【作者】 刘莹；

【导师】 宾建平；

【作者基本信息】 南方医科大学 ， 内科学， 2011， 硕士

【摘要】 背景和目的动脉粥样硬化(atherosclerosis, AS)导致的心脑血管疾病是危害人类健康的主要原因之一,AS的致病机理一直是心血管领域的研究热点,粥样斑块常出现于动脉特定部位(血流低速区,回流区等),最主要的脂质浸润学说、血栓形成学说、单克隆学说和损伤反应学说等并不能完全解释AS发生的局灶性(localization of atherosclerosis),而血流动力学因素在AS发生条件中具有主要的作用,血流动力学成因理论为认识AS的局灶性和发病机制提供了新的视角,与作用时间相关的血流剪切应力,尤其是低剪切应力(low shear stress)大小、方向的改变,可通过引起血管重建(vascular remodeling)和内皮黏附分子(endothelial adhesion molecule)表达的上调,导致内皮炎症的发生,从而在AS的发生、发展中起着关键作用。正常范围的血流剪切应力对于维持血管稳态包括抗血栓形成、屏障功能和血管本身的动态平衡有重要的调节作用,血流剪切应力改变后血管稳定性受到破坏,将致使血管重建的发生。血管重建是指为适应内、外环境(尤其是力学环境)的变化而发生的血管形态结构和功能的一系列适应性改变,包括早期组分不变的适应性重排和晚期结构与功能改变的病理性重建,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常见心脑血管病变共同的病理基础,但是造成血管重建的细胞分子生物学机制尚不清楚。剪切力学说认为,均匀的层流剪切力可选择性地诱导内皮抗动脉粥样硬化基因的表达,作用于局部以抵消全身危险因素的有害作用,当剪切力的性质和大小出现异常,内皮分泌抗动脉粥样硬化分子减少,促炎因子表达增加,从而激活相应的信号转导通路,参与诱导炎症进展,导致血管重建和AS的发生、发展。炎症细胞与内皮细胞的粘附和跨壁转移是AS形成机制的第一步,正常情况下白细胞很难与内皮黏附,只有当外来因素如细胞因子、氧化脂蛋白、切应力等与内皮相互作用后使内皮表面不表达或低表达的粘附分子如：细胞间粘附分子-1 (ICAM-1)、VCAM-1、P-选择素和E-选择素等表达增加,有助于各种白细胞的粘附。其中P-选择素(P-selectin)在动脉病变早期对活化的血小板、白细胞与内皮细胞的初次黏附起决定性作用,是启动内皮炎症反应并维持炎症状态的关键成分,正常的内皮细胞仅表达极少量无介导白细胞活性的P-选择素,在血流低切应力等因素持续作用下,通过进一步的脂质堆积、内皮功能改变等引起炎症刺激内皮,内皮才表达具有介导炎症细胞活性的P-选择素；而血管内皮黏附分子-1 (vascular cell adhesion molecule-1, VCAM-1)主要在后续的白细胞与内皮细胞的紧密黏附及介导白细胞渗出、迁移过程中起作用,正常条件下内皮不表达VCAM-1,理化因素(如血流剪切应力异常等)刺激损伤内皮后表达增加,并有延迟表达或持续长久的特点,可促进对内皮的损伤,亦是维持内皮炎症状态的关键成分之一。因此,如果血流剪切应力异常是AS发生的始动因素,内皮表达的P-选择素、VCAM-1等粘附分子则可能是AS形成的枢纽之一。如能尽早检测出反映血流动力学变化的综合指标,深入研究剪切力变化早期引起的血管生物学效应以及其与AS形成之间的关系和作用机制,对进一步揭示AS的本质、探索防治AS的方法有重要的理论和实际意义。目前已有研究发现,大小、形态异常的血流剪切应力作用可通过影响多种炎症分子的表达而导致不同性质的粥样斑块形成,但有关血流动力学变化早期,低剪切应力作用对动脉病变早期血管重建的影响及其相关分子生物学机制尚不清楚。为此,本研究拟通过制备小鼠腹主动脉局部狭窄模型,在同一段平直血管上建立近似层流和振荡流的低剪切应力区域,观察血流动力学改变短时间内不同形态低剪切应力对动脉重建和内皮P-选择素表达的影响,并在此基础上较长时间观察近似层流低剪切应力作用下动脉病理形态学与内皮P-选择素、VCAM-1等黏附分子表达的动态变化,以进一步了解低剪切应力引起的血管生物学效应与早期AS发生、发展之间的关系及其作用机制。材料和方法1、实验动物分组：①短时间观察组：20只昆明小白鼠,性别不限,体重(18.0-2.0)g,随机分为短时间对照组和短时间观察组(狭窄1h,4h,24h组),每组5只；②长时间观察组：25只昆明小白鼠,性别不限,体重(18.0-2.0)g,随机分为长时间对照组和长时间观察组(狭窄1d,7d,14d,28d组),每组5只。2、腹主动脉局部狭窄模型制备：狭窄组小鼠腹腔注射戊巴比妥钠麻醉后,仰卧位固定四肢,腹部去毛后75%酒精消毒皮肤,取正中线剪开皮肤及腹壁,移开肠腔并用温盐水纱布保湿,分离左肾动脉分支与髂动脉分叉之间腹主动脉,平直段放置内径0.1mm动脉银夹,小心回置肠腔,无菌缝合切口关闭腹腔,对照组组小鼠只分离,未放置银夹。对照组于术后即刻,短时间组于术后1、4、24h,长时间组于术后1、7、14、28d行彩超检查,再从原切口开腹,从心脏采血1ml置抗凝管待测血粘度,暴露腹主动脉,用冰丙酮灌注固定后,分别取出短时间组银夹近心端及远心端、长时间组银夹近心端及对照组腹主动脉组织各约5mm,液氮速冻待冰冻切片染色观察。3、血流动力学参数测定：将超声高频探头置于小鼠腹部,分别测量短时间组银夹近心端及远心端、长时间组银夹近心端5mm内血流动力学参数：舒张末期血管内径(D)、最大血流速度(Vm),每只小鼠测10次最后取平均值。抗凝血于4h内用毛细血管黏度计测量血液粘度(η)(低切)。根据公式τm=η×4×Vm/D计算血流剪切应力(τm)。4、动脉HE染色与形态学分析：采用冰冻切片常规HE染色方法,5μm不连续切片,4℃冰丙酮固定30 s稍水洗,苏木素染色40s-60s后水洗5s-10s(必要时1%盐酸酒精分化温水返蓝),伊红滴后速洗,梯度酒精脱水、二甲苯透明,中性树胶封片,每条动脉组织随机抽取2张切片,光镜下观察血管壁形态结构,AutoCad图像分析软件测量血管内径与壁厚。5、内皮粘附分子免疫组织化学染色与量化分析：短时间组检测内皮P-选择素,长时间组与对照组检测内皮P-选择素与VCAM-1。各组在相同条件下采用二步法检测,5μm冰冻切片置室温30mmin,4℃冰丙酮固定10min (PBS洗5 minx3次),加过氧化氢室温10 min阻断内源性过氧化物酶活性(PBS洗5 minx3次),5%-10%山羊血清封闭10min倾去勿洗,滴加一抗置4℃冰箱过夜后室温复温30min (PBS洗5 minx3次),加二抗工作液室温30min, DAB显色,苏木精复染,脱水透明后封片观察并摄片。每个标本随机抽取2张不连续切片,用IPP图像分析系统进行阳性染色吸光度的量化分析。结果1、血流动力学参数检测结果显示：各实验组动物腹主动脉内径(D)和血液黏度(η)之间无统计学差异；狭窄组银夹近心端血流近似层流,其最大血流速度(Vm)和血流剪切应力(τm)较对照组减低,差异有统计学意义,银夹远心端血流形成了振荡性涡流区,血流速度极低,超声的分辨率未能检测到具体波形,剪切应力趋向于O。提示成功制备低剪切应力模型,并在同一段血管上形成了不同形态的低剪切应力血流区。2、腹主动脉病理形态学观察与分析结果显示：对照组血管内皮光整,长梭形平滑肌细胞排列均匀；狭窄组动脉管壁不均匀增厚,内中膜增厚凸向管腔,可见脱落细胞核,平滑肌细胞增生明显,细胞核缩短呈层多形状,且随狭窄时间的增加内皮与平滑肌细胞核形态与分布逐渐紊乱,炎性细胞浸润逐渐增多。与对照组比较,短时间组近心端及远心端内-中膜均显著增厚,且以近心端为甚；长时间组狭窄1d已有动脉壁增厚、内径减少、壁厚内径比增大,随着时间的延长,狭窄7、14、28d组动脉内径逐渐减小,动脉壁增厚与壁厚内径比进一步增大。提示不同形态的低剪切应力导致的病变程度是不同的,所引起的血管生物学效应与作用时间和强度有关。3、内皮P-选择素与VCAM-1的表达结果显示：对照组血管内皮无明显P-选择素与VCAM-1表达；短时间组近心端及远心端内皮均有明显P-选择素表达,而且近心端内皮染色更为显著；长时间组近心端内皮P-选择素的表达在1d开始明显上调并持续至28d,但随作用时间的延长在7d达高峰后呈下降趋势,而内皮VCAM-1表达在7d才开始显著上调,14d达高峰并持续保持高水平表达。提示各种黏附分子对血流剪切应力的敏感性可能不同。结论1、血流低剪切力较短时间内即可引起动脉重建和上调内皮P-选择素表达；2、不同形态低剪切力导致的动脉病变不同,近似层流较振荡流引起的动脉病理改变更为显著；3、低剪切力通过引起动脉重建和上调内皮P-选择素、VCAM-1等黏附分子的表达在AS的发生机制中起重要作用。更多还原

【Abstract】 Background and objectiveAtherosclerotic disease is one of the major hazards to human health. The pathogenic mechanism of atherosclerosis has been the focus of cardiovascular research field. Atherosclerotic plaques often appear in the low velocity blood flow and recirculation zone. Theory of lipid infiltration, thrombosis theory, doctrine and injury response of monoclonal theory can not well explain localization of atherosclerosis. Hemodynamic factors have a major role in incidence of atherosclerosis, and hemodynamic theory provides a new perspective for recognizing the the causes and pathogenesis of focal atherosclerosis. The time-related blood flow shear stress, especially low shear stress, plays a key role in the occurrence and development of atherosclerosis by causing vascular remodeling, upregulation the expression of endothelial adhesion molecules and lead to endothelial inflammation.Normal blood flow shear stress has an important regulatory role for the maintenance of vascular homeostasis, including anti-thrombosis, its barrier function and vascular homeostasis, etc. Changes in blood flow will lead to the occurrence of revascularization due to the destruction of vascular homeostasis. Vascular reconstruction is occurring changes in vascular structure and functions to meet internal or external environment, especially the mechanical environment changes, including adaptive rearrangement in early stage and pathological reconstruction in advanced stage which is the shared pathological basis of cardiovascular and cerebrovascular diseases. However, vascular remodeling mechanism is not clear in the field of cellular and molecular biology. Hemodynamic theory hold that, uniform laminar shear stress can induced endothelial anti-atherogenic gene expression selectively to offset the harmful effects of systemic risk factors, yet the abnormal shear stress will reduce the atherosclerosis resistant molecules and promote the expression of inflammatory cytokines of endothelium. Then accelerate the development of artery inflammation through activating the appropriate signal transduction pathways.Leukocyte is hard to adhesion to endothelium under normal circumstances, only when the interaction between external factors and endothelial cell increased endothelial adhesion molecules expression could help leukocyte adhesion to endothelium and transfer across the wall, which is the first step in the formation mechanism of atherosclerosis. In which P-selectin plays a decisive role in the initial adhesion and maintain the inflammatory state of early arterial lesions. Vascular cell adhesion molecule-1(VCAM-1) mainly play a role in the subsequent close adhesion of leukocytes and endothelial cells, and mediated leukocyte exudative and migration process, is also critical to maintain the endothelial inflammation by promote endothelial damage. To detect hemodynamic indicator early and study the vascular biological effects induced by shear stress deep will be great theoretical and practical significance for further revealing the nature of atherosclerosis and exploring the prevention and treatment methods.In the present study, we will create an animal model for local change of shear stress to analyze the arterial wall response at different patterns of fluid shear stress and evaluate the dynamic changes of vascular structure and adhesion molecules in endothelial cell under low shear stress. Methods1. Experimental animals and groups.20 Kunming mice were randomly divided into four groups:three short time stenosis groups (n=15) and sham operated group (n=5).25 Kunming mice were randomly divided into five groups:four long time stenosis groups (n=20) and sham operated group (n=5).2. Prepartion of mice model of abdominal aortic stenosis.we used a cast to induce standardized changes in shear stress. The cast imposes a fixed geometry on the vessel wall and thereby causes a stenosis, resulting a decrease in blood flow and consequently a lowered shear stress region upstream from the cast, and a vortex downstream from the cast (oscillatory blood flow). The upstream reduction in blood flow is caused by a device-induced. The downstream vortex is generated by a boundary separation immediately downstream from the cast, which is induced by a combination of velocity acceleration at the beginning of the cast, inertia of the blood, and the angle of the streamline at the end of the cast. For surgery, the test group mice were anesthetized with isoflurane, and the cast were placed around the abdominal aortic and fixed with a suture. The control group underwent sham operation without a cast. Artery samples were taken at 1 hour to 28 days after surgery and Determination of hemodynamic parameter, and reserved in -80℃.3. Determination of hemodynamic parameter.The shear stress values have been derived from measurements of vessel diameter and the velocity of blood flow which detected by Color Doppler Ultrasound, and capillary viscometer measured the viscosity of blood in 4 hours. The wall shear stress was calculated by Poiseiulle hydrodynamics formula (τm=η×4×Vm/D).4. Hematoxylin and eosin staining and morphological analysis of arterial.Pathological examination were performed to observe the arterial morphological changes. Using routine H&E staining of frozen sections. Sections were stained with hematoxylin and eosin. The intimal-media thickness and adventitia thickness of the aorta were measured. Morphology’s analysis was made on the HE’s photomicrograph by AutoCad picture analysis system. 5. Immunohistochemical staining and quantitative analysis.The immunohistochemical staining were performed for P-seletin and VCAM-1. All groups used two-step test:under the same conditions. The distribution of the positive cells was observed under the optical microscope, and were analyzed by Image-pro plus auto picture analysis system. One-way ANOVA was performed by using of SPSS 13.0 statistical package, data were expressed as mean±standard error.ResultsRegions of low shear stress and oscillatory shear stress were created upstream and downstream of the cast respectively. Vascular remodeling and P-selectin expression in the upstream arterial wall were much more severe than those in the downstream in all observed time-points (P<0.05). As compared with the control, with the increase of observed time, the changes of both wall thickness and the ratio of wall thickness to inner diameter were gradually greater at the area of low shear stress (P<0.05). The up-regulated expression of endothelial P-selectin was seen from 1 to 28 days at the area of low shear stress, the peak was at 7 days and above that the up-regulation attenuated (P<0.05). While the up-regulation of VCAM-1 was noted until 7 days (P<0.05), above 14 days the plateau was reached.Conclusions1. Vascular remodeling and endothelial P-selectin expression could happened in a relative short time when it exposured under low shear stress.2. Variant patterns of fluid shear stress may play different roles in the pathological process of artery.3. Low shear stress may play significantly initial roles in the pathological process of atherosclerosis resulting from endothelial inflammation更多还原