小鼠异基因角膜移植术后调节性T细胞与相关因子的表达研究

目的 探讨在小鼠异基因角膜移植免疫排斥中,调节性T细胞(Treg)及其下游细胞因子的表达变化.方法 实验研究.实验组以BALB/c(H-2~d)小鼠为受体、C3H/He(H-2~k)小鼠为供体,建立小鼠异基因角膜移植模型48只;对照组的受体与供体均为BALB/c,建立小鼠同基因角膜移植模型48只.观察术后植片的存活率、排斥反应发生时间;组织病理学检查术后3 d、7 d,4周及8周各时间点角膜植片中炎症细胞浸润与角膜新生血管生成;流式细胞仪检测术前、术后3 d、7 d、4周及8周各时间点受体外周血和脾脏中CD4~+CD25~+Treg及CD103~+CD8~+Treg的表达变化;同时酶联免疫吸附试验检测血清与房水中白细胞介素10(IL-10)、IL-4、γ干扰素(IFN-γ)及II-1β的表达变化.应用Kaplan-Meier生存曲线、两独立样本t检验、非参数秩和检验进行统计学分析.结果 实验组角膜植片免疫排斥发生时间为7 d至4周,平均存活时间为(14.79±1.02)d,对照组术后植片保持透明,观察期(8周)内未发生排斥反应,存活时间显著长于实验组,差异有统计学意义(x~2=46.934,P=0.000).流式细胞仪检测显示对照组外周血CD4~+CD25~+Treg术后各时间点的表达分别为(3.36±0.29)%、(4.09±0.44)%、(5.44±0.35)%、(5.73±0.53)%,显著高于实验组的(2.50±0.39)%、(3.24±0.25)%、(4.20±0.45)%、(4.18±0.14)%,差异有统计学意义(t=3.828、2.898、3.780、4.892,均P＜0.05),两组脾脏中CD4~+CD25~+Treg的表达上调先于外周血;术后各时间点实验组外周血内CD8+CD103+Treg的表达分别为(2.20±0.33)%、(2.79±0.57)%、(4.55±1.03)%、(4.31±0.07)%,显著高于对照组的(0.73±0.12)%、(1.10±0.19)%、(1.43±0.14)%、(2.10±0,14)%,差异有统计学意义(t=7.133、4,876、5.196、19.960,均P＜0,05),两组脾脏中CD8~+CD103~+Treg的表达上调先于外周血.实验组术后各时间点血清内IL-10与IL-4水平均显著低于对照组,差异有统计学意义(t=3.203、3.141、3.012、2.869与2.340、6.681、8.839、8.574.P=0.011、0.012、0.013、0.019与0.053、0.000、0.000、0.000);实验组术后血清内IFN-γ与IL-1β含量上调,各时间点的表达水平均高于对照组(除术后4周、8周的IL-1β,余差异有统计学意义,t=3.508、3.265、4.402、5.539与3.630、5.796、1.728、0.660,P=0.006、0.011、0.002、0.000与0.005、0.000、0.115、0.524).房水中各细胞因子的表达改变与血清中变化相似.结论　小鼠同种异基因角膜移植免疫排斥中,CD4~+CD25~+Treg表达下调并伴随血清与房水中IL-10、IL-4表达下调,而CD8~+CD103~+Treg的表达上调伴随IFN-γ、IL-1β的含量增加,可能参与了角膜移植免疫排斥反应的过程.

Expression of regulatory T cells and related cytokines in mice allogenic corneal transplantation rejection

Objective To study the changes of CD4~+CD25~+ and CD8~+CD103~+ regulatory T cells and their relevant cytokines in corneal allograft transplantation in mice.Methods BABL/c (H-2~d) mice received corneal allografts from C3H/He (H-2~k) mice were served as the experimental group.BABL/c (H-2~d) mice received corneal from BABL/c (H-2~d) mice were used as the control group.Corneal graft survival time was recorded pre-,3rd day,7th day,4th week,8th week post-operatively.Infiltration of inflammatory cells and corneal neovascularization were evaluated by histopathologic examination.The percentage of CD4~+CD25~+ and CD8~+CD103~+T cell in peripheral blood and spleen was determined by flow cytometry.The expression of interleukin-10 (IL-10),IL-4,γ-interferon (IFN-γ) and IL-1β in serum and aqueous humor was measured by enzyme-linked immunospecific assay (ELISA).Results The graft rejection in experimental group occurred at 7 days to 4 weeks after the operation,averaged (14.79±1.02) days.The grafts in the control group remained clear within 8 weeks observation and the survival time is much longer than that of the allografts (x~2=46.934,P=0.000).Flow cytometry showed that the percentage of CD4~+CD25~+ T cell in peripheral blood in the control group after surgery was (3.36±0.29)%,(4.09±0.44)%,(5.44±0.35)%,(5.73±0.53)% at day 3,day 7,4th week,and 8th week,respectively,which was significantly higher than that in the experimental group [(2.50±0.39) %,(3.24±0.25)%,(4.20±0.45)%,(4.18±0.14)%;t=3.828,2.898,3.780,4.892;P＜0.05].On the other hand,CD8~+CD103~+T cell in peripheral blood in the experimental group after surgery was (2.20±0.33)%,(2.79±0.57)%,(4.55±1.03)%,(4.31±0.07)% at different periods,which was much higher than that in the control group (t=7.133,4.876,5.196,19.960;P＜0.05).The changes of CD4~+CD25~+ and CD8~+CD103~+ T cells in the spleen was earlier than those in peripheral blood.ELISA showed the expression of IL-10 and IL-4 in the serum in experimental group after surgery was much lower than that in the control group (t=3.203,3.141,3.012,2.869 and 2.340,6.681,8.839,8.574;P=0.011,0.012,0.013,0.019 and 0.053,0.000,0.000,0.000).The serum levels of IFN-γ and IL-1β in experimental group were higher than that in the control group (t=3.508,3.265,4.402,5.539 and 3.630,5.796,1.728,0.660;P=0.006,0.011,0.002,0.000 and 0.005,0.000,0.115,0.524).Furthermore,the cytokine levels in the aqueous humor behaved similarly with those in the serum.Conclusion The down-regulation of CD4~+CD25~+ Treg,IL-10 and IL-4 and the up-regulation of CD8~+CD103~+ Treg,IFN-γ and IL-1β in mice allograft corneal rejection may play an important role in allograft rejection.