摘要
目的 研究晚期糖基化终末产物 (AGE)与糖尿病 (DM)皮肤微血管病变的关系 ,探讨DM创面新生血管化障碍或延迟的机制。方法 应用链脲佐菌素 (STZ)将SD大鼠诱导成速发型DM大鼠模型 ,并以正常SD大鼠作为对照 ,于致病后 12周获取大鼠背部中央的皮肤标本。应用F 30 10荧光分光光度计和免疫组织化学技术测定皮肤中AGE含量的变化 ;应用透射电镜观察皮肤微血管超微结构的改变。再选用人脐静脉内皮细胞株ECV30 4细胞与不同浓度的AGE修饰人血清白蛋白 (AGE HSA)在体外共同培养 ,采用四甲基偶氮唑蓝(MTT)比色法和流式细胞仪检测AGE对人血管内皮细胞活性的的影响。结果 DM大鼠皮肤胶原提取液的荧光值明显高于相应年龄的正常大鼠 (P <0 .0 1) ;免疫组织化学检测显示DM大鼠皮肤中AGE呈片状或块状沉积 ,其表达阳性率和强阳性率显著高于正常大鼠 (P <0 .0 1) ;DM大鼠皮肤普遍存在微血管病变 ,主要表现为血管内皮细胞变性和基底膜增厚 ;内皮细胞经 10 0 μg/mlAGE HSA干预 6h后 ,采用MTT法测定其吸光度值显著低于正常大鼠 (P <0 .0 1) ,流式细胞仪测定的各时相点凋亡细胞百分率亦明显高于正常大鼠 (P <0 .0 1) ,且均呈时间和剂量的依赖性。结论 AGE是DM皮肤微血管病变的重要致病因素 。
Objective To explore the relationship of advanced glycation end products (AGE) and diabetic skin with microangiopathy and the mechanism of impaired wound healing. Methods 18 male Sprague-Dawley(SD) rats weighing 200～220 g were randomized into the control and STZ-induced diabetic groups. The shaved skin specimens from the back of rats were collected at 12 w after STZ-inducing. Skin AGE concentrations were assessed by detecting the total fluorescence in tissue collagen with F-3010 spectrofluorometer and immunohistochemistry assay. The ultrastructural changes of skin microvessels were observed by electronmicroscopy. Human umbilical vein endothelial cells ECV304 were cultured in vitro with AGE modified human serum albumin (AGE-HSA) at the concentrations of 12.5, 25, 50, and 100 μg/ml for 6, 12, 24, and 48 hours. The cells incubated with HSA of the same concentrations were used as controls. The cell vitality at each time point was analyzed by MTT assay and fluorescence microscopy. Results Skin fluorescence of diabetic rats increased significantly in comparing with age-matched controls thoughout the course of the study (P<0.05). Consistent with collagen fluorescence, the positive expression of skin AGE in diabetic animals was much stronger than the weak expression in healthy controls. Furthermore, skin microangiopathy was easily detected in the diabetic group, manifesting as the degenerated vascular endothelial cells and thickening of basement membrane. The absorption of ECV304 cells cultured with AGE-HSA at the concentration of 100 μg/ml for 6 h was significantly lower than that in the control group (P<0.01). Meanwhile, flow cytometry and fluorescence microscopy showed higher proportions of apoptotic cells among the ECV304 cells cultured with 100 μg/ml AGE-HSA than those among the control cells at each time point (P<0.01). Conclusion AGE is an important cause of diabetic skin microangiopathy. Furthermore, AGE modification-induced pathobiological cascade may be involved in the mechanism of impaired wound heali
出处
《上海医学》
CAS
CSCD
北大核心
2004年第11期832-835,i002,共5页
Shanghai Medical Journal
基金
国家 973重点基础研究发展规划项目资助 (G19990 5 42 0 5 )
