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目的:研究同型半胱氨酸(Homocysteine,Hcy)对内皮细胞形态及细胞骨架肌动蛋白的影响,并探讨骨架变化与内皮损伤的关系。方法:将体外培养的ECV-304内皮细胞株随机分为正常对照组、低剂量Hcy组、中剂量Hcy组和高剂量Hcy组。倒置相差显微镜观察各组培养24h的内皮细胞的形态变化;并用Rhodamine-Phalloidin标记纤维状肌动蛋白(F-actin),Alexa flour488-DnaseⅠ标记球状肌动蛋白单体(G-actin),激光扫描共聚焦显微镜(LSCM)观察各组内皮细胞骨架肌动蛋白的不同变化。结果:正常对照组内皮细胞基本为梭形,呈典型的鹅卵石样外观;各Hcy组内皮细胞形态则明显不同,随着Hcy浓度的增加,胞体出现收缩,细胞变圆、渐至不规则,且轮廓增强,细胞间隙增大,甚至坏死脱落。LSCM下发现,正常对照组内皮细胞F-actin在周边形成致密周围带,胞质内有部分应力纤维;随着Hcy浓度的递增,外周致密带逐渐出现断裂、甚至消散;胞质内应力纤维变短、增粗,最终消失。而G-actin正常时仅局限在核周,在受到不同浓度Hcy作用后,明显向外周扩散,且荧光强度增大。结论:内皮细胞骨架肌动蛋白的形态变化与重排,可能是Hcy损伤内皮细胞的重要机制之一。
OBJECTIVE: To study the effect of homocysteine (Hcy) on the morphology of endothelial cells and actin in cytoskeleton, and to explore the relationship between skeletal changes and endothelial injury. Methods: ECV-304 endothelial cell lines were randomly divided into normal control group, low dose Hcy group, medium dose Hcy group and high dose Hcy group. Inverted phase contrast microscope was used to observe the morphological changes of endothelial cells cultured for 24h. The expression of F-actin, G-actin and Alexa flour488-DnaseⅠ were detected by Rhodamine-Phalloidin, Confocal microscopy (LSCM) was used to observe the changes of actin in endothelial cells of each group. Results: The endothelial cells in the normal control group were basically spindle-shaped with a typical pebble-like appearance. The morphology of endothelial cells in each Hcy group was significantly different. With the increase of Hcy concentration, the somatic cells appeared contracted, the cells became round and gradually irregular, Contour enhancement, cell gap increases, or even necrosis. LSCM found that the normal control group of endothelial cells F-actin formed in the periphery of the dense surrounding bands, the cytoplasm of some stress fibers; with increasing concentration of Hcy, the gradual emergence of the peripheral zonula fracture, or even disappear; cytoplasmic stress fibers Short, thick, and eventually disappear. While G-actin normal only confined to the perinuclear weeks, after being exposed to different concentrations of Hcy, significantly to the periphery, and the fluorescence intensity increased. Conclusion: The morphological changes and rearrangements of actin in endothelial cells may be one of the important mechanisms of Hcy injury to endothelial cells.