课题背景：角膜移植免疫排斥反应是一个多因素参与的调节过程，近年来研究表明细胞因子在角膜移植免疫中起重要作用，角膜移植免疫排斥反应是主要由Th1因子介导的迟发型超敏反应。但也有研究表明，Th2因子亦与移植排斥有关，应用Th2因子抑制剂可延长移植器官的存活。

应用要点：目前临床上尚未出现针对角膜移植术后局部免疫状态变化、排斥反应发生、临床表现的早期诊断及发生排斥反应时免疫抑制治疗效果等的有效检测手段。因此进一步观察分析各种细胞因子间的相互作用及其在排斥反应发生前后的表达情况，不仅能为阐明角膜移植排斥反应机制提供理论基础，还将为临床尽早发现排斥反应并给予早期干预以降低角膜移植排斥率、提高存活率提供重要的辅诊检测手段。文章结果表明CD25、γ-干扰素和白细胞介素4在角膜移植免疫排斥反应过程中发挥重要的作用。

同行评价：文章观察了γ-干扰素（Th1因子）、白细胞介素4（Th2因子）及CD25等细胞因子在大鼠角膜移植术后排斥反应过程中局部及全身的表达情况，并分析其作用，具有一定的创新性，为临床治疗角膜移植术后排斥反应提供了实验依据。

摘要
目的: 角膜移植免疫排斥反应是一个多因素参与的调节过程，近年来研究表明细胞因子在角膜移植免疫中起重要作用。实验拟验证γ-干扰素、白细胞介素4及CD25在大鼠角膜移植免疫排斥反应中的作用。
方法：①实验于2006-10/2007-05在南方医科大学南方医院实验动物中心完成。选用Wistar大鼠24只，SD大鼠54只，实验方法符合动物伦理学要求。②将54只SD大鼠按随机数字表法分为3组，正常组6只，角膜移植组及地塞米松组均24只。后两组制备Wistar→SD角膜移植大鼠模型，受体选择右眼手术，供体提供双眼角膜。③用裂隙灯观察移植排斥情况；利用反转录-聚合酶链反应检测植片内γ-干扰素、CD25 mRNA的表达；应用酶联免疫吸附实验检测房水和血清中γ-干扰素、白细胞介素4含量；应用流式细胞术检测移植前后不同时相外周血淋巴细胞CD25的表达；术后第11天，应用免疫组织化学方法检测角膜CD4+，CD8+和CD25+ T细胞的表达。
结果：54只SD大鼠全部进入结果分析。①地塞米松组大鼠发生角膜移植排斥反应的时间长于角膜移植组(P=0.000)。②正常角膜植片无CD25、γ-干扰素mRNA表达，角膜移植组术后第11天角膜植片内γ-干扰素、CD25 mRNA的表达较地塞米松组增强(P < 0.05)。③角膜移植组角膜移植术后6，11 d血清γ-干扰素含量高于正常组（P < 0.05）；术后11 d含量高于术后6，24 d（P < 0.01）；地塞米松组术后6，11 d低于同期角膜移植组（P < 0.05）。④角膜移植组角膜移植术后6，11，24 d房水γ-干扰素、白细胞介素4含量均高于正常组（P < 0.05）；地塞米松组术后6，11 d均低于同期角膜移植组（P < 0.05~0.01）。⑤地塞米松组术后6，11 d外周血CD3+CD25+T/CD3+T比值低于同期角膜移植组（P < 0.05）。⑥术后第11天角膜移植组植片内CD4，CD8及CD25明显表达，地塞米松组CD4，CD8及CD25表达明显减弱。
结论：CD25、γ-干扰素和白细胞介素4在角膜移植免疫排斥反应过程中发挥重要的作用；促进白细胞介素4表达，抑制γ-干扰素及CD25产生，对降低角膜移植免疫排斥反应具有积极的作用。术后动态检测外周血CD25和γ-干扰素的表达有助于临床了解局部免疫反应程度并预测角膜移植排斥反应的发生。
关键词：角膜移植；干扰素类；白细胞介素4；受体，白细胞介素2；移植免疫

宫玉波，陆晓和，周瑾，袁伟，柯晓云，崔国华.γ-干扰素、白细胞介素4与CD25在角膜移植免疫排斥反应中的作用[J].中国组织工程研究与临床康复，2008，12(5):861-867 [www.zglckf.com/zglckf/ejournal/upfiles/08-5/5k-861(ps).pdf]

Effects of interferon-gamma, interleukin-4 and CD25 in rat corneal allograft immunological rejection

Abstract

AIM：Corneal allograft rejection is a complicated adjusting process which multiple factors participate in. Recent researches showed that the cytokine plays an important role in corneal transplantation immunity. This study was designed to investigate the role of interferon (IFN)-γ, interleukin-4 (IL-4) and CD25 in the allograft rejection of rat penetrating keratoplasty (PKP).
METHODS: ①Experiments were performed at the Experiment Animal Center of Nanfang Hospital of Southern Medical University from October 2006 to May 2007. A total of 24 Wistar rats and 54 SD rats were selected. Experimental procedures met the animal ethical standards. ②The 54 SD rats were randomly divided into three groups, normal control group (n=6), corneal graft group (n=24) and dexamethasone group (n=24). Rats in the latter two groups received Wistar→SD corneal transplantation. Receptors received right eye surgery, and donors offered cornea of both eyes. ③Rejection reaction was observed with a slit lamp microscope. Levels of IFN-γand CD25 mRNA in grafts were detected by using reverse transcription polymerase chain reaction (RT-PCR). Aqueous humor and serum levels of IFN-γand IL-4 were measured by enzyme linked immunosorbent assay (ELISA). The expression of CD25 in peripheral lymphocytes was measured by flow cytometry (FCM) before and after surgery. Immunohistochemical method was performed to examine corneal CD4+，CD8+ and CD25+T expressions at day 11 after surgery.
RESULTS: Totally 54 SD rats were included in the final results. ①The rejection time was longer in the dexamethasone group than in the corneal graft group (P =0.000). ②There was no expression of IFN-γ and CD25 mRNA in the normal cornea. Compared to the dexamethasone group, the expressions of IFN-γ and CD25 mRNA in grafts were markedly increased in corneal graft group at day 11 after PKP (P < 0.05). ③The IFN-γlevels in serum were higher in the corneal graft group than in the normal control group at days 6 and 11 after PKP (P < 0.05). IFN-γlevels in serum were higher at day 11 than at days 6 and 24 after PKP (P < 0.01). IFN-γlevels in serum were lower in the dexamethasone group than in the corneal graft group at days 6 and 11 (P < 0.05). ④IFN-γand IL-4 levels in aqueous humour were higher in the corneal graft group than in the normal control group at days 6, 11 and 24 after PKP （P < 0.05）. IFN-γand IL-4 levels were lower in the dexamethasone group than in the corneal graft group at days 6 and 11 (P < 0.05-0.01). ⑤Peripheral blood CD3+CD25+/CD3+ ratio was lower in the dexamethasone group than in the corneal graft group at days 6 and 11 (P < 0.05). ⑥At day 11, the expression of CD4, CD8 and CD25 on grafts was obvious, but weak in the dexamethasone group.
CONCLUSION: IFN-γ, IL-4 and CD25 play important roles during corneal allograft rejection. Increasing the expression of IL-4 and decreasing the expression of IFN-γand CD25 may help to inhibit corneal allograft rejection. Monitoring the expression of IFN-γand CD25 in peripheral blood after surgery can help to indicate the degree of local immune reaction and predict the occurrence of corneal allograft rejection.

Gong YB, Lu XH, Zhou J, Yuan W, Ke XY, Cui GH.Effects of interferon-gamma, interleukin-4 and CD25 in rat corneal allograft immunological rejection.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(5):861-867(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-5/5k-861(ps).pdf]