摘要
目的研究晚期糖基化终产物修饰蛋白对内皮细胞细胞骨架肌动蛋白的形态学影响及特异的糖基化终产物受体和氧化应激在此病理过程中的作用。方法用不同浓度的糖基化终产物修饰人血清白蛋白与人脐静脉内皮细胞株ECV304在体外共同培养不同时间,并设立对照组进行比较,采用免疫荧光染色法显示细胞骨架的形态学改变。结果与对照组相比,糖基化终产物修饰人血清白蛋白以时间和剂量依赖的方式影响内皮细胞骨架肌动蛋白聚合丝状和可溶性单体(或球状)形态的改变。随着糖基化终产物修饰人血清白蛋白作用浓度和时间的增加,丝状肌动蛋白所形成的外周致密带边缘出现锯齿样断裂,趋于变细崩解消散,最终形成由非极性单行排列的肌动蛋白丝组成的应力纤维。可溶性单体表现为向细胞浆和胞膜移位,胞浆区出现点状和丝状染色,细胞间距离明显增大。可溶性糖基化终产物受体的抗体和烟酰胺腺嘌呤二核苷酸磷酸盐氧化酶抑制剂均可阻断糖基化终产物对细胞骨架的影响。结论糖基化终产物修饰蛋白对细胞骨架的影响是通过与内皮细胞上的糖基化终产物受体结合并引起细胞内的氧化应激所介导的,这一作用可能与糖基化终产物所致血管通透性升高有关。
Aim To investigate the effect of advanced glycation end products （AGE） modified protein on morphological changes of actin cytoskeleton in endothelial cell and the role of receptor for AGE （RAGE） and oxidant stress in this pathological procedure. Methods Human umbiheal vein endothelial cells （hUVEC）-derived cell line （ECV304） were incubated with AGE modified human serum albumin （AGE-HSA） of concentrations of 12.5, 25, 50, and 100 mg/L respectively, for 2, 4, 8, 12 and 24 h. As control, HSA of the same concentration was administrated to ECV304 cells. To visualize the morphological changes of actin cytoskeleton, the treated cells were incubated with rhndamine-phalloidin or Oregon Green-Dnase I to stain F-actin or G-actin. ECV304 were treated with anti-RAGE IgG before AGE-HSA applications or both together. Apocynin, a specific inhibitor of NADPH oxidase, was pre-administrated to the endothelial ceils in concentrations of 10, 100,250, and 500μmol/L respectively, for 30 min, then the cells were rinsed with DMEM for three times and exposed to 50 mg/L AGE-HSA for 8 h. 31ae morphological changes of actin cyteskeleton were observed after the above-mentioned procedure. Results Morphology of Facfin and G-acfin in endothelial cells were changed greatly under the stimulation of AGE-HSA in a concentration and time-dependent manner. Exposure of ECs to AGE-HSA caused a shift in F-actin distribution from web-like structure to polymerized stress fiber. Cells subjected to higher-concentration and longer-time AGE-HSA exposure showed more and more stress fiber aceumnlation.Also the edge of G-actin became coarse and illegible and merged with each other, even formed tufts of flock extending all around nuclear area. And the changes can be inhibited by not only pretreatment with anti-RAGE IgG or NADPH oxidase inhibitor Apocynin, but also co-administrated with anti-RAGE IgG and AGE-HSA. The unmodified HSA, anti-RAGE IgG and Apocynin did not affect morphology of actin cytoskeleton. Conclusion AGE induced morphological c
出处
《中国动脉硬化杂志》
CAS
CSCD
2005年第3期282-286,共5页
Chinese Journal of Arteriosclerosis
基金
广东省自然科学基金(10717)
国家自然科学基金(30028008)
国家重点基础研究发展规划973项目(G2000057004)
