Experimental Study On Effects And Mechanism Of A Inhibitor (J2) Of CD4/MHC-Ⅱ Molecule In Prevention Of Mouse Corneal Allograft Rejection

Objective: To observe characteristics of corneal rejection in mouse model and discuss the standard of graft rejection.Methods: The C57BL/6 and BALB/c were used as donors and recipients to establish corneal transplant model. Group A, autograft, Group B, allograft. The two groups were divided into 4 subsets by the index of comeal opacity on twelfth day after comeal transplantation, the index of comeal opacity of Group A1 and Group B1 was less than 2, when the index of comeal opacity was more than 3, they were grouped into Group A2 and Group B2;The variation of index of comeal opacity and comeal neovascular had been compared. We also compared the difference of histological examination at different time between these groups.Result: There were no difference of the index of comeal opacity and CNV between Group A and Group B on 7th day and 12th day after transplantation. Histological examination had also found no disparity at that time between the two groups. When the index of comeal opacity reached 3 in group B1, histological section showed the rejection feature by mononuclear cells infiltrated into grafts.Conclusion: Index of comeal opacity exceeds 3 from 7^th day to 12^th day after comeal transplantation could not be judged as graft rejection;As rejected graft could recover clear, we should pay more attention to variation of comeal opacity to make judgment of graft rejection since 12^th day after comeal transplantation;Theanimal that index of comeal opacity still exceeds 3 should be discarded on 12^th day after transplantation,Part TwoObjective: To investigate the effects of J2 on preventing of allograft rejection in mouse skin transplantation and corneal transplantation;To observe the drug safety for mouse.Methods: The C57BL/6 and BALB/c were used as donors and recipients to establish skin allograft model, and divided into 3 groups randomly. Group A, allograft control (this group was given placebo only);Group B and group C, allograft groups, were treated with orally CsA and J2, respectively. The drugs were delivered for 12 days beginning at the day of transplantation. Graft survival was assessed by Kaplan-Meier analysis. On day 14 after transplantation, histological examination was performed to confirm the clinical diagnosis of rejection. The weight of mouse and the organ coefficient were compared, histological examination of liver and renal were performed;The C57BL/6 and BALB/c were used as donors and recipients to establish corneal allograft model, and divided into 6 groups randomly. Group A, autograft control;Group B, allograft control (the control groups were given placebo only);Group C, allograft groups, Was treated with orally CsA (lOmg/kg), Group D,E,F, allograft groups, were treated with orally J2 by different dose , respectively. The drugs were delivered for 12 days at the beginning day of transplantation. Graft survival was assessed by Kaplan-Meier analysis, the index of corneal opacity and CNV were compared according to the method of part one. On day 18 after transplantation, histological examination was performed to confirm the clinical diagnosis of rejection.Result: The average transplant survival time in the skin allograft control (Group A) was (13.7±1.4)d. Treatment with J2 (Group C) led to a statistically prolongation of transplant survived (17.7±2.4)d (P < 0.01). Compared with treatment with CsA (Group B) (18.2±2.1)d, there was no statistically significant difference between the two groups(P > 0.05). Fewer inflammatory cells were found in the skin allograft of J2 group. The curve of the weight between differentgroups had no different. The histological section of liver and renal showed J2 have no harm, the organ coefficient of J2 group had no difference with normal mouse. The average transplant survival time in the corneal allograft control (Group B) was (17.77±2.10) d. Treatment with J2 (30mg/kg) led to a statistically prolongation of transplant survival to (40.55±8.27) d (P < 0.01). Compared with treatment with CsA (Group C) (38.14±9.13) d and group E (38.14±9.13) d, there were no statistically significant difference between the three groups(P > 0.05), the survival time of Group F was fewer than Group E and Group D, there was no statistically significant difference between Group E and Group D. Fewer inflammatory cells were found in the corneal allograft in group D. By step regression stastistics. The corneal neovascular had relationship to suture on day 7 and day 12 after corneal transplantation.Conclusion: J2 can prolong skin and corneal graft survival from rejection in mouse model. The short term outcome shows J2 do no harm to important organ;J2 could not inhibit corneal neovascular initially.Part ThreeObjective: To discuss the mechanism of J2 on preventing corneal rejection.Methods: Balb/c mousse had been divided into A, B, C, D, E groups randomly, Group A did no management, Group B, autograft control, Group C, D and E, allograft groups (The C57BL/6 were used as donors), Group B and group C were given placebo, Group D and group E were treated with orally CsA (10mg/kg)and J2 (15mg/kg), respectively. We observed the variation of lymphocyte subgroup of peripheral blood mononuclear cells in different groups by flow cytometer analysis every week after corneal transplantation. DTH assay had been done at week three, Cytokines including IL-2, IL-10, INF-y Secreting level of mouse spleen cells detected by ELLISPOT assay on day 18 after corneal transplantation.Result: The results of flow cytometer analysis showed: Lymphocyte subgroup of peripheral blood mononuclear cells hadn't proliferated specifically inGroup E;DTH assay showed low reaction to both donor's and third strain mouse spleen cells in Group E;The results of ELLISPOT assay indicated: the spot level of IL-2-. INF-y in Group E raised slightly compared to Group A, but compared to Group B and Group D, there were no statistically significant difference, the spot level of IL-10 between all groups had no statistically significant difference.Conclusion: J2 could block up the process of antigen presentation by inhibiting CD4/MHC-II moleculeonania. We guess Thl and Th2 cells haven't proliferated specifically by analyzing the level variation of IL-2, IL-10 and INF-y;we now couldn't conclude that J2 could induce recipient mouse tolerance to donor antigen.