Study On The Effects Of Indoleamine 2,3 Dioxygenase On Rat Penetrating Keratoplasty Rejection

PartⅠStudy on Expansion and Biological Characteristics of Dendritic Cells from Rat Bone MarrowObjective: To investigate the isolation, purification, expansion and biological characteristics of dendritic cells (DCs) derived from bone marrow in vitro.Methods: The rat bone marrow cells were collected and the rIL-4 and rGM-CSF were added into the fresh medium, cultured for 48h the floating cells were removed, and fresh media added every 48h. Typical dendritic morphology of the cultured DCs was observed under light microscope and transmission electron microscope. Expressions of MHC classⅡand CD86 were detected by flow cytometry. The cultured DCs were cocultured with allogenetic T cells derived from rat spleen, and T cell proliferation was measured by CCK8.Results: The cultured DCs had the typical morphological characterization of dendritic cell, and the expression rates of MHC classⅡand CD86 were 23.6% and 6% at the 6th day of the culture; by the 14th day of the culture the rates reached 74.3% and 66.5%. The cultured DCs could notably stimulate the proliferation of allogenetic T cells at the 14th day of culture.Conclusion: The adherent culture of rat bone marrow cells, removal floating cells and co-culture with rIL-4 and rGM-CSF could obtain a number of high purity of DCs, which lay the foundation for study on DC's function. PartⅡThe Expression of IDO in DC and its Effects on Allogenetic T Lymphocyte ProliferationObjective: To investigate the expression of IDO on DCs and its effects on allogenetic T lymphocyte proliferation.Methods: The plasmid PGCTLA4Ig and PEGFP were transfected into dendritic cells of F344 rat mediated by LipofectamineTM 2000. Expression of CTLA4Ig was detection by immuno-fluorescence. The expression of IDOmRNA and IDO protein in DCs were analyzed by RT-PCR method and Western Blot method respectively, and IDO activity was determined by high-performance liquid chromatography (HPLC). MLRs cultures were set up with mitomycin C treated F344 derived DCs as stimulators and Lewis-derived T lymphocyte as responder cells with or without L- tryptophan. T lymphocytes proliferation was determined by CCK8 assays and IDO activity in coculture supernatant was determined by HPLC. The flow cytometry assay in Annexin V/PI was used to detect the apoptosis of T lymphocyte after mixed lymphocyte reaction.Results: The expression of IDO mRNA and protein in DCs were increased gradually after the transfection of gene CTLA4Ig(P<0.01), and IDO functional activity in DCs was also increased significantly(P<0.01). CTLA4Ig-transferred dendritic cells could induce allogeneic T cell apoptosis, and so T lymphocytes proliferation decreased apparently in vitro(P<0.01). Added L-tryptophan in the MLR, the apoptosis rate decreased and T lymphocytes proliferation raised lightly (P<0.01).Conclusion: IDO activity in DCs is relation to T lymphocyte proliferation, and DCs may perform their immunosuppressive function by IDO activity in vitro. PartⅢEstablishment of the Model of Rat Penetrating KeratoplastyObjective: To establish stable rat penetrating keratoplasty model for advanced study in vivo.Methods: Corneal transplantation was performed from F344 rats to Lewis rats, as described by Williams and Coster. The immune rejection was observed by slit lamp biomicroscope and immunohistochemistry methodResults: The immune rejection took place on all the models of rat allogenetic penetrating keratoplasty and the average survival time was about 7.92±1.69 days. In the isogenic group, the inflammation response was obvious at about one week after operation, but the edema and opacity disappeared after one week. Immunohistochemistry showed: in allogenetic group corneal epithelium was integrated , the endothelium was absent and there were a lot of phlogistic cells. In the isogenic group corneal endothelium was integrated phlogistic cells, and there were few phlogistic cells.Conclusion: The model of rat penetrating keratoplasty was successfully established.PartⅣThe Inhibition Effects of DCs with High Expressing Functional IDO on the Penetrating Keratoplasty Rejection in RatObjective: To study the effects of donor derived DCs with high expressing functional IDO on the penetrating keratoplasty rejection in rat.Methods: Corneal transplantation was performed from F344 rats to Lewis rats, as described by Williams and Coster. Then divided recipient into five goups: group PBS, group CsA, group DC, group induced DC and group induced DC+ L- tryptophan . At 0 and 3rd day after operation injected PBS 50μl, 106 BMDC(50μl), 106 induced BMDC(50μl), 106 induced BMDC(50μl, with 250μmol/l L- tryptophan) respectively, group CsA 10g/l CsA 50μl OD tid. The movement of the DCs were be observed by immunofluorescence. T cell apoptosis in the submandibular lymph node (SLMN) was determined by TUNEL assay. Mixed lymphocyte cell reaction of receptor to donor was performed by CCK8 on 7th day after operation. A rejection reaction was observed using a slit lamp, and the survival of corneal allograft was evaluated by Holland criterion.Results: There are many TUNEL+ cells in the rats'submandibular lymph node in the group induced DC seven days after the operation. The survival time of the above groups were 8.19±2.03days, 22.36±2.67days, 7.05±1.26days, 18.33±3.12days and 9.21±1.85 days. The survival time of corneal allografts was prolonged significantly in group induced DC and group CsA (P<0.01), and the responsiveness of receptor's T cell to donor's antigen was significantly lower in group induced DC and group CsA (P<0.05).Conclusion: Donor derived DCs with high expression of IDO could specifically suppress receptor's immune response to donor's antigen and significantly prolong the survival of corneal allografts.