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Rho/ROCK 信号通路参与晚期糖基化终产物诱导的人皮肤微血管内皮细胞骨架结构改变

王吉萍, 郭晓华, 王陵军, 李强, 陈波, 吴炜, 黄绪亮, 黄巧冰^*

南方医科大学基础医学院病理生理教研室，广东省医学休克微循环重点实验室，广州 510515 摘

摘要

本文旨在探讨Rho 信号转导通路在晚期糖基化终产物(advanced glycation end products, AGEs)诱导的人皮肤微血管内 皮细胞(human dermal microvascular endothelial cells, HMVECs)形态及功能改变中的作用及机制。体外培养HMVECs 细胞 株，分别以不同浓度的AGEs 修饰的人血清白蛋白(human serum albumin, HSA)处理不同时间，并设立对照组进行比较。 用免疫荧光染色法、激光共聚焦显微镜观察细胞骨架肌动蛋白F-actin 的结构及分布；用Rho 激酶(Rho kinase, ROCK)抑制 剂Y-27632 或H-1152 预处理细胞30 min 后，与前者进行对比，同时用免疫印迹法检测Rho、ROCK 及其磷酸化水平的变 化；用FITC 荧光标记蛋白漏出法测定单层内皮细胞的通透系数(apparent permeability coefficient, Pa)值。结果显示，AGEs- HSA以剂量和时间依赖的方式引起HMVECs 细胞骨架蛋白F-actin 结构和分布的改变，未经修饰的HSA 无此作用；ROCK 特异性抑制剂Y-27632 和H-1152 均可抑制AGEs 对细胞骨架的影响。AGEs-HSA 引起Rho、ROCK 磷酸化水平的增加(P< 0.05)，但对总蛋白没有明显影响。与对照组相比，AGEs-HSA 使内皮细胞的通透性升高(P<0.01)，Y-27632 和H-1152 均 能抑制这种改变(P<0.01)。以上结果提示，Rho 信号通路在AGEs 介导的HMVECs 形态和功能改变中发挥了重要作用。

关键词： Rho 信号通路; 晚期糖基化终产物; 人皮肤微血管内皮细胞; 细胞骨架肌动蛋白

分类号：R587.1

[Effects of Rho/ROCK signal pathway on AGEs-induced morphological and functional changes in human dermal microvascular endothelial cells.] [Ariticle in Chinese]

WANG Ji-Ping, GUO Xiao-Hua, WANG Ling-Jun, LI Qiang, CHEN Bo, WU Wei, HUANG Xu-Liang, HUANG Qiao-Bing^*

Department of Pathophysiology, Southern Medical University, the Key Laboratory of Shock and Microcirculation of Guangdong Province, Guangzhou 510515, China

Abstract

The present study aimed to determine the role of Rho/Rho kinase (Rho/ROCK) phosphorylation on advanced glycation end products (AGEs)-induced morphological and functional changes in human dermal microvascular endothelial cells (HMVECs). HMVECs were respectively incubated with different concentrations of AGEs-modified human serum albumin (AGEs-HSA) for different time. In some other cases, HMVECs were pretreated with ROCK inhibitors (H-1152 or Y-27632). The morphological changes of F-actin cytoskeleton were visualized by rhodamine-phalloidin staining and the phosphorylation of Rho and ROCK were determined by Western blot. Endothelial monolayer permeability was assessed by measuring the flux of FITC-albumin across the endothelial cells. The results showed that the distribution of F-actin was significantly altered by AGEs-HSA in time and dose-dependent patterns. These effects were inhibited by ROCK inhibitors. The phosphorylation of Rho and RCOK was remarkably increased by AGEs-HSA treatment while total Rho and ROCK protein levels were not affected. The permeability of endothelial monolayer was dramatically increased by AGEs-HSA, and both ROCK inhibitors (H-1152 or Y-27632) attenuated these hyperpermeability responses. The results obtained suggest that the phosphorylation of Rho/ROCK plays an important role in AGEs-induced morphological and functional alterations in HMVECs.

Key words: Rho kinase; advanced glycation end products; endothelial cell; actin cytoskeleton

收稿日期：2008-11-19　　录用日期：2009-01-12

通讯作者：黄巧冰　　E-mail: bing@fimmu.com

引用本文：

王吉萍, 郭晓华, 王陵军, 李强, 陈波, 吴炜, 黄绪亮, 黄巧冰. Rho/ROCK 信号通路参与晚期糖基化终产物诱导的人皮肤微血管内皮细胞骨架结构改变[J]. 生理学报 2009; 61 (2): 132-138.

WANG Ji-Ping, GUO Xiao-Hua, WANG Ling-Jun, LI Qiang, CHEN Bo, WU Wei, HUANG Xu-Liang, HUANG Qiao-Bing. [Effects of Rho/ROCK signal pathway on AGEs-induced morphological and functional changes in human dermal microvascular endothelial cells.] [Ariticle in Chinese] . Acta Physiol Sin 2009; 61 (2): 132-138 (in Chinese with English abstract).