【作者】 陈雯艾；

【导师】 王建安；

【作者基本信息】 浙江大学 ， 内科学， 2012， 博士

【摘要】 实验背景：随着社会经济的发展和人民生活水平的提高,近年来,动脉疾病的发病率、致残率、致死率明显上升。动脉疾病已经成为危险人类健康的定时炸弹,其中动脉瘤和动脉粥样硬化均是慢性炎症性动脉疾病,随着超声、CTA、MRA的普及,动脉瘤与动脉粥样硬化得以及时发现。大的动脉瘤可以行开放性外科手术和微创腔内隔绝术,小的动脉瘤患者只能消极等待与检查,目前尚无任何有效的药物可逆转或者是预防动脉瘤的发生与发展。虽然动脉粥样硬化从基础理论到临床特征,已经被研究多年,但是目前动脉粥样硬化的发病速度大于治疗速度,它仍然是危险人类健康的主力军。因此,继续寻找有效药物,预防、逆转与治疗动脉瘤与动脉粥样硬化,已经是医学研究者迫在眉睫的工作。众所周知,口服3-羟-3-甲戊二酰辅酶A还原酶抑制剂(他汀)对冠心病的病人是一种非常有效的治疗方法。随着他汀的治疗,心血管病的发病率与死亡率下降归功于血浆胆固醇浓度的降低和其他非脂机制,例如内皮功能改善和抗炎效果。罗苏伐他汀和阿托伐他汀是两种最广泛的他汀,用以大大地降低血浆总胆固醇浓度和低密度脂蛋白胆固醇浓度。在动脉粥样硬化斑块中,他汀能通过抑制NF-κB信号,激活可诱导的NO合酶的表达来减轻炎症趋化因子的表达,减少MMP-9容量,增加胶原和纤维帽的厚度。基于他汀类药物的多效性,尤其是抗炎的作用,他汀类药物有望在抗动脉粥样硬化的基础上成为一种抑制、逆转动脉瘤有效的药物。他汀是否能阻止人类腹主动脉瘤的扩张和破裂尚有争议。有研究提示：在弹性酶诱导的大鼠腹主动脉瘤模型上,阿托伐他汀通过抑制巨噬细胞的聚集来抑制腹主动脉瘤的发展,但是还尚没有瑞苏伐他汀抑制腹主动脉瘤的确切疗效报导。实验目的：研究他汀类药物(瑞苏伐他汀和阿托伐他汀)对血管紧张素Ⅱ诱导的apoE基因敲除雄性小鼠的腹主动脉瘤与主动脉粥样硬化的影响,并对其相关的作用机制进行探讨。实验方法：我们选用了血管紧张素Ⅱ诱导apoE基因敲除雄性小鼠而获得的腹主动脉瘤与主动脉粥样硬化的实验动物模型。60只apoE基因敲除雄性小鼠,随机分成三组,每组20只。安慰剂组：给予不含药物的饮用水；瑞舒伐他汀组：给予含瑞舒伐他汀10mg/kg/d的饮用水；阿托伐他汀组：给予含阿托伐他汀20mg/kg/d的饮用水。1周后,60只apoE基因敲除雄性小鼠均皮下植入含有血管紧张素Ⅱ1000ng/kg/min的ALZET(?)渗透泵诱导4周而成。我们每周测量小鼠体重。应用Kent无创血压监测系统监测小鼠血压变化。应用小动物活体超声成像系统探测肾上主动脉的最大内直径和最大横断面积,以监测不同组别小鼠的肾上主动脉内直径和横断面积大小的变化；按照Daugherty分型法,根据肾上主动脉形状对腹主动脉瘤进行分型,观察各组腹主动脉瘤不同型的构成情况。为了明确apoE基因敲除雄性小鼠体内存在有效血药浓度,我们采用了高效液相色谱-质谱联用法相继测定了瑞苏伐他汀和阿托伐他汀的血清浓度。应用了Wako LabAssayTM Clolesterol试剂盒测定了三组的血清总胆固醇浓度；应用小鼠单核细胞趋化蛋白-1ELISA试剂盒测定了三组的血清单核细胞趋化蛋白-1浓度；应用小鼠巨噬细胞移动抑制因子ELISA试剂盒测定了三组的血清巨噬细胞移动抑制因子浓度；应用En Face技术分析三组主动脉弓粥样硬化面积的绝对值与占内膜面积的百分比。应用图像分析软件Image Pro5.0分析三组主动脉根部每10um与每80um的粥样硬化斑块面积,比较瑞苏伐他汀与阿托伐他汀对血管紧张素Ⅱ诱导的apoE基因敲除雄性小鼠的动脉粥样硬化斑块的影响。应用MicroProbe手动染色系统对主动脉根部的粥样硬化斑块进行免疫组织化学染色的方法,以人工免疫染色分级的方法显示其中的血管平滑肌细胞、巨噬细胞、B淋巴细胞、T淋巴细胞的变化,从而阐述瑞苏伐他汀与阿托伐他汀对粥样硬化斑块内炎性细胞组分的不同影响。应用原位杂交检测了主动脉根部粥样硬化斑块内的PPAR-α mRNA、PPAR-γ mRNA、MCP-1mRNA、TNF-α mRNA、 NF-KappaB p50mRNA、NF-KappaB p65mRNA,从分子水平探讨瑞苏伐他汀与阿托伐他汀对抗炎因子与促炎因子的影响,从而说明动脉粥样硬化斑块内炎症状态的改变。实验结果：血管紧张素Ⅱ诱导的apoE基因敲除雄性小鼠的腹主动脉瘤与动脉粥样硬化的动物模型是一个成功的模型。在本次研究中,三组小鼠的体重、血压在同时段比较无显著性差异。比较植入含有血管紧张素Ⅱ的ALZET(?)渗透泵前后,血压有明显升高,说明血管紧张素Ⅱ使小鼠的血压升高,这可能对apoE基因敲除雄性小鼠的动脉瘤与动脉粥样硬化的形成有协助作用,但是该血压不受瑞苏伐他汀和阿托伐他汀的影响。通过用高效液相色谱-质谱联用法测定瑞苏伐他汀和阿托伐他汀的血清浓度,我们得出瑞苏伐他汀组与阿托伐他汀组均有相应的血药浓度。通过腹主动脉瘤的发生率与破裂的情况,用高频超声成像系统测定肾上主动脉的最大内直径与最大横断面积,分析腹主动脉瘤的分型构成比。我们认为瑞苏伐他汀与阿托伐他汀均未能改变血管紧张素Ⅱ所诱导的腹主动脉瘤的进展。通过测量三组的主动脉弓与主动脉根部的动脉粥样硬化斑块面积,在血管紧张素Ⅱ诱导的apoE基因敲除的主动脉粥样硬化斑块的模型上,阿托伐他汀有抗动脉粥样硬化的效果,却未见瑞苏伐他汀抗动脉粥样硬化的效果。我们从细胞与分子水平来探讨了阿托伐他汀在本模型中抗动脉粥样硬化斑块的机制。通过对主动脉根部的粥样硬化斑块内细胞组分进行免疫组化染色,我们发现：阿托伐他汀改变了粥样硬化斑块内的细胞组分,增加了中膜的血管平滑肌细胞的含量,减少了巨噬细胞与T淋巴细胞的聚集。通过原位杂交方法检测动脉粥样硬化斑块内促炎因子的转录因子NF-κB p50mRNA、NF-κB p65mRNA、MCP-1mRNA、TNF-α mRNA,及抗炎因子的转录因子PPAR-αmRNA和PPAR-γmRNA,阿托伐他汀显著下调了促炎因子的转录因子NF-B p50mRNA、NF-κB p65mRNA、MCP-1mRNA、TNF-α mRNA的丰度,上调了抗炎因子的转录因子PPAR-αmRNA和PPAR-γ mRNA的丰度。但是瑞苏伐他汀与阿托伐他汀对循环中的炎症因子MCP-1与MIF均无影响,对循环中的血清总胆固醇浓度亦无影响,这说明在本研究中阿托伐他汀的抗动脉粥样硬化斑块的效果是直接影响粥样硬化斑块内炎症状态而发挥作用的,与循环中的炎症状态无关,独立于调脂作用。实验结论：1.瑞苏伐他汀与阿托伐他汀均未能阻止与逆转血管紧张素Ⅱ诱导apoE基因敲除雄性小鼠的腹主动脉瘤的发生与进展。2.阿托伐他汀对血管紧张素Ⅱ诱导的apoE基因敲除雄性小鼠有明确的抗动脉粥样硬化斑块的效果,而瑞苏伐他汀的效果却不明显。3.阿托伐他汀抗动脉粥样硬化斑块的效果与增加了血管中膜的平滑肌细胞的含量,减少了斑块内巨噬细胞与T淋巴细胞的聚集有关。4.阿托伐他汀抗动脉粥样硬化斑块的效果与斑块内的抗炎因子上调、促炎因子下调有关,与循环中的炎症因子无关,且独立于调脂作用。5.血管紧张素Ⅱ诱导的apoE基因敲除雄性小鼠的腹主动脉瘤与动脉粥样硬化斑块的实验动物模型是一个稳定的模型,可进一步推广使用。更多还原

【Abstract】 Background:With the development of the social economy and the improvement of the people’s living standard, morbidity and mortality of arterial diseases have increased significantly in recent years. Arterial diseases are the time bomb which have been harmful to human health. Aortic aneurysms and atherosclerosis are all chronic inflammatory artery diseases. The popularity of ultrasound, CTA and MRA contributes to detect aneurysms and atherosclerosis. The patients with large aneurysms can be made open surgical operations or be performed minimally invasive endovascular aortic repair, but the patients with small aneurysms wait passively for repeating examination. There are no effective medicines to prevent or reverse the occurrence and development of aneurysms. Atherosclerosis has been studied for many years from basic theory to clinical feature. Although we have made great progress in antiatherosclerotic theory. Atherosclerotic disease is faster than the treatment speed. It is still a danger to human health. Therefore, searching for effective medicines to prevent and to reverse aneurysms and atherosclerosis is an imminent medical work for researchers.As is well known, administration of3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) inhibitor (statin) is a highly validated therapeutic approach for patients with coronary heart disease. Reductions of cardiovascular morbidity and mortality with statin therapy have been attributed to lowering of plasma cholesterol concentrations and other non-lipid-based mechanisms, such as improved endothelial function and anti-inflammatory effects. Rosuvastatin and atorvastatin are two of the most widely prescribed statins that profoundly reduce plasma concentrations of total cholesterol and low-density lipoprotein cholesterol (LDL-C). Statins reduced inflammatory chemokines expression by inhibition of NFκB signaling and activation of inducible NO synthase expression, decreased MMP-9content, and increased collagen and fibrous cap thickness in atherosclerotic lesions.Based on its pleiotropic effects, especially the anti-inflammatory effect, statins are expected to become an effective medicine to reverse aneurysm on the basis of anti-atherosclerosis. Now it is controversial whether satins can prevent the expansion and rupture of human abdominal aortic aneurysms (AAAs). Atorvastatin had been demonstrated to suppress the development of AAAs in elastase-induced rats through inhibition of macrophage migration. No studies have defined the effects of rosuvastatin on AAAs.Objectives:The objective of this study is to evaluate statins (rosuvastatin and atorvastatin) on angiotensin Ⅱ-induced abdominal aortic aneurysms and atherosclerosis in apoE knockout male mice, and to explore their mechanisms.Methods:We selected angiotensin Ⅱ-induced abdominal aortic aneurysms and atherosclerosis in apoE knockout male mice as the experimental animal models. Sixty apoE konckout male mice fed a normal diet were randomly divided into three groups: control group, rosuvastatin group and atorvastatin group. The mice in the rosuvastatin group were administered drinking water with rosuvastatin1Omg/kg/day, while the mice in the atorvastatin group with atorvastatin20mg/kg/day for1week prior to initiating28-day angll infusion (1000ng/kg/min). We measured body weight in mice weekly. We used a computerized tail cuff to measure the blood pressure of the mice. We measured maximum suprarenal diameters and maximum cross-sectional areas using a high frequency ultrasound imaging system to monitor suprarenal change in each goup. Aneurysm severity was scored as professor Alan Daugherty. We analysed the constituent conditions of AAAs types. In order to identify the effective serum therapeutic medicine concentrations in apoE knockout male mice, we used liquid chromatography with electrospray ionization tandem mass spectrometry to detect rosuvastatin and atorvastatin concentrations. Serum total cholesterol concentrations were determined using enzymatic assay kits. Serum concentrations of monocyte chemoattractant protein-1and macrophage migration inhibitory factor were measured with ELISA kits according to manufacturer’s recommendation. Atherosclerosis was assessed on the intimas of aortic arches by en face technique and on cross-sections of aortic roots. Quantitative analysis of aortic root atherosclerotic cross-sections (10μm and80um thick) was performed using Image-Pro software. We compared the effects of rosuvastatin and atorvastatin on angiotensin II-induced atherosclerotic plaques in apoE knockout male mice. Immunostaining of aortic root atherosclerosis plaque was performed with a commercially available system (Fisher Microprobe). Cellular components consisting of atherosclerotic lesions were graded. We identified the change of vascular smooth muscle cell、macrophages、B-lymphocytes、T-lymphocytes in lesions. We used in situ hybridization to detect PPAR-amRNA、PPAR-ymRNA、MCP-1mRNA、TNF-αmRNA、NF-κB p50mRNA、NF-κB p65mRNA in aortic root atherosclerotic lesions. From the molecular levels, we explored the effects of rosuvastatin and atorvastatin on inflammatory factors and anti-inflammatory factors. Thus we confirmed inflammatory changes in the atherosclerotic lesions.Results:The experimental animal model of angiotensin-induced abdominal aortic aneurysms and atherosclerosis in apoE knockout male mice is a successful model. In the study, body weight and blood pressure had no significant difference at the same time among the three groups. Blood pressure had increased after ALZET(?) osmotic pumps containing angiotensin Ⅱ implanted. That would be benefit to aortic aneurysms and atherosclerosis in apoE knockout male mice. But the blood pressures were not affected by rosuvastatin and atorvastatin. Serum concentrations of rosuvastatin and atorvastatin were measured by liquid chromatography with electrospray ionization tandem mass spectrometry. We found that statins (rosuvastatin and atorvastatin) administration led to therapeutic serum drugs concentrations. We calculated the occurrence rate of abdominal aortic aneurysms. We measured maximum suprarenal diameter and cross-sectional area using a high frequency ultrasound imaging system. We analysed the constituent ratio of abdominal aortic aneurysm types. Finally we made a decision that neither rosuvastatin nor atorvastatin altered development of angiotensin Ⅱ-induced AAAs. We measured atherosclerotic plaque areas induced by angiotensin Ⅱ of aortic arch and aortic root in the three groups. We found that atorvastatin had anti-atherogenic effects, but rosuvastatin had no effects.We explored the mechanisms of atorvastatin suppressing atherosclerotic plaque from the cell and molecular levels in the animal model. Atorvastatin changed cellular component in atherosclerotic plaque. It increased the content of vascular smooth muscle cells in the medial membrane and significantly decreased accumulation of macrophages and T lymphocytes in the lesions. We detected NF-κB p50mRNA、NF-κB p65mRNA、 MCP-1mRNA、TNF-amRNA as major inflammatory factors and PPAR-amRNA and PPAR-ymRNA as major anti-inflammatory factors in lesions using in situ hybridization. As a result, Atorvastatin administration decreased NF-κB p50mRNA, NF-κB p65mRNA, MCP-1mRNA and TNF-a mRNA abundances and increased PPAR-a mRNA and PPAR-y mRNA abundances in atherosclerotic lesions. But serum inflammatory markers MCP-1and MIF concentrations were not altered by rosuvastatin and atorvastatin, and serum total cholesterol were not changed too.Atherosclerosis attenuated by atovastatin was independent of reductions in serum total cholesterol concentrations. Atherosclerosis was not related to circulating inflammatory state, while to related to inflammatory state in atherosclerotic plaque.Conclusions:1. Administration of either rosuvastatin or atorvastatin all failed to suppress angⅡ-induced abdominal aortic aneurysms in apoE knockout mice.2. Atorvastatin significantly reduced angⅡ-induced atherosclerotic lesion areas in apoE knockout mice, but rosuvastatin had no effects.3. Effect of atorvastatin on atherosclerotic plaque was related to the increase of vascular smooth muscle cells and significant decrease of macrophages and T lymphocytes in lesions.4. Effect of atorvastatin on atherosclerotic plaque was related to upregulation of anti-inflammatory factors and downregulation of inflammatory factors in lesions, while it was independent of circulating inflammatory factors and serum total cholesterol.5. The experimental animal model of angiotensin Ⅱ-induced abdominal aortic aneurysms and atherosclerosis in apoE knockout mice is a stable model. It can be used widely in the future.更多还原