Advanced glycation end products in diabetic corneas.
Advanced glycation end products in diabetic corneas.
复制标题
DOI：
--
复制
发表时间：
2000-02
影响因子：
4.4
通讯作者：
Y. Kaji;T. Usui;T. Oshika;M. Matsubara;H. Yamashita;M. Araie;T. Murata;T. Ishibashi;R. Nagai
中科院分区：
文献类型：
--
作者：
Y. Kaji;T. Usui;T. Oshika;M. Matsubara;H. Yamashita;M. Araie;T. Murata;T. Ishibashi;R. Nagai
目的角膜并发症常与糖尿病相关，并可危及视力。糖尿病患者的角膜尽管具有无血管特性，但仍暴露在葡萄糖浓度升高的环境中，这种情况可能会导致晚期糖基化终产物(AGEs)的积累。本研究的重点是探讨AGEs在糖尿病角膜病变发病机制中的作用。方法制备以N(Epsilon)-羧甲基赖氨酸(CML)蛋白加合物为表位的抗AGE单抗(6D12)。对年龄匹配的糖尿病患者和非糖尿病患者(各8例)进行CML免疫组织化学定位检测。在体外，I型胶原、IV型胶原或层粘连蛋白包被的96孔板被葡萄糖-磷酸糖化。在一些实验中，孵化混合物中存在氨基胍。用6D12双抗体夹心法测定细胞外基质(ECM)中CML-蛋白加合物的含量。将SV40-永生化人角膜上皮细胞接种于96孔板中修饰或未修饰的ECM上，贴壁3小时。固定贴壁细胞，测量每种状态下贴壁细胞的面积。取出未固定的贴壁细胞，计数细胞数。结果8例糖尿病患者角膜上皮基底膜均可见CML免疫反应，而8例非糖尿病患者中7例未见相应区域的CML免疫反应。在体外，层粘连蛋白在培养皿上的非酶糖基化减少了角膜上皮细胞的黏附和铺展。在糖基化过程中，孵育混合物中的氨基胍以剂量依赖的方式抑制慢性粒细胞白血病的形成，促进角膜上皮细胞的黏附和铺展。结论AGEs在基底膜上的积聚，尤其是层粘连蛋白的积聚，可能是糖尿病患者角膜上皮病变的原因之一。
PURPOSE Corneal complications are often associated with diabetes mellitus and can be vision threatening. Corneas in diabetic patients are exposed to increased glucose concentration despite cornea's avascular property, and this condition may contribute to the accumulation of advanced glycation end products (AGEs). The focus of this study was to examine the role of AGEs in the pathogenesis of diabetic keratopathy. METHODS An anti-AGE monoclonal antibody (6D12), which recognizes a N(epsilon)-carboxymethyl lysine (CML)-protein adduct as an epitope, was prepared. Immunohistochemical localization of CML was examined in human age-matched diabetic and nondiabetic corneas (8 of each). In vitro, type I collagen-, type IV collagen-, or laminin-coated 96-well plates were glycated by glucose-phosphate. In some experiments, aminoguanidine was present in the incubation mixture. The amounts of CML-protein adducts in the extracellular matrix (ECM) were determined by enzyme-linked immunosorbent assay using 6D12. SV40-immortalized human corneal epithelial cells were seeded onto modified or unmodified ECM in 96-well plates and allowed to attach for 3 hours. Attached cells were fixed, and the areas of attached cells in each condition were measured. Attached cells without fixation were removed, and cell number was counted. RESULTS In all of the 8 diabetic corneas, CML immunoreactivity was observed in the epithelial basement membrane, whereas CML immunoreactivity was not found in the corresponding area in 7 of 8 nondiabetic corneas. In vitro, nonenzymatic glycation of laminin on the culture dish attenuated adhesion and spreading of corneal epithelial cells. The presence of amninoguanidine in the incubation mixture during glycation inhibited CML formation and promoted the adhesion and spreading of corneal epithelial cells in a dose-dependent manner. CONCLUSIONS The accumulation of AGEs on the basement membrane, particularly on laminin, may play a causative role in the corneal epithelial disorders of diabetic patients.