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目的研究人肺微血管内皮细胞丝状肌动蛋白(F-actin)在缺氧前后的变化,探讨在血管内皮细胞水平研究新生儿肺出血机制的方法。方法人肺微血管内皮细胞常规培养,分为对照组和1、2、4、6、12 h和24 h缺氧培养组,每组设8个复孔,异硫酸氢荧光素-鬼笔环肽与胞浆内F-actin结合而发出红色荧光,共聚焦显微镜扫描观察F-actin的变化情况并记录相应荧光值。结果缺氧1 h组F-actin平均荧光强度明显降低,为对照组的66.3%±5.3%,差异有统计学意义(P<0.05),缺氧24 h F-actin含量下降至对照组的47.1%±6.9%。缺氧后,细胞变得狭长,细长的丝状伪足明显可见,核周F-actin的分布明显减少,皮质状结构消失,应力纤维排列紊乱或部分消失,随着缺氧时间的延长F-actin逐渐解聚断裂。结论通过观察缺氧前后肺血管内皮细胞F-actin变化,可以为在内皮细胞水平研究新生儿肺出血发病机制提供实验研究基础。
Objective To study the changes of F-actin in human pulmonary microvascular endothelial cells before and after hypoxia, and to explore a method to study the mechanism of pulmonary hemorrhage in neonates with vascular endothelial cells. Methods Human pulmonary microvascular endothelial cells were cultured routinely and divided into control group and 1, 2, 4, 6, 12 h and 24 h hypoxia groups. Each group consisted of 8 duplicate wells, fluorescein isothiocyanate-phalloidin F-actin binding with the cytoplasm and emit red fluorescence, confocal microscopy changes observed F-actin and record the corresponding fluorescence value. Results The average fluorescence intensity of F-actin in 1 h hypoxia group was significantly lower than that in control group (66.3% ± 5.3%, P <0.05), and the content of F-actin in hypoxia group decreased to 47.1 % ± 6.9%. After hypoxia, the cells become slender, slender filopodia visible, the distribution of F-actin in the perinucleus significantly reduced, the cortical structure disappeared, the stress fibers arranged disorganized or partially disappeared, with the extension of hypoxia time F -actin gradually depolymerize the fracture. Conclusion Observing the changes of F-actin in pulmonary vascular endothelial cells before and after hypoxia can provide the experimental basis for studying the pathogenesis of neonatal pulmonary hemorrhage at the level of endothelial cells.