CD25单克隆抗体对大鼠角膜移植术后房水中细胞因子表达的影响

Effects of anti-CD25 monoclonal antibody on the level of cytokines in aqueous humour after rat penetrating keratoplasty

目的研究CD25单克隆抗体对大鼠角膜的毒性以及对同种异体大鼠角膜移植后房水中细胞因子的影响,探讨其对大鼠角膜移植免疫排斥反应的防治效果。方法①将12只SD大鼠随机分为生理盐水对照组、50μg CD25单克隆抗体结膜下注射组、100μg CD25单克隆抗体结膜下注射组、200μg CD25单克隆抗体结膜下注射组,每组3只。各组大鼠均右眼用药,分别于0、2、4、6、8 d结膜下注射生理盐水及不同剂量的CD25单克隆抗体,共5次。每日行裂隙灯显微镜检查,观察角膜有无水肿、混浊发生,并于第9天取右眼角膜行病理及透射电镜观察角膜各层的变化。②以Wistar大鼠为供体,SD大鼠为受体建立角膜移植实验模型。将93只SD大鼠随机分为五组,A组:正常组;B组:角膜移植对照组;C组:CD25单克隆抗体治疗组;D组:CD25单克隆抗体联合地塞米松治疗组;E组:地塞米松治疗组。B组术后给予生理盐水;C组术后给予CD25单克隆抗体100μg;D组术后给予CD25单克隆抗体100μg联合地塞米松50μg治疗;E组术后给予地塞米松100μg;各共用5次,分别于术后0、2、4、6、8 d经球结膜下注射给药。用裂隙灯观察移植排斥情况。利用逆转录-多聚酶链反应检测植片内IFN-γmRNA的表达,酶联免疫吸附法检测房水中IFN-γ和IL-4的浓度。结果①50μg CD25单克隆抗体和100μg CD25单克隆抗体对角膜基本无影响;200μg的CD25单克隆抗体组透射电镜检查发现角膜基质细胞及内皮细胞有不同程度的肿胀。②C、D、E组发生排斥反应时间分别为(13.167±1.169),(17.333±2.160),(16.417±1.379)d较B组发生排斥反应时间(10.583±1.084)d明显延迟,差异有统计学意义(P<0.05)。正常角膜无IFN-γmRNA的表达,术后11天,B组移植角膜植片内IFN-γmRNA的表达较C、D、E组明显增强(P<0.05)。C、D、E组术后6 d及11 d房水IFN-γ的含量明显低于同期的B组(P<0.05);同C组相比,术后11d D、E组房水IFN-γ的含量明显降低(P<0.05)。术后6 d和11 d,与B组相比,同期C组IL-4含量明显升高(P<0.05),而同期D、E组IL-4含量明显降低(P<0.05);术后6 d和11 d,与C组相比,同期D、E组IL-4含量明显降低(P<0.05)。结论50μg CD25单克隆抗体和100μg CD25单克隆抗体对角膜基本无影响。IFN-γ和IL-4在角膜移植免疫排斥反应过程中发挥重要的作用。CD25单克隆抗体通过抑制Th1因子(IFN-γ)、促进Th2因子(IL-4)表达降低角膜移植免疫排斥反应的发生率,从而延长角膜植片存活时间;而CD25单克隆抗体联合地塞米松治疗通过同时抑制Th1因子(IFN-γ)、Th2因子(IL-4)表达,却更能延长角膜植片的存活时间,具有重要的临床应用价值。

Objective To investigate the toxicity ofanti-CD25 monoclonal antibody （mAb） to rat cornea and its effects on the cytokines in the aqueous humour after penetrating keratoplasty （PKP）, thereby evaluating the effect of anti-CD25 mAb in preventing corneal allograft rejection. Methods The corneal toxicity ofanti-CD25 mAb at 50, 100 and 200 μg administered via subconjunctival injection was evaluated in 12 SD rats by histological examination and transmission electron microscopy. Another 93 SD rats were randomized into 5 groups, and transplantation of corneal allograft from Wistar rats was performed in 4 groups with the other group as the normal control. The 4 allograft groups were treated with saline, 100 μg anti-CD25 mAb, 100 μg anti-CD25 mAb with 50 μg dexamethasone, and 50μg dexamethasone, respectively. The graft rejection was observed, the aqueous humour levels of IFN-γ and IL-4 were measured with ELISA, and IFN-γ mRNA expressions in the grafts detected with RT-PCR. Results anti-CD25 mAb at 50 or 100 μg did not show significant toxicity on .the cornea, 〈 but at 200 μg, the mAb caused swelling of the corneal stromal cells and endothelial cells. After corneal allograft transplantation, a significant delay in allograft rejeetion was observed in the 3 groups with mAb or dexamethasone treatment as compared with that in saline group （P〈0.05）. IFN-γ mRNA expression in the allografl on days 11 after PKP and in the aqueous humour on days 6 and 11 was markedly increased in saline group compared with that in the 3 treatment groups （P〈0.05）. The mean IL-4 level in the aqueous humour was significantly higher in the mAb group than in saline group （P〈0.05）, but markedly lower in anti-CD25 mAb＋dexamethasone and dexamethasone groups than in anti-CD25 mAb group （P〈0.05）. Conclusions Anti-CD25 mAb at 20 and 100 μg does not obviously affect the rat corneas. Anti-CD25 mAb inhibits IFN-γ expression and promotes IL-4 the expression to reduce corneal allografl rejection, whereas anti-CD25 mAb with low-dose dexamethasone inhibits both IFN-γand IL-4 expressions to more effectively promote the graft survival.