晚期糖基化终产物受体在脂多糖介导小鼠肺微血管内皮细胞骨.PDFVIP

·6 · doi 10.3969/j.issn. 1673-4254.2015.01.02 J South Med Univ, 2015, 35(1): 6-11 基础研究 晚晚期糖基化终产物受体在脂多糖介导小鼠肺微血管内皮细胞骨期糖基化终产物受体在脂多糖介导小鼠肺微血管内皮细胞骨 架变架变化中的作用化中的作用 周晓燕，张伟金，黄巧冰，郭晓华 南方医科大学病理生理学教研室//广东省医学休克微循环重点实验室，广东 广州 510515 摘要：目的 建立一种可以得到高纯度肺微血管内皮细胞的分离方法，并分别观察野生型小鼠及晚期糖基化终产物受体 （RAGE）敲除小鼠细胞在脂多糖（LPS）刺激下细胞骨架F-actin 变化情况。方法 取6~8周龄野生型C57小鼠及RAGE 基因敲除 小鼠的肺，经I 型胶原酶消化后，尼龙细胞筛过滤，然后采用免疫磁珠法分离原代小鼠肺微血管内皮细胞（PMVEC），经形态学及 免疫荧光法鉴定后，以LPS（1 mg/L）刺激不同时间后用激光共聚焦显微镜观察细胞骨架F-actin 的变化情况。结果 镜下可见原 代培养的PMVECs 呈梭形或多角形，经细胞抗Ⅷ因子相关抗原（ⅧF-Ag）染色鉴定纯度高，LPS 刺激后野生型小鼠PMVEC 骨架 重排，形成应力纤维，而RAGE 敲除小鼠PMVEC 骨架破坏不明显。结论 采用免疫磁珠法可以获得高纯度的小鼠肺微血管内 皮细胞，且RAGE 参与了LPS 介导的小鼠肺微血管内皮细胞骨架破坏。 关键词：小鼠肺微血管内皮细胞；脂多糖；晚期糖基化终产物受体；细胞骨架 Role of RAGE in lipopolysaccharide-induced cytoskeletal changes in mouse pulmonary microvascular endothelial cells ZHOU Xiaoyan, ZHANG Weijin, HUANG Qiaobing, GUO Xiaohua Department of Pathophysiology of Southern Medical University, Key Laboratory of Shock and Microcirculation of Guangdong Province, Guangzhou 510515, China Abstract: Objective To investigate lipopolysaccharide (LPS)-induced changes of cytoskeletal filamentous actin in primary isolated pulmonary microvascular endothelial cells (PMVECs) from wild-type and RAGE knock-out mouse. Methods The lungs of wild-type and RAGE knock-out mice were digested with collagenase type I to obtain endothelial cells purified by anti-CD31-coupled magnetic beads. The PMVEC identified by factor VIII labeling were stimulated with LPS at different concentrations and the changes of filamentous actin were observed by confocal microscopy. Results The cultured primary cells showed typical endothelial cell phenotype as examined with factor VIII labeling. LPS stimulation caused rearrangement of the cytoskeletal filament F-actin in wild-type mouse PMVECs with stress fiber formation, but such changes