利用荧光蛋白示踪脑微血管内皮细胞微丝的动态变化

Application of fluorescent protein-based technique in tracing the dynamic changes of actin microfilaments in brain microvascular endothelial cells

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-p EGFP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-p EGFP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。

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-p EGFP 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.