【摘要】 目的：探讨肿瘤坏死因子α（TNFα）在角膜移植免疫排斥反应中的作用以及抗TNFα单克隆抗体（TNFα mAb）对排斥反应的抑制作用。方法：以近交系大鼠30只制作穿透性角膜移植模型（F344→Lou），随机等分入移植治疗组（每日结膜下注射0.5g/L抗TNFα mAb 0.1mL）和移植模型组（结膜下注射等量生理盐水）。利用酶联免疫吸附实验（ELISA）技术分别检测术后3，7 d及发生排斥反应时大鼠外周血清中TNFα的水平，并观察移植角膜的组织病理学变化及植片存活情况。结果：角膜移植术后早期各组大鼠血清TNFα浓度均有升高，与移植治疗组相比，移植模型组升高速度更快，水平更高（P<0.05）；但当发生排斥反应时，二者已无显著性差异（P>0.05）。移植模型组大鼠植片存活时间为10.2±1.9d，而抗体治疗组平均13.8±2.2 d，二者相比差异有显著性（P<0.01）。结论：TNFα在角膜移植免疫排斥反应中发挥一定作用。动态检测外周血TNFα浓度有助于预测、诊断排斥反应。应用抗TNFα mAb治疗可部分延长植片存活时间。
【关键词】 角膜移植；排斥反应；肿瘤坏死因子α；单克隆抗体；大鼠
Inhibition of antitumor necrosis factoralpha monoclonal antibody on rat corneal graft rejection
ShiSheng Zhang, GuiRen Li, HuaPing Liao, KangSun Wang
Foundation item: Funded Construction Projects of Key Disciplines of Shanghai, China(No.S30205)
1Department of Ophthalmology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine; New Vision Eye Clinic, Shanghai 200025, China;2Eye Center, Affiliated Hospital, Weifang Medical College, Weifang 261031, Shandong Province, China
AbstractAIM: To investigate the role of tumor necrosis factoralpha (TNFα) in the allograft rejection of rat corneal transplantation and to evaluate the inhibition effect of anti TNFα monoclonal antibody (mAb) on it.METHODS: After received allogeneic grafts (F344→Lou rat), 30 inbred Lou rats were divided equally into two groups: the transplantation model group and the transplantation treatment group. While the treatment group were treated daily by subconjunctival injection of antiTNFα mAb (0.1mL, 0.5 g/L), the model group received the same volume injection of physiologic saline. Then, the serum levels of TNFα were measured by using enzyme linked immunosorbent assay (ELISA) at 3 and 7 day, and the time when graft rejection occurred after corneal transplantation respectively. The corneal histopathological changes and the allograft survival times were also bserved.
RESULTS: The serum levels of TNFα in all rats elevated in the early stage after surgery, especially to the model group. AntiTNFα mAb decreased the levels and made them increase slowly (P<0.05). When the rejection occurred, the significant difference between the two groups didn't exist any more (P>0.05). The treatment group had a significant delay in corneal allograft rejection with mean survival time of 13.8± 2.2 days vs 10.2±1.9 days for the model group (P<0.01). CONCLUSION: TNFα may play an important role in the pathogenesis of corneal allograft rejection. Sequential determinations of serum TNFα after operation may be of value in the prediction and diagnosis of acute rejection. AntiTNFα antibody treatment can prolong the allograft survival time to some extent.
KEYWORDS: corneal transplantation; graft rejection; tumor necrosis factoralpha; monoclonal antibody; rat
0引言
角膜病是致盲的主要眼病之一，目前使角膜病致盲者重见光明的最有效治疗手段依然是角膜移植。自从1905年Zirm等[1]首开角膜移植手术以后，随着显微手术和眼库技术的不断提高和完善，角膜移植免疫排斥反应已成为移植失败的重要原因。因此探讨排斥反应发生机制，并采取相应措施治疗以进一步提高手术成功率无疑仍是眼科领域关注的焦点之一。近年来不少学者发现，肿瘤坏死因子α（tumor necrosis factoralpha, TNFα）在肝脏、肾脏及心脏等器官移植排斥反应中发挥着重要作用，应用其拮抗剂可不同程度地延长移植物存活时间。我们通过大鼠角膜移植动物模型，观察角膜移植排斥反应过程中外周血清TNFα的水平变化以及应用抗TNFα mAb治疗后的植片存活情况。
1材料和方法
1.1材料
以近交系F 344大鼠（RT1e）为供体， Lou大鼠（RT1y）30只为受体，随机进行单眼穿透性角膜移植手术。所有大鼠均为健康雄性，体质量200～250g，购自中国医学科学院医学实验动物研究所。
1.2方法
手术参照Williams等[2]方法进行。术前15min以10g/L阿托品和托品酰胺充分散瞳。肌注氯胺酮（60mg/kg）和氯丙嗪（40mg/kg）麻醉，并用10g/L地卡因点眼进行局部麻醉。50丝线牵引上下直肌，3mm环钻制作植床植片，100尼龙线间断缝合8针。术中以林格氏液保持晶状体湿润，勿用粘弹性物质。术后注入空气形成前房，涂10g/L阿托品眼膏。将30例手术后大鼠随机等分入移植模型组和移植治疗组，其中因晶状体损伤、前房出血导致手术失败5眼，其中移植模型组2眼、移植治疗组3眼，不列入统计结果。治疗组自手术之日起，每日结膜下注射0.5 g/L抗基因重组人TNFα（rhTNFα）mAb（中国医学科学院医学研究所）0.1 mL；模型组同时点注入等量生理盐水。另取4只正常Lou大鼠作为正常对照。术后每日裂隙灯显微镜下观察术眼，并依据Holland标准分别评分，3项评分之和为当日排斥反应指数（rejection index, RI），当RI≥6时为排斥反应发生时间[3]。术后5d内出现严重的角膜植片水肿、混浊及破坏不能归入角膜移植免疫排斥反应。出现白内障、前房出血、前房不形成等并发症者亦不列入统计结果。分别对正常对照大鼠及手术后3，7d，排斥反应时大鼠进行眼眶静脉取血约0.5 mL，4℃下2000rpm离心10min，取血清，70℃储存。采用TNF酶免定量检测试剂盒（华美生物工程公司）进行外周血清TNFα浓度测定。分别于手术后7d及发生排斥反应时完整摘出眼球，中性甲醛固定，常规病理切片，HE染色后光学显微镜下观察组织病理学变化。正常Lou大鼠眼球作为对照。
统计学分析：用SAS 6.12软件包对植片存活时间进行成组设计两样本均数的t检验，对外周血清TNFα浓度进行成组设计多个样本均数的方差分析及两两比较。P<0.05为差异有统计学意义。
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