低密度脂蛋白对血管内皮细胞骨架及粘附能力的影响

Effect of low density lipoprotein on cytoskeleton and adhesion of vascular endothelial Cells

目的研究低密度脂蛋白(LDL)诱导的血管内皮细胞脂质及细胞骨架的改变及其对内皮细胞粘附的影响。方法不同浓度LDL(0mg/L,50mg/L,100mg/L,150mg/L)孵育内皮细胞24h。采用高效液相色谱法检测细胞内胆固醇酯;油红O检测细胞内脂质;FITC标记的鬼笔环肽进行F-actin纤维荧光标记;流室系统检测生理剪切流场下(1.5Pa)内皮细胞的粘附。结果不同浓度LDL(50mg/L,100mg/L,150mg/L)均导致细胞内胆固醇酯含量增加,其结果分别为(80.3±1.5)μg/g,(115.5±2.2)μg/g和(160.6±3.2)μg/g,与对照组(61.3±1.6)μg/g相比P<0.05;油红O染色内皮细胞内脂质含量明显增加。荧光染色发现,对照组内皮细胞的F-actin纤维主要富集在相邻细胞膜的周边,细胞质中未见密集的F-actin纤维;随着LDL浓度的增加,内皮细胞内F-actin纤维形态和分布发生明显的改变,出现应力纤维;在高浓度LDL下,大部分细胞周边的F-actin纤维消失,胞质中出现密集的应力纤维。在生理剪切流场下,高浓度LDL降低内皮细胞的粘附与保留,150mg/L组内皮细胞的粘附与保留仅50%±10%,对照组为93%±5%。结论LDL增加内皮细胞中脂质含量,损伤细胞骨架,降低内皮细胞的顺应性,降低内皮细胞的粘附。

Objective To investigate the Effect of low density lipoprotein （LDL） on endothelial cell adhesion in shear stress and its relationship with lipid and filamentous actin （F-actin）. Methods The endothelial cells （ECV-304） were incubated with different concentrations of low density lipoprotein （0 mg/L, 50 mg/L,100 mg/L,150 mg/L） for 24h. Thereafter, the content of cholesterol ester in endothelial cells was assayed with High Performance Liquid Chromatography （HPLC）. The lipid within endothelial cells was detected with oil red O. F-actin was examined with fluorescence staining of rhodamine-phalloidin. After treated with LDL, endothelial cells were subjected to flow shear stress of 15Pa for 24h. Results Compared to control LDL significantly increased the ester level in endothelial cells（80.3±1.5μg/g,115.5±2.2μg/g and 160.6±3.2μg/g to control 61.3±1.6μg/g,p〈0.05）. Observation by fluorescence microscopy showed that the fibers of F-actin in normal endothelial cells concentrated along the cell membrane and no dense fibers were observed in the cytoplasm. The abnormal F-actin were appeared in LDL-treated endothelial cells, while most of the peripheral fibers disappeared and dense stress fibers were observed in the cytoplasm with high dose LDL After exposed to 1.5 Pa shear stress the conformance and the adhesion of endothelial cells which incubated with 150 mg/L LDL decreased comparing with control. Conclusion LDL causes lipid accumulation and the cytoplasm damaged, which may relate to endothelial cell conformance and adhesion.