晚期糖基化终产物引起的RhoA和ROCK在人皮肤微血管内皮细胞的分布变化

Advanced Glycation End Products Induced the Redistribution of RhoA and Phosphorylated ROCK in Human Dermal Microvascular Endothelial Cells

目的观察在晚期糖基化终产物修饰的人血清白蛋白作用下,小G蛋白RhoA及其激活的激酶ROCK在人微血管内皮细胞中分布的变化。方法培养人皮肤微血管内皮细胞株,分别以糖基化修饰的人血清白蛋白(AGE-HSA)处理不同的浓度和时间,用免疫荧光染色法、激光共聚焦显微镜观察RhoA和磷酸化ROCK在细胞中的分布变化;并用活性型RhoA L63或失活型的RhoA N19转染细胞后再给予AGE-HSA刺激,观察磷酸化ROCK细胞内分布的变化。结果 AGE-HSA刺激可以导致RhoA在胞浆中分布增多,其变化随着AGE-HSA作用时间的延长和剂量的增加更加明显。RhoA的下游激酶被活化的磷酸化ROCK在人微血管内皮细胞中的分布与RhoA类似;用失活型RhoAN19先处理细胞后,AGE-HSA介导的ROCK分布变化被抑制。结论晚期糖基化终产物刺激引起小G蛋白RhoA在细胞内分布变化,并导致其下游激酶ROCK磷酸化和定位变化。

Aim To observe the changes of distribution of RhoA and phosphorylated RhoA kinase（ROCK） induced by advanced glycation end products（AGE） in human dermal microvascular endothelial cells（HMVEC-1）. Methods Cultured HMVEC-1 were treated with AGE-modified human serum albumin（AGE-HSA）.The distribution of RhoA and phospho-ROCK were detected by immunohistochemistry using anti-RhoA and anti-phospho-ROCK antibodies and observed under confocal microscope. Results The data revealed that RhoA located around the nucleus in quiescent HMVEC-1.The stimulation of AGE-HSA evoked an increased distribution of RhoA in cytoplasma,even an accumulation at the cellular edge.The administration of AGE-HSA also triggered a significant increase of phospho-ROCK localization in HMVEC-1 cytoplasma.The transfection of dominant negative RhoA N19 attenuated this AGE-induced phosphor-ROCK spreading while constitutive active RhoA L63 mimiced the AGE-induced response in phospho-ROCK distribution. Conclusion AGE stimulation causes the redistribution of RhoA and results in the phosphorylation of ROCK in human microvascular endothelial cells.