材料表面化学和流体剪切力共同刺激对人脐静脉内皮细胞的影响

Effects of co-stimulation of material surface chemistry and fluid shear stress on human umbilical vein endothelial cells

背景:组织工程骨内缺乏完善的脉管系统,其中血管化是限制其广泛应用的关键,适当的基质化学和流体剪切力刺激能促进血管内皮细胞增殖分化和发挥功能,指导支架材料的设计制作。目的:探究不同化学官能团和流体剪切力对人脐静脉内皮细胞的影响,以期找到材料化学与流体剪切力对人脐静脉内皮细胞调控的高效组合。方法:在玻片表面分别制备以OH、CH3和NH2为末端化学官能团的自组装单分子膜,以空白玻片作为对照;将人脐静脉内皮细胞分别接种于4组玻片表面,培养15 min内检测ATP的释放量,培养1 h内检测NO的释放量,培养1 h后Western blotting检测内皮型一氧化氮合酶的表达,培养48 h后激光共聚焦显微镜观察黏着斑及F肌动蛋白的形成。将人脐静脉内皮细胞分别接种于4组玻片表面,当细胞融合至80%时给予加载1.5 N/m2流体剪切力1 h(分别为OH-FSS组、CH3-FSS组、NH2-FSS组和FSS组),加载15 min内检测ATP的释放量,加载1 h内检测NO的释放量,加载1 h后Western blotting检测内皮型一氧化氮合酶的表达。结果与结论:(1)单纯化学刺激对人脐静脉内皮细胞的ATP、NO释放与一氧化氮合酶表达无明显影响,加载FSS刺激可增加ATP、NO的释放与内皮型一氧化氮合酶表达;当材料化学和流体剪切力同时作用时,人脐静脉内皮细胞释放的ATP和NO量及内皮型一氧化氮合酶表达与材料化学密切相关,其中NH2-FSS组中的ATP和NO释放量及内皮型一氧化氮合酶表达最高,其次为FSS组,CH3-FSS组和OH-FSS组最低;(2)激光共聚焦显微镜下可见,NH2组有大量黏着斑和细胞骨架蛋白F肌动蛋白,其次为对照组,CH3组和OH组最少;(3)结果表明,NH2官能团和流体剪切力共同作用时人脐静脉内皮细胞对剪切力刺激产生高效应答,其机制可能是在NH2表面上形成了最佳的黏着斑和F肌动蛋白。

BACKGROUND:There is a lack of perfect vascular system in tissue-engineered bone,among which vascularization is the key to limit its wide application.Appropriate matrix chemistry and fluid shear stress stimulation can promote the proliferation,differentiation and function of vascular endothelial cells,and guide the design and fabrication of scaffold materials.OBJECTIVE:To explore the effects of different chemical functional groups and fluid shear stress (FSS) on human umbilical vein endothelial cells,in order to find an efficient combination of material chemistry and fluid shear stress on human umbilical vein endothelial cells.METHODS:Self assembled monolayers with OH,CH3 and NH2 as end functional groups were prepared as matrix materials and blank glass as control.Human umbilical vein endothelial cells were seeded on the surface of four groups of glasses.The release of ATP within 15 minutes after culture and NO within 1 hour after culture was detected.The expression of endothelial nitric oxide synthase protein was detected by western blotting.The formation of focal adhesion and F-actin was observed by laser confocal icroscopy.Human umbilical vein endothelial cells were seeded on the surface of four groups of glasses.When the cells fused to 80%,they were loaded with 1.5 N/m2 FSS for 1 hour.ATP release was detected within 15 minutes after loading,and NO release was detected within 1hour after loading.The expression of endothelial nitric oxide synthase was detected by western blotting after loading for 1 hour.RESULTS AND CONCLUSION:(1) Chemical stimulation had no effect on the release of ATP or NO,but FSS could increase the release of ATP and NO,and the expression of endothelial nitric oxide synthase.When FSS and material chemistry acted simultaneously,the expression of ATP and NO was closely related to material chemistry.The release of ATP and NO and the expression of endothelial nitric oxide synthase were highest in NH2-FSS group,followed by FSS group,and those in the CH3-FSS and OH-FSS groups were lowest.(2) Laser confocal microscope showed that a large number of focal adhesions and cytoskeleton protein F-actin could be detected in the NH2 group,followed by control group;those were least in the CH3 and OH groups.(3) The results showed that the combination of NH2 functional group and FSS produced high-efficiency response to shear stress,and the mechanism may be the formation of optimal focal adhesion and F-actin on NH2 surface.