Rho激酶在烧伤大鼠血清诱导的血管内皮细胞骨架变化中的作用  

Role of Rho kinase in reorganization of the vascular endothelial cytoskeleton induced by rat burn serum

作　　者：郑惠珍[1] 赵克森[1] 黄巧冰[1] 

机构地区：[1]南方医科大学病理生理学教研室

出　　处：《中华烧伤杂志》2005年第3期181-184,共4页Chinese Journal of Burns

基　　金：国家重点基础研究发展规划资助项目 (G2000057004;G1999054202 );国家自然科学基金海外青年学者合作研究资助项目(30028008)

摘　　要：目的　观察烧伤血清刺激下血管内皮细胞骨架的变化及Rho信号转导通路所起的作用。　方法　常规培养人脐静脉血管内皮细胞系ECV304,随机分为空白对照组、实验对照组、烧伤组、Y -27632组、烧伤+Y -27632组、Y- 27632+烧伤组、溶血磷脂酸(LPA)组和LPA+Y- 27632组,分别用正常大鼠血清、烧伤大鼠血清、30μmol/LRho激酶抑制剂Y -27632、13μmol/LRhoA激动剂LPA单独刺激或联合刺激。采用HE染色于光镜下观察各组内皮细胞形态。于荧光倒置相差显微镜下观察内皮细胞骨架变化,除Y -27632组外,各组均在刺激后6、7、8h3个时相点进行观察。用流式细胞仪检测实验对照组、烧伤组、Y -27632组、烧伤+Y- 27632组、LPA组和LPA+Y 27632组刺激6h的肌动蛋白含量。　结果　空白对照组、实验对照组细胞呈梭形或多边形,生长良好;丝状肌动蛋白主要分布在细胞周边,形成周边肌动蛋白丝带,细胞生长融合为单层后呈网状结构;球状肌动蛋白集中在细胞中央。烧伤组烧伤血清刺激6h,细胞贴壁差,丝状肌动蛋白重组,应力纤维形成,周边肌动蛋白丝带模糊;球状肌动蛋白松散,胞浆中可见散在分布的絮状球状肌动蛋白,且这些反应随着刺激时间延长而增强。烧伤+Y- 27632组或Y -27632+烧伤组细胞生长、贴壁良好,丝状肌动蛋白的分布与空白对照组。Objective To investigate the changes in endothelial cytoskeletal reorganization and the role of Rho in the signal transduction pathway. Methods ECV304 cells were cultured and randomly divided into following groups: i. e. sham(with normal rat serum treatment), burn (with burn rat serum treatment), Y( with 30 μmol/L Rho kinase inhibitor Y-27632 treatment), burn plus Y (pretreatment of cells with burn serum before treated with 30 μmol/L Y-27632), Y plus burn (pretreatment of cells with Y-27632 for 1 hour before treated with burn serum), LPA (with normal rat serum and 13 μmol/L LPA), and LPA plus Y (pretreatment of cells with LPA before treated with Y-27632) groups. The indices were examined at 6,7 and 8 posttreatment hours (PTH) in all groups except in Y group. The endothelial morphology was observed with HE staining. Endothelial cytoskeleton was observed by dual-fluorescence labeling of filamenta (F) with Rhodamind-phalloidin and monomer (G) with oregon green labeled DNAase. The actin content in the cells in all groups was measured with flow cytometry. Results In sham and control group, the cells were in fusiform or polygonal shape, with satisfactory growth; filamentous actin (F-actin) was mainly distributed in the peripheral site of the cytoplasm and formed peripheral filamental band. The cells became confluent to form a single layer with reticular structure. Globular actin (G-actin) was concentrated in the nucleus and perinucleus. In burn group, after 6 hours of burn serum treatment, the ability of cells to adhere to vessel wall was weakened, and a striking reorganization of the actin cytoskeleton and the formation of the stress fibers were found. Furthermore, the fluorescent intensity of the peripheral filament bands was weakened, and dispersed actin monomers were seen in the cytoplasm. This reaction was enhanced along with elapse of stimulation time. In burn plus Y or Y plus burn group, the cells grew and adhered well to the wall of culture vessel. The distribution of the filamentous actin was the s

关 键 词：内皮细胞骨架 RHO激酶 血清诱导 肌动蛋白骨架重组 血管内皮细胞系 流式细胞仪检测 肌动蛋白丝 信号转导通路 烧伤血清 ECV304 内皮细胞形态 显微镜下观察 蛋白含量 对照组 大鼠血清 细胞生长 溶血磷脂酸 激酶抑制剂 LPA 

分 类 号：R644[医药卫生—外科学] R363[医药卫生—临床医学]