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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30min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。
This article aims to investigate the role and mechanism of Rho signaling pathway in the morphological and functional changes of human dermal microvascular endothelial cells (HMVECs) induced by advanced glycation end products (AGEs). The HMVECs were cultured in vitro. Human serum albumin (HSA) modified with different concentrations of AGEs were treated with different time, and the control group was set up for comparison. The structure and distribution of actin F-actin in cytoskeleton were observed by immunofluorescence staining and laser confocal microscopy. The cells were pretreated with Rho kinase (ROCK) inhibitor Y-27632 or H-1152 for 30 min, The changes of Rho, ROCK and phosphorylation were detected by Western blotting. The permeability coefficient (Pa) of monolayer endothelial cells was measured by FITC fluorescence-labeled protein leakage method. The results showed that the AGEs-HSA induced a change in the structure and distribution of F-actin in HMVECs in a dose-and time-dependent manner, while the unmodified HSA did not. ROCK-specific inhibitors Y-27632 and H-1152 Inhibition of AGEs on the cytoskeleton. AGEs-HSA caused an increase of phosphorylation of Rho and ROCK (P <0.05), but had no significant effect on total protein. Compared with the control group, AGEs-HSA increased endothelial cell permeability (P <0.01), Y-27632 and H-1152 inhibited this change (P <0.01). The above results suggest that Rho signaling pathway plays an important role in the AGEs-mediated morphological and functional changes of HMVECs.