【作者】 王燕华；

【导师】 姜宗来；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2009， 博士

【摘要】 血管内皮细胞(endothelial cells, ECs)迁移(migration)是血管重建(remodeling)的主要特征之一,对于维持血管功能和结构的稳定具有重要作用。在血管发育、血管新生以及血管内膜损伤(如球囊损伤和动静脉移植)修复等过程中,均出现ECs的迁移。ECs迁移受生物、化学和物理等多种体内外因素的调控,研究ECs的迁移行为及其调控机制,阐明ECs迁移的力学生物学(mechanobiology)机制对于深入了解心血管活动和疾病发生的本质有重要意义,且可以为发展心血管疾病有效的防治措施提供理论依据。ECs和血管平滑肌细胞(vascular smooth muscle cells, VSMCs)是构成血管壁的主要细胞成分,生理状态下,这两种细胞本身相互影响,同时又不断的受到血液动力学的作用。因此,研究ECs的迁移行为,应该考虑相关的力学因素及VSMCs的影响。本文首先应用ECs与VSMCs联合培养系统,通过细胞粘附实验,观测了VSMCs对ECs粘附的影响及其机制。用免疫荧光法观测了ECs在粘附过程中,粘着斑(focal adhesions, FAs)的聚集;用全内反射荧光显微镜观测了粘附ECs的FAs面积变化;同时应用免疫印迹法检测了VSMCs对ECs的细胞骨架成分微管(microtubule)聚合程度、桩蛋白(paxillin)和胞外信号调节蛋白激酶(extracellular signal-regulated protein kinase,ERK)磷酸化的影响。在此基础上,应用ECs与VSMCs联合培养的平行平板流动腔系统,对ECs面施加15 dynes/cm2切应力,以Transwell法检测ECs的迁移能力,免疫印迹法定量检测相关蛋白,研究了切应力对与VSMCs联合培养ECs迁移的影响及其细胞骨架机制,即组蛋白去乙酰化酶(histone deacetylase,HDAC)/乙酰化微管信号途径在其中的作用。结果发现:①与VSMCs联合培养明显促进了ECs的粘附和迁移,降低了ECs的微管骨架聚集,诱导了ECs的HDAC6、paxillin和ERK的磷酸化表达增高;②HDAC6抑制剂曲古抑菌素A (trichostatin A,TSA)和三丁酸甘油酯(tributyrin,TB)可改变微管的聚集状态,抑制VSMCs诱导的ECs粘附、迁移及HDAC6及ERK激酶、paxillin的激活;③生理范围切应力同样可以促进ECs迁移,诱导ECs的HDAC6表达,同时改变了微管骨架的乙酰化修饰水平;④生理切应力加载降低了与VSMCs联合培养ECs的迁移能力和HDAC6的表达及与ECs联合培养的VSMCs的CTGF的表达。上述结果表明,VSMCs能够促进ECs的粘附与迁移,而生理大小的切应力作为血管壁结构和功能的稳定因素,同样可以调控ECs的迁移,对血管壁起保护作用。切应力和VSMCs通过HDAC6影响ECs微管骨架动态聚集,经由ERK/paxillin信号通路,调控ECs迁移。更多还原

【Abstract】 Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress, which behaviors are strongly influenced by the neighboring vascular smooth muscle cells (VSMCs). The adhesion and migration of ECs are essential functions to keep the vascular homeostasis. Thus, the research in the area of EC biology will require considering mechanical factors and cell-to-cell interaction between VSMCs and ECs.In this paper, using a coculture system, we firstly investigated the effect of interaction of ECs and VSMCs on adhesion of ECs. And then the difference of EC migration of VSMC/EC co-culture or cultured alone under static conditions and in response to shear stress was assessed by a parallel-plate co-culture flow chamber system. The results showed that ECs co-cultured with VSMCs exhibited a significant increase in the number of adherent cells, which was correlated with enhanced assembly of FA and increased focal adhesion area. At the same time, VSMCs also prompted ERK and paxillin phosphorylation of ECs and downregulated its microtubule polymerization state. Using Trichostatin A which can reverse microtubule polymerization state, it was demonstrated that the adhesion and activation of ERK and paxilllin were impacted by microtubule polymerization state.With Transwell migration assays, we also demonstrated that VSMCs induced cocultured EC migration; when laminar shear stress (1.5 Pa, 15 dynes/cm2, NSS) applied to ECs cultred alone for 12 h, the EC migration was also significantly elevated; when NSS and VSMCs impacted ECs at the same time, the migration of ECs co-cultured with VSMCs was not prompted more under NSS. The changes in EC migration had a consisitent correlation with the level of acetylated microtubule and HDAC6. While HDAC6 was inhibited by tribytyrin or specific small interference RNA, EC migration was impacted, and followed hyperacetylation of tubulin.These results indicate that NSS and VSMCs regulate EC migration, which may be an atheroprotective function on the vessel wall to keep the vascular homeostatis. Meanwhile, microtubule/ERK/paxillin pathway may play a key role in EC migration modulated by NSS and VSMCs.更多还原