased miRNA and 7 miRNA 7 significantly(P<0.01).Among them,miR-155-5p,miR-142-3p,miR-142-5p and miR-223-3p are at the same time in the above 2 comparison groups.GO and KEGG analysis showed that the function of miRNAs was mainly involved in metabolic pathway,cytokine secretion and tumor immunity.PPI and MetaCore analysis showed that miR-155-5p was an important gene in the regulatory network of miRNA-mRNA gene.MetaCore analysis showed that C/EBP,p53 and SP1 were the key transcription factors in the network.Conclusion MiRNAs are involved in corneal allograft rejection.MiR-155-5p,miR-142-3p,miR-142-5p and miR-223-3p may simultaneously regulate corneal loss and corneal graft rejection.C/EBP beta,p53 and SP1 may be the key proteins.