摘要： 目的 利用活细胞显微成像术对脑微血管内皮细胞内肌动蛋白的动态变化实现实时观察。方法 构建绿色荧光蛋白（GFP）标记Lifeact的真核表达载体即Lifeact-pEGFP，利用脂质体转染法将Lifeact-pEGFP质粒转染至脑微血管内皮细胞中，利用共聚焦激光扫描显微镜对Lifeact-pEGFP在细胞内的分布进行分析，并检测其在细胞伸展和迁移过程中的动态变化。结果 成功将Lifeact-pEGFP质粒转染至人脑微血管内皮细胞，细胞存活未受到影响。Lifeact-pEGFP能将活细胞中的肌动蛋白丝标记为绿色荧光，并能够有效监测细胞伸展和迁移过程中肌动蛋白丝的动态变化。结论 Lifeact-pEGFP转染结合活细胞显微成像术可以用于实时记录和分析脑微血管内皮细胞内肌动蛋白丝的动态变化。

利用荧光蛋白示踪脑微血管内皮细胞微丝的动态变化

杜书嵩^1,2, 赵伟东¹

1. 中国医科大学生命科学学院发育细胞生物学教研室, 教育部医学细胞生物学重点实验室暨卫健委细胞生物学重点实验室, 沈阳 110122;
2. 中国医科大学附属第一医院肛肠外科, 沈阳 110001

收稿日期:2020-04-17出版日期:2020-08-30发布日期:2020-08-04
通讯作者:赵伟东E-mail:wdzhao@cmu.edu.cn
作者简介:杜书嵩(1988-),男,医师,硕士.
基金资助:国家自然科学基金（31670845，31870832）


关键词: Lifeact, 肌动蛋白, 脑微血管内皮细胞, 活细胞显微成像术
Abstract: Objective To detect the dynamic changes of actin in brain microvascular endothelial cells (BMECs) using real-time live-cell imaging. Methods The eukaryotic expression vector encoding green fluorescent protein (GFP) -labeled Lifeact peptide was constructed. The Lifeact-pEGFP plasmid was transfected into BMECs using a liposome reagent. The intracellular distribution and dynamic changes of the plasmid during cell spreading and cell migration were analyzed by confocal microscopy. Results The Lifeact-pEGFP plasmid was successfully transfected into BMECs without affecting cell survival. Lifeact-pEGFP can label the actin filaments as green fluorescence in living cells and can effectively monitor the dynamic changes of actin filaments during the process of cell spreading and cell migration. Conclusion Lifeact-pEGFP transfection combined with live-cell imaging can be used for the real-time recording and analysis of the dynamic changes of actin filaments in BMECs.
Key words: Lifeact, actin, brain microvascular endothelial cells, live-cell imaging
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