肿瘤坏死因子-α在角膜移植免疫排斥反应中作用的实验研究

Experimental Study of Tumor Necrosis Factor-alpha in the Rejection of Rat Corneal Transplantation

目的 探讨肿瘤坏死因子 α在角膜移植免疫排斥反应中的作用。方法 以近交系大鼠制作穿透性角膜移植模型 4 4例 ,利用逆转录 多聚酶链反应和酶联免疫吸附实验技术分别检测不同时间供、受体角膜组织内TNF αmRNA的表达和外周血清中TNF α的水平 ,同时观察移植角膜组织的病理形态学变化 ,并观察应用抗TNF α单克隆抗体治疗后上述各项指标的变化。结果 同基因移植大鼠角膜轻度增厚 ,浅基质层见轻度炎性反应。在发生排斥反应的异基因移植鼠 ,角膜明显变厚 ,各层弥漫散在炎性细胞。术后早期各组大鼠血清TNF α浓度均有升高 ,而后同基因组逐渐下降 ,术后第 7天即已降至正常水平。异基因组渐趋升高 ,治疗组升高时间后延 ,而当发生排斥反应时 ,二者差异无显著性 (P <0 .0 5 )。异基因组自术后第 3天以及治疗组自术后第 7天 ,在供、受体角膜组织中均检测到TNF αmRNA的表达 ,而在同基因移植大鼠和正常大鼠角膜中则未检测到。异基因移植大鼠植片存活时间为 10 .2± 1.94d ,而抗体治疗组 13.8± 2 .17d ,二者相比差异有显著性 (P <0 .0 1)。结论 TNF α在角膜移植免疫排斥反应中发挥重要作用 ,术后监测TNF α浓度变化对排斥反应的发生有一定预测和早期诊断作用 。

Objective To investigate the role of tumor necrosis factor-alpha in the allograft rejection of rat penetrating keratoplasty.Methods Forty-four inbred rats received an allo or isogeneic graft,and half allograft rats were treated by subconjunctival injection of anti-TNF-α monoclonal antibody.The serum levels of TNF-α were measured and the expression of TNF-α mRNA in corneas were detected by using enzyme linked immunosorbent assay and reverse transcription PCR respectively at different time points after corneal transplantation.The changes of corneal histopathy were also observed with microscope.Results Examination of the syngeneic grafts showed thin corneas with moderate inflammatory reaction in the superficial stroma.In therapy group,the changes were similar to the syngeneic group in the early phase,and to the allogeneic group when the rejection reaction occurred.The serum levels of TNF-α in all rats elevated in the early stage after surgery,then decreased gradually to the normal on post-operative day 7 in isogeneic group,while increased sharply in allogeneic group.Anti-TNF-α mAb decreased the levels and made them increase later,but there was no significant difference between the therapy group and allogroup when the rejection occurred (P>0.05).RT-PCR analysis revealed that TNF-α mRNA was present in allografts,while no TNF-α mRNA expression in isogeneic grafts and normal corneas.The group receiving anti- TNF-α mAb had a significant delay in corneal allograft rejection with mean survival time of 13.8±2.17 days vs 10.2±1.94 days for the control group (P<0.01).Conclusions It is suggested that TNF-α plays an important role in the pathogenesis of corneal allograft rejection,sequential determination of serum TNF-α after operation may be of value in the prediction and diagnosis of acute rejection,and anti-TNF-α antibody therapy can prolong the allograft survival time.