【作者】 石蕊；

【导师】 洪晶；

【作者基本信息】 中国医科大学 ， 眼科学， 2004， 硕士

【摘要】 前言 角膜病是我国常见的致盲眼病之一，占致盲眼病的第二位。在角膜疾病愈合修复过程中，许多细胞因子和生长因子参与组织修复调控，促进成纤维细胞增殖和新生血管形成，终致角膜新生血管(corneal neovascularization，CNV)形成。而CNV严重影响角膜透明性，成为角膜病致盲最常见的原因之一，同时又成为角膜炎、角膜移植排斥反应、角膜化学烧伤等疾病治疗的一个难题。因此，研究CNV的发病机制和通过不同途径干预其形成是各国学者关注的一大热点。大量研究表明，血管内皮生长因子(vascular endothelial growth factor，VEGF)是眼部新生血管形成最重要的生长因子，研究其致病机制、抑制其表达将是21世纪治愈新生血管性疾病的希望。目前的研究发现白介素-18(interleukin-18，IL-18)在抑制肿瘤新生血管的形成中具有重要的作用，而在眼科领域IL-18与新生血管的形成中的作用国内外尚未见报道。鉴于此，本研究采用免疫组化的方法检测IL-18、VECF在正常角膜和血管化角膜、无血管病变角膜的表达，并对二因子进行相关性分析，试从细胞因子的角度探讨CNV的发病机制，为临床CNV的治疗提供理论依据。 实验材料 病例来源于2003年1月至2004年1月中国医科大学附属一院眼科角膜移植手术钻取下的血管化角膜23例，无血管化角膜15例，角膜移植取材后的供体剩余角膜10例；主要试剂和器材有兔抗人IL-18多克隆抗体、兔抗人VEGF多克隆抗体，SABC(兔IgG)试剂盒，免疫组化计算机图象采集及分析系统。 实验方法 将收集到的人角膜经抗冻剂处理后放人深低温冰箱(-80℃)编号保存，实验时将标本放人4O℃恒温水浴箱复温，进行现及免疫组化染色，检测IL一18、vEGF在正常角膜、血管化角膜、无血管化角膜的表达，分析二因子在每组表达的相关性。结果 免疫组化显示正常角膜组织中IL一18主要分布在上皮层、前弹力层、内皮层，以上皮层的表达最明显，VEGF在上皮层、前弹力层、和内皮层均有表达，前弹力层表达最强烈，基质层可见弱表达，IL一18在血管化的角膜标本中表达降低，而VEGF在该标本中上皮层、基质层均强烈表达，新生血管化区域、炎性细胞均呈现强阳性，IL一18在无血管病变角膜中有阳性染色，VEGF出现强表达，二者在各组中的表达均呈负相关，以血管化角膜标本中二者相关性最显著。讨论 透明、无血管是角膜的重要特征，这与角膜组织致密的结构和角膜本身含有血管生长抑制因子有关。既往对CNV发病机制的认识有炎症、缺氧、水肿等学说。最近有学者认为角膜组织中含有许多血管生成抑制因子，同时也含有血管生成刺激因子，二者的平衡表达维持角膜的透明和无血管。角膜损伤致平衡破坏，血管形成因子上调，最终可致角膜新生血管形成，透明性丧失，出现血管化，严重影响视功能。我们研究显示，正常角膜中都存在IL一18和VEGF一定量的基本表达，并且生理情况下IL一18抑制VEGF的过量表达。因此，二者间的平衡对于维持角膜自稳状态具有重要的作用。在圆锥角膜、角膜变性、大泡性改变等未出现新生血管的角膜疾病中IL-18的含量略微减少，虽然VEGF上调，仅参与以上疾病的修复，并不引起血管形成，提示在这些疾病中IL一18仍然对VEGF存在一定作用，抑制过VEGF度表达，从而抑制了新生血管的生成。而在血管化的角膜病变中，IL一18对VEGF的抑制作用完全被破坏，IL一18下调，VEGF迅速大量释放，促进内皮细胞迁移、增殖及血管渗漏，终致新生血管形成。由此可见，角膜复杂的因子网络中IL一18与VEGF之间的抑制关系扮演调节角膜生理、病理过程的重要角色。因而，IL一18抑制CNV深人研究为临床治疗角膜新生血管化提供了新途径。但VEGF在角膜具有的正常生理功能提醒我们不能完全抑制VEGF，如何合理地选择IL一18的浓度，调整VEGF生理和病理之间的平衡尚待进一步的研究。结论 一、IL一18、VEGF在正常角膜均有表达，且二者呈负相关; 二、IL一18在血管化角膜中表达降低，而VECF分泌显著升高，二者呈显著负相关;IL一18在无血管化角膜中表达略减低，VEGF升高，二者呈负相关:三、角膜新生血管的病理过程与IL一18及更多还原

【Abstract】 PerfaceKeratopathy is one of the common blind ophthalmopathies in our country , which constitutes second among them. Many cytokines and growth factors are in-voloved in tissue repair and control . During the keratopathy healing and repair , they promote fibroblast multiplication and angiogenesis , finally lead to corneal neovascularization ( CNV). CNV severely affects corneal transparency and becomes one of the common causes of keratopathy - induced blindness , which brings a difficult problem to the theraphy of keratitis , keratopathy rejection , corneal chemical injuries and so on . So every country s scholars think it is a focus on studying the mechanism of CNV and interfering its formation. According to many researches , vascular endothelial growth factor (VEGF) may have important effects on CNV . Recently it is found that IL -18 has an inhibitory action that have not still been reported between IL - 18 and angigenesis in the ophthalmology field on tumor angiongenesis . So in the study the expression of IL18 and VEGF are detected in normal corneas and pathological corneas (including vascular corneas and avascular corneas ) by immunohistochemistry ( IHC ) technique and the correlatin analysis are made. Investigating the mechanism of CNV would provide a theory basis for clinical theraphy.Experimental materialsTen cases of normal corneas and thirty - eighty cases of morbid corneas were collected during corneal transplantation in China medical college from Janto Oct 2003. The main reagents and equipments are rabbit polyclonal anti - human IL -18, rabbit polyclnal anti - human VEGF, SABC reagent box, computer collection and analysis system of immunohistochemistry.MethodsThe corneal specimens were conserved in profound hypothermia ice box( -80t ) after anti - freezing and rewarmed in thermostat water bath ( + 40 C ). The next step is to test the expression of IL - 18 and VEGF in group (normal corneas, vascular corneas, avascular corneas) and to analysis the correlation between them.ResultsIn normal corneas, the positive expression pf IL - 18 and VEGF were all presented in corneal epithelis, Bowman’s membrane, endothelia. Spearman relationship analysis showed the IL -18 expression levels were negatively correlated with that of VEGF in every group. In vascular corneal specimens, the expression of IL - 18 decreased but that of VEGF increased and stronger expression was presented in the epithelia, stroma, the area of corneal neovascularization, inflammatory cells. The positive staining of IL - 18 in avascular corneal specimens has no statistic difference than in normal corneas, but the stronger expression of VEGF were found.DiscussionTransparent and avascular are important characters in cornea, which are related to compact instruction and inhibitory factors existence.Recently, scholars think that there are many angiogenesis factors and an-giogensis inhibition factors and the stabilization between them would keep corneal transpareat and avascular in cornea. Cornea injury would lead to the balance damage and angiogenesis factors’up -regulation. Finally CNV forms and obvi-ously decreases diminution of vision.This study showed that in normal corneas the basic expression of IL - 18 and VEGF was presented and IL - 18 might inhibit VEGF over - expression in physiological condition. So the expressional balance plays an important role in corneal self - stabilization state. In avascular morbid cornea, the IL - 18 levels decreased slightly, the up - regulated VEGF only took part in the morbid corneal repair but not caused angiogenesis. In vascular cornea, the inhibition action was thorough damaged and VEGF had rapidly high production and promoted endothe-lial cells migration , proliferation and blood vessel effusion. Expression of VEGF increasing led to CNV. From it we see that inhibition relation between IL -18 and VEGF plays an important role which modulates physiological and pathological course in complicated factor net of cornea. The further study which IL -18 inhibits CNV will p更多还原