【作者】 郭子义；

【导师】 姜宗来；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2007， 博士

【摘要】 正常的血流切应力对于维持血管正常功能包括抗血栓形成、屏障功能和血管本身的动态平衡都是至关重要的。血流切应力变化将导致血管重建(remodeling),而病理性的血管重建是动脉粥样硬化、高血压和血管再狭窄等病变共同的病理基础。了解切应力变化对血管重建的影响及其机制,对探讨心血管疾病的发病机制、诊断与治疗有重要的理论和实际意义。本文采用了结扎一侧颈总动脉部分分支,造成两侧颈总动脉的血流切应力显著改变的大鼠模型,研究血流切应力变化对颈总动脉重建的影响并初步探讨其机制。以不结扎动脉分支的假手术动物作为对照组。采用光镜和透射电镜观察动脉显微组织结构、几何形态学和超微结构的变化;观测动脉零应力状态下张开角的变化;应用TUNNEL法观察了血管平滑肌细胞(VSMC)凋亡;用激光共聚焦显微镜观测动脉内弹力膜(IEL)重建;并用大分子示踪剂辣根过氧化物酶(HRP)示踪法研究了IEL通透性的变化;用免疫组化和免疫印迹法检测了动脉calponin, fibronectin, p-Akt, p21等分子表达;用明胶酶谱法研究了基质金属蛋白酶(MMPs)活性,并在应用了MMPs的抑制剂“强力霉素”对血管重建进行干预后,再观察calponin, p-Akt, p21, MMPs表达的变化。结果显示:①结扎大鼠左颈总动脉(LCA)的远侧部分分支,造成同一动物LCA的低平均切应力状态,对侧右颈总动脉(RCA)高平均切应力状态。在切应力显著降低7天后,LCA管径减小,壁厚内径比增加,张开角减小,VSMC凋亡和去分化增加,内皮下层增厚;而RCA的管径和壁厚内径比未见明显变化,但VSMC的凋亡和去分化也显著增加;②切应力的降低导致LCA的IEL窗减小,通透性改变,大分子示踪剂在IEL下层的显著聚积,并伴有fibronectin表达的增加;明显区别于高切应力RCA的变化;③切应力的显著变化导致血管内MMPs的活性增加,并伴有Akt / p21信号通路的活化;应用MMPs的抑制剂“强力霉素”可以显著抑制MMPs的活性,同时也在一定程度上抑制了切应力诱导的Akt / p21信号通路的活化及VSMC的去分化。上述结果表明,在血流切应力降低的较早阶段即出现动脉壁的结构性重建,其中以IEL的重建最为显著,并可导致大分子物质在动脉壁内的异常聚积。血流切应力变化增加了MMPs的活性,而MMPs的抑制剂可能在多个环节上拮抗切应力变化所诱导的血管重建,以维持血管结构和功能的稳定。更多还原

【Abstract】 Physiological flow shear stress plays a central role in anti-thrombus, maintaining barrier function and homestasis of blood vessel. The alterated flow shear stress would induce vessel remodeling, which was the common pathological foundation of vascular diseases such as atherosclerosis, hypertension and restenosis. Therefore, it is very important to eluciate the underlying mechanisms of the vascular remodeling induced by flow shear stress alterations for the prevention, diagnosis and treatment of cardiovascular diseases.In present study, the ligation of partial distal branches of the left common carotid (LCA) was performed, which resulted in different changes of flow shear stress in both carotids of the same rat. The sham-operation animals without arterial ligation were as a control group. The effects of shear stress alteration on remodeling of carotid arteries and underlying mechanisms were inspected. The morphometry, histology, and ultrastructures of the vessels were examined by both light microscopy and transmission electron microscopy. The changes of vascular opening angle at zero-stress state were detected. Apoptotic cells of the vascular wall were examined by TdT-mediated dUTP-biotin nick end labeling (TUNNEL) staining. The vascular internal elastic lamina (IEL) was insighted with horseradish peroxidase (HRP), a large molecular tracer, by laser confocal scanning microscopy. The expressions of calponin, fibronectin, p-Akt and p21 in the vascular wall were examined by immunohistochemistry and Western blotting. The activity of matrix metalloproteinases (MMPs) in the vascular wall was revealed by gelatin zymography.Our results revealed that: (1) The ligation of partial distal branches of LCA induced low shear stress of LCA and high shear stress of the contralateral right carotid arteries (RCA). At 7th day of post-operation both the inner diameter and opening angle of LCA were decreased, while the ratio of wall thickness to inner diameter was increased. RCA did not show such changes. The apoptic and dedifferentiated vascular smooth muscle cells were increased due to changes of shear stress. The thickness of sub-endothelial layer was increased in LCA significantly. (2) Low shear stress induced the decrease of IEL fenestrae, which resulted in abnormal molecular accumulation in the interstitial space underlying IEL with augmented expression of fibronectin, significantly different from RCA. (3) Changes of shear stress increased the MMPs activities and p-Akt expressions, while reduced expressions of p21 and calponin in vessels, which was reversed by Doxycycline, an inhibitor of MMPs.These results revealed that low shear stress induced structural changes of artery, especially IEL remodeling, and resulted in macromolecular accumulation in the arterial wall at more early in course of the alternation of shear stress. The alteration of shear stress induced MMPs activities in the vascular wall. Otherwise, the inhibitor of MMPs might benefit to the vascular remodeling induced by the alternation of shear stress.更多还原