| Objective: To investigate the survival time of corneal rejection in allogene mice model and the effects of J2 on prevention of allograft rejection; To determine the effect of J2 on the immunal systems of both normal mice and those after allogeneic corneal transplantation. Methods:1. The C57BL/6 and BALB/c mice were used as donors and recipients to establish coreal allograft model, and divided into A, B, C and D groups randomly. Groupe A, autograft control; Group B, allograft control (the control groups were given placebo only); Group C, allograft group, were treated with intraperitoneal CsA (10mg/kg/d) and Group D, allograft group, were treated with intraperitoneal J2(15mg/kg/d). Continuously administrated 12 days, beginning at the day of transplantation. Graft survival index, paraphin sections with HE staining and immunohistochemical staining of frozen section were observed in each group.2. BALB/c mice were divided into 3 groups randomly and were treated with intraperitoneal injection of placebo, CsA and J2, respectively. The drugs were delivered for 12 days consecutively. Murine splenocytes were stimulated by ConA and anti-CD3mAb. IL-2, IFN-γ , IL-4 and IL-10 of cyto-supernatant were measured by ELISA. Measure the index of cell proliferation and production of cytokine.3. Corneal transplantation model mice were randomly divided into A, B, C, D and E. Group A, control group; Grope B, autograft control; Group C, allograft control (the control groups were given placebo only); Group D and Group E allograft, treated with intraperitoneal CsA (10mg/kg/d) and J2(15mg/kg/d), respectively. Continuously administrated the drugs for 12 days, beginning at the day of transplantation. Observed the changes of lymphocyte subgroup of peripheral blood mononuclear cells in different groups by flow cytometer, analysised every week after corneal transplantation. Murine splenocytes of each group werestimulated by ConA, anti-CD3mAb and corneal protein. IL-2,IFN-γ ,IL-4 and IL-10 of the cyto-supernatant were measured by ELISA. The index of cell proliferation and production of cytokine were calculated.4. Corneal transplantation model mice were randomly divided into five groups. Group A, control group; Grope B, autograft control; Group C, allograft control given placebo only; Group D, allograft group, treated with intraperitoneal CsA (10mg/kg/d) and Group E, allograft group, treated with intraperitoneal J2(15mg/kg/d), respectively. Continuously administrated 12 days, beginning at the day of transplantation. At 1, 2, 3 and 4 weeks after operation, corneal cytokine expression was detected by RT-PCR. Result:The average transplant survival time in the corneal allograft control was 18.87 ± 4.19d. Treated with J2 led to a statistical prolongation of grafts' survival time (33.12 ± 6.80d) (P < 0.01). But there was no significant difference between J2 and CsA Groups. Histological examination showed that, lots of lymphocyte infiltrated into graft after operation 21 days. Very mild lymphocyte infiltration was found in both CsA and J2 groups. Immunohistochemistry showed that there were, large amount of CD4~+ and CD8~+ T lymphocytes infiltrates in the grafts of the placebo group at 21 days; However, J2 and CsA groups only showed a small number of CD4~+ and CD8~+ T lymphocytes in the grafts.After ConA stimulation, the average index of cell proliferation in the control group was 9.61±2.20. Treatment with J2 led to statistical decreases of index of cell proliferation (6.53 ± 3.21, P < 0. 01) and production of IL-2 and IFN-γ . But compared with treatment with group CsA there was no significant difference between the two groups (P > 0. 05) statistically. After anti-CD3mAb stimulation, the average index of cell proliferation in the control group was 9.52 ± 2.44. Treatment with J2 led to a significant decreas of index of cell proliferation (3.27±1.33, P < 0. 01) and production of IL-2 and IFN- γ . But compared with treatment with group CsA there was no significant difference between the two groups (P > 0. 05) statistically. There were no significant differences of IL-4 and IL-10 among all groups statistically.The flow cytometer analysis showed that the number of CD3~+ and CD4~+ T lymphocytes of autografting group in peripheral blood lymphocytes is higher than those of normal mice at 1 week after transplantation; A downward trend and CD8~+ T cells did not change significantly at 3 weeks after the transplantation. The number of total CD3~+ lymphocytes, CD4~+ T cells and CD8~+ T cells in placebo group after 1 to 2 weeks keratoplasty is similar to that of autografting group, tended to increase. 3 weeks after keratoplasty, the number of total CD3~+ lymphocytes, CD4~+ T cells and CD8~+ T cells was significantly higher than those of the control group and autografting group. 4 weeks after keratoplasty, total CD3~+ lymphocytes, CD4~+ T cells and CD8~+ T cells were decreased. At the J2 treated group , the total CD3~+ lymphocytes, CD4~+ T lymphocytes, CD8~+ T lymphocytes with the the basically same changing trends of autografting group and CsA treated group. ConA significantly stimulated the proliferation of spleen cells of pro-keratoplasty mice. The quantity of IL-2 and IFN-γ in spleen cells of mice 3 weeks after allografting, and that of IL-4 and IL-10 were not significantly change. After intraperitoneal injection of J2, the quantity of IFN-γ was significantly lower. After stimulated by specific and non-specific antigen, the ELISA results showed that the quantity of IFN-γ in spleen cells of mice after allografting significantly increased, J2 inhibited antigen-induced IFN-γ production.RT-PCR results showed that there were no corneal RANTES, IFN-γ, IL-2, IL-10 and IL-4 gene expressed in normal mice. In the first week, few RANTES, IFN-γ, IL-2, IL-10 and IL-4 gene expressed in autologous transplantation groop, and in the other three weeks no significantly expressed; Various kinds of gene expressed from the first week in allogeneic transplantation groop and significantly increased in the third week; Various kinds of gene expressed from the first week in CsA group J2 groop, the expression reduced in the second and the third week. The expression of the fourth week was stronger than the other three weeks. Conclusion:J2 can resistant corneal rejection in allogene mice model and its effect is similar to CsA; J2 inhibit T cell activation, reduce the cytokine secretion of Th1. when the rejection occurs, peripheral blood CD4~+ T lymphocytes have noticeably increased, J2may inhibit the occurrence of corneal rejection by inhibiting CD4~+ T cells activation; J2 can inhibit the proliferation of spleen cells stimulated by ConA and anti- CD3mAb of mice after corneal transplantation; Th1 cells in mouse corneal allograft rejection play a major role, while Th2 cells play a secondary role. J2 can inhibit Th1 cytokine production, but didn't induce immune tolerance. RANTES gene was involved in mouse corneal allograft rejection, J2 can inhibite its expression.
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