小鼠角膜移植排斥反应中植片内细胞因子表达水平的变化

Expression of cytokine in mouse graft of corneal allograft rejection

背景研究表明细胞因子在角膜移植免疫调节中起重要的作用，但关于角膜移植术后角膜植片内细胞因子表达的变化报道较少。目的探讨角膜移植术后不同时间点细胞因子的表达变化。方法采用雌性6～8周龄BALB／c小鼠及C57BL／6小鼠分别建立小鼠自体角膜移植和同种异体角膜移植模型，每组各10只，自体角膜移植术组供体和受体同为BALB／c小鼠，同种异体角膜移植术组供体为C57BL／6小鼠，受体为BALB／c小鼠。于术后1d开始临床观察角膜植片并对炎症程度进行评分，当植片评分总和≥5分或植片混浊≥2分时为发生植片排斥反应。将BALB／c小鼠随机分为正常对照组3只和同种异体角膜移植术组15只，后者分别于术后6h及1、3、7、14d对角膜植片行逆转录PCR（RT—PCR）检测植片中细胞因子白细胞介素4（IL-4）、γ-干扰素（IFN-γ）、IL-10、肿瘤坏死因子α（TNF—α）表达的变化。结果在60d的观察期中，自体角膜移植术后小鼠角膜植片混浊评分均〈2分，植片的炎症评分之和均〈5分，植片存活率为100％，但同种异体角膜移植术后角膜植片水肿、混浊，伴有角膜新生血管形成，至术后24d角膜植片排斥反应评分之和均≥5分，植片混浊评分≥2分，全部发生排斥反应，角膜植片平均存活时间为（17．80±4．66）d。RT—PCR检测结果表明，正常角膜中IL-4和IFN-γ呈阳性表达，IL-10和TNF-α表达阴性。同种异体角膜移植术后6h可检测到IL-4呈阳性表达，IFN-γ和IL-10表达呈强阳性，而未检测到TNF—α的表达。术后1～3d角膜植片中IL-4呈强阳性表达，IFN-γ阳性表达，TNF—α表达增强，IL-10表达逐渐消失。角膜移植术后7d，可见IL-4表达阴性，而IFN-γ、IL-10和TNF—α仍呈强阳性表达。至术后14d，角膜植片中IL-4、IFN-γ和TNF—α．均未见表达，仅检测到IL-10呈阳性表达。结论TNF-α是角膜移植术后局部参与调控的主要因子。

Background Cytokines play a crucial role in mediating immune tolerance or immune rejection of corneal transplantation. However,the study on the expression of cytokines in corneal graft is seldom. Objective The purpose of the study is to investigate the change of cytokine expression in allografts at different time points after corneal transplantation. Methods BALB/c and C57BL/6 mice aged 6 to 8-week old were used to establish autologous and allografts keratoplasty models. BALB/c mouse was use as donor and receipt in autologous group, and the cornea of C57BL/6 mouse was used to graft on the BALB/c mouse in the allografts group. The graft inflammation was clinically scored,and graft inflammatory scores of 1〉5 or opaeification scores of ≥ 2 were identified as rejection. BALB/e mice were randomized into normal control group（3 mice）and allografts group（ 15 mice）. Reverse transcript- PCR （RT-PCR） was used to detect the expression of interleukin-4 （ IL-4 ） mRNA, interferon-γ （IFN-γ） mRNA, IL-10 mRNA and tumor necrosis faetor（TNF-α） mRNA in graft 6 hours and 1 day,3,7,14 days after operation. Results The corneal graft opacification score was 〈2 and inflammatory score was 〈5 in the 10 mice with autologous keratoplasty until 60 days with the survival rate 100%. The edema, opacification and new blood vessel were seen in the BALB/c mice received allografts keratoplasty. The inflammatory score was ≥5 and the opacifieation score was ≥2 24 days after surgery with the rejection rate 100% ,in the allografts group,and the graft survival time was（17.80±4.66） days. RT-PCR showed that IL-4 and IFN-γ were positively expressed, and IL-10 and TNF-α were absently expressed in normal mouse cornea. In the allografts keratoplasty mice,positive responses for IL-4,IFN-γ and IL-10 were found in 6 hours after operation,but TNF-α was absent. From 1 day through 3 days after operation,the expressions of IL-4,IFN-γ and TNF-α were enhanced but IL-10 was disappeared in the graft. IL-10 ,IFN-γ and TNF-α were expressed till the 7th day,but on the 14th day,only IL-10 was detected in graft in the allografts keratoplasty mice. Conclusions TNF-α is a main factor among the variety of cytokines that may influent corneal allograft rejection locally.