| Objective: To investigate the survival time of corneal rejection in allogene mice modeland effects of Sphingosine-1Phosphate receptor1modulators(S1P1) were applied incorneal allograft in mice.To study the effects of prolonging corneal allograft survival byS1P1in combination with other immunosuppressants application. To determine theeffect of S1P1on the immune systems of those mice that after allogeneic cornealtransplantation, in order to prove the novel immunosuppresent mechanism of preventionof allograft rejection.Methods:1. To establish corneal allogeneic penetrating keratoplasty model, theC57BL/6and BALB/C mice were used as donors and recipients, and divided into A、B、C、D and E groups randomly(n=7). GroupA, allograft control(the control group weregiven placebo only); GroupB, allograft group,were treated with intraperitionalS1P1(5mg/kg/d); GroupC, allograft group, were treated with intraperitionaldexamethasone (1mg/kg/d); GroupD, allograft group,were treated with intraperitionalrapamycin (2mg/kg/d) and GroupE, allograft group,were treated with intraperitionalCsA(5mg/kg/d).Continuously administrated14days, beginning at the day oftransplantation. Nylon corneal sutures were removed on day10after transplantation.The state of corneal allograft was evaluated by microscope daily in the two weeks aftertransplantation procedure, and that were evaluated every other day beyond two weeks.At the time of rejection in the end of the study, mice were sacrificed.2. BALB/c mice were divided into7groups randomly after transplantation. TheC57BL/6and BALB/C mice were used as donors and recipients, and divided into A、B、C、D、E、F、G groups randomly (n=7). GroupA, allograft control(the control groupwere given placebo only); GroupB, allograft group,were treated with intraperitionalS1P1alone(5mg/kg/d)；GroupC, allograft group,were treated with intraperitional dexamethasone alone(1mg/kg/d); GroupD, allograft group, were treated withintraperitional in combination S1P1(5mg/kg/d) with rapamycin (2mg/kg/d) and GroupEwere treated in combination S1P1(5mg/kg/d) with CsA (5mg/kg/d); GroupF, allograftgroup,were treated with intraperitional rapamycin alone(2mg/kg/d); GroupG, allograftgroup,were treated with intraperitional CsA alone (5mg/kg/d).Continuouslyadministrated14days, beginning at the day of transplantation. Mice corneal sutureswere removed on day10after operations.3. BALB/c mice were divided into7groups randomly after transplantation. TheC57BL/6and BALB/C mice were used as donors and recipients, and divided into A、B、C、D、E、F、Ggroups randomly(n=5). The cervical lymph nodes, mesentery lymphnodes, spleen and peripheral bloods of mice in each group were collected by flowcytometry on day14after transplantation for measuring CD4+and CD4+CD25+Foxp3+T cells.4. BALB/c mice were divided into7groups randomly after transplantation. TheC57BL/6and BALB/C mice were used as donors and recipients, and divided into A、B、C、D、E、F、G groups randomly(n=10). The expressions of IL-2, IL-10, IFN-γ,TGF-β1of the cyto-supernatant were measured by ELISA. The index of cellproliferation and production of cytokine were calculated. Corneal cytokine expressionsuch as IL-2、IL-10、IFN-、TGF-1、Foxp3mRNAin each group mice corneas weredetected by real-time quantitative PCR. Moreover, corneas in each group were stainedwith hematoxylin and eosin for histological examination. The levels of CD4+T cellsand cytokines in corneal grafts were identified by immunofluorescence staininganalysis.Results:1. Control group (mean survival time (MST)15.14±2.5days), corneal graftssurvival was prolonged by intraperitoneal injections in S1P1systemic application alonegroup (MST37.85±0.8days) or combination with S1P1and rapamycin group (MST38.71±9.2days) or S1P1and Cyclosporin A (MST,32.71±7.2days) respectively (bothP<0.01).2. S1P1systemic application alone significantly increased the percentages of CD4+T cells (p<0.01) and Treg cells (p<0.01) in the cervical lymph nodes and in the mesenterylymph nodes(p<0.01) compared with the control group. Combination with S1P1andrapamycin significantly increased the percentages of CD4+T (p＜0.05) and Treg cells(p＜0.05) in the cervical and mesentery lymph nodes compared with controls.3.Transforming growth factor-1(TGF-β1) and interleukin-10(IL-10) mRNAtranscription in corneal grafts after combination with S1P1and rapamycin treatmentsignificantly increased (both p＜0.01), there play a role of synergic effort; whileInterleukin-2(IL-2) and interferon-(IFN-) mRNA expression in corneal grafts treatedwith Cyclosporin A systemic application alone group decreased compared withcontrols(both p＜0.05). The similar results of cytokines in corneal samples wereparalleled identified by immunohistochemical staining. S1P1systemic application aloneand combination with S1P1and rapamycin reduced the infiltration of CD4+T cells incorneal grafts.4.The serum levels of TGF-β1and interleukin-10(IL-10) in S1P1systemic applicationalone group increased (both p＜0.01) compared with controls, IFN-γincreased (p＜0.05) in S1P1systemic application alone group, while IL-2in the serum were identifiedhave no significantly differences between the groups(both p＞0.05).Conclusion:1.S1P1combined with rapamycin or Cyclosporin A play synergic effort,.and effectively prolong allogeneic corneal graft survival in mice.2. S1P1systemic application alone significantly affect the distribution of the lymphocyte,increased the percentages of CD4+T and Treg cells in the cervical and mesentery lymphnodes. S1P1in combination with rapamycin treatment can increase the local lymphdrainage lymphocytes.3. S1P1in combination with rapamycin treatment increased TGF-β1and IL-10mRNAexpression and decreased infiltration of CD4+Tcells in corneal grafts. There play a roleof synergic effort.4. S1P1systemic application alone resulted in high serum levels of TGF-β1and IL-10,followed by IFN-γ increased. |
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