晚期糖基化终产物诱导内皮细胞紧密连接改变及其机制

目的探讨晚期糖基化终产物修饰蛋白对内皮细胞紧密连接带状闭合蛋白1的形态学影响,并对其机制作初步研究。方法培养人脐静脉内皮细胞,用免疫荧光染色方法显示带状闭合蛋白1在内皮细胞分布的形态和部位。观察不同浓度和不同时间晚期糖基化终产物修饰蛋白作用下内皮细胞带状闭合蛋白1的形态学变化。研究了细胞外信号调节激酶、p38丝裂原活化蛋白激酶通路抑制剂及相关特异蛋白突变体重组腺病毒转染对晚期糖基化终产物介导的内皮细胞闭合蛋白1形态学变化的影响。结果正常对照组的内皮细胞闭合蛋白1存在于细胞周边呈环状,线条清晰连续,边缘光滑流畅,晚期糖基化终产物以时间和剂量依赖的方式引起内皮细胞紧密连接带状闭合蛋白1结构形态的改变;细胞外信号调节激酶通路抑制剂或p38丝裂原活化蛋白激酶通路抑制剂均可减轻晚期糖基化终产物对闭合蛋白1的影响;转染显性失活的细胞外信号调节激酶上游激酶MEK1和p38丝裂原活化蛋白激酶上游激酶MEK6b的重组腺病毒,均可减轻晚期糖基化终产物对带状闭合蛋白1形态的影响,而转染组成性激活的MEK1和MEK6b的重组腺病毒本身即可引起内皮细胞带状闭合蛋白1形态的变化。结论晚期糖基化终产物刺激可以引起内皮细胞紧密连接蛋白带状闭合蛋白1分布和形态的变化,这一作用可能是由细胞外信号调节激酶及p38丝裂原活化蛋白激酶信号通路介导的。

The Mechanism of Advanced Glycation End Products-Induced Morphological Changes of Tight Junction in Endothelial Cell

Aim To investigate the effect of advanced glycation end products modified human serum albumin(AGE-HSA) on morphological changes of tight junction associated protein ZO-1 in endothelial cell and the mechanism in this pathological procedure. Methods Human umbilical vein endothelial cell(hUVEC)-derived cell line(ECV304) were incubated with AGE-HSA in different concentrations and timing. To visualize the morphological changes of tight junction protein ZO-1,the treated cell were incubated with mouse anti-ZO-1 primary antibody and then FITCanti-mouse IgG secondary antibody.The morphological changes of ZO-1 were observed with confocal microscope.The cell were pre-administrated with PD98059,a specific inhibitor of MEK1(ERK upstream kinase)or SB203580,a specific inhibitor of p38 MAP kinase,respectively,before exposed to AGE-HSA,then the cell were rinsed with DMEM for three times and exposed to AGE-HSA. The cell were transfected with dominant negative MEK1 or MEK6b(p38 upstream kinase) mutant adenoviruse,then exposed to AGE-HSA.And the cell were transfected with constitutively active MEK1 or MEK6b mutant adenoviruse. Results In normal control group,ZO-1 staining appeared as a continuous and smooth line along the regions of cell cell contact.Under the stimulation of AGE HSA,morphology of ZO-1 in endothelial cell were changed greatly in a concentration and time-dependent manner.The changes were partially blocked by PD98059 and SB203580.The transfection of dominant negative MEK1 and MEK6b mutant adenoviruse had the similar effects.The transfection of constitutively active MEK1 and MEK6b disrupted the structure of ZO-1. Conclusion AGE modified proteins can induce morphological changes of ZO-1 in endothelial cell.Activations of ERK and p38 MAP kinase pathways play an important role in this procedure.