豚鼠动脉粥样硬化模型的建立及机理探讨

发布时间：2018-01-14 17:18

本文关键词：豚鼠动脉粥样硬化模型的建立及机理探讨　出处：《中国协和医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 背景动脉粥样硬化及其并发症严重危害人类健康。动脉粥样硬化的发病机制复杂,是多种因素长期共同作用的结果。探明动脉粥样硬化发病机理、筛选研究有效治疗药物是国内外研究的重点和难点,而建立一种与人类动脉粥样硬化发病机制、临床表现相一致或接近的整体动物模型则是进行上述研究的关键。近年来,国内外学者使用鸟类、鼠类、兔、猪、非人灵长类动物和转基因动物等用于实验性动脉粥样硬化的研究,在取得诸多进展的同时,也逐步发现模型的缺陷和不足,限制了其应用。研究资料提示,豚鼠是极少数通过LDL携带和转运大部分胆固醇的动物之一,这点与人类极为相似。其次,豚鼠血浆脂质构成、胆固醇和脂蛋白代谢方式上也与人类存在诸多相似之处,对药物治疗的反应性与临床研究结果一致。提示将豚鼠作为由脂代谢紊乱造成的动脉粥样硬化的动物模型,可能具有一定的优势。目的探索建立豚鼠动脉粥样硬化模型的方法,使模型具有典型的动脉粥样硬化病变特征。从整体、细胞和分子水平探讨豚鼠动脉粥样硬化的发病机制,并与另一种啮齿类动物大鼠模型进行平行比较,阐明豚鼠作为一种新型动脉粥样硬化动物模型的特点及优势所在。方法将豚鼠和大鼠分别随机分为正常、模型组1组和模型2组。正常对照组饲常规饲料,模型1组饲含0.5%胆固醇、10%猪油的高脂饲料,模型2组饲含1%胆固醇、10%猪油的高脂饲料,连续10周的方法建立动脉粥样硬化模型。观测动物体重、摄食量和状态变化。诱导结束后首先检测血清脂质和肝脏脂质的变化；酶联免疫吸附法测定血清CETP、LCAT、hs-CRP和Ox-LDL浓度；取主动脉弓至胸主动脉下端进行苏Ⅳ染色,观察主动脉壁内脂质沉积、动脉粥样硬化病灶面积百分比；取主动脉根部至主动脉弓部的动脉进行石蜡切片,Masson染色法观察动脉内膜胶原纤维的变化,HE染色法分析内膜面积／中膜面积、内膜炎性细胞浸润和内膜表层斑块的形成情况；免疫组化检测血管内膜ABCA1、CD36、ICAM-1蛋白表达的变化；实时荧光定量法检测肝脏PPARa、LCAT、LDL-R、SR-BI mRNA相对表达的变化,对豚鼠SR-BI基因进行克隆测序。结果 1.血脂和肝脂的变化高脂饲料诱导10周后,与正常组相比,豚鼠两个模型组血浆TC、TG、LDL-C水平均显著升高,形成了典型的高脂血症。而大鼠只有模型1组LDL-C和模型2组TC、LDL-C出现显著升高,且升高幅度明显低于相同条件诱导的豚鼠。豚鼠和大鼠各模型组肝脏TC和TG均显著性升高,且升高幅度近似。豚鼠模型1组和模型2组的血清HDL-C水平显著升高了9.9和10.8倍,HDL的两个主要亚型HDL3/ HDL2比值也都显著升高,HDL组成和分布发生变化,小颗粒的HDL3堆积。大鼠两个模型组的血清HDL-C水平和HDL3/ HDL2比值与正常组相比都未发生变化。 2.动脉粥样硬化病变正常豚鼠和大鼠主动脉壁光滑、平坦,内皮细胞完整、连接紧密、紧贴于平直的内弹力板上,平滑肌细胞呈长梭形、排列整齐,无炎症细胞聚集,无斑块形成。豚鼠经高脂饲料诱导10周后,主动脉壁大体染色出现斑块(脂质斑块)；高倍镜下观察(×400),内膜明显增厚(表现为大量单核／巨噬细胞来源的泡沫细胞堆积于内膜表层)；内膜单核细胞、巨噬细胞等炎性细胞浸润与聚集增加,大量的泡沫细胞聚集形成斑块；泡沫细胞内富含脂质,细胞外形成脂滴,破坏内膜平滑肌细胞之间的正常连接,使平滑肌细胞被这些脂质包绕分割。少数病变严重的豚鼠主动脉出现细胞外脂质融合和纤维帽,形成纤维斑块。但病灶内未发现明显组织坏死、钙化、斑块破裂出血和血栓形成。按AHA的动脉粥样硬化分期标准,豚鼠模型组形成了Ⅱ-Va型动脉粥样硬化病变,介于动脉粥样硬化的早期和晚期之间。对豚鼠动脉粥样硬化病理变化进行半定量分析,模型1和模型2组斑块的发生率分别为70%(7／10)和75%(6／8),主动脉斑块面积%、纤维面积%、内膜面积%,内膜面积／中膜面积比值较正常对照组都明显升高。而相同条件诱导下大鼠动脉管壁未形成明显的动脉粥样硬化病变,半定量分析也无显著变化。 3.分子机制研究与正常组相比,豚鼠模型组肝脏LDL-R mRNA表达下调,血浆Ox-LDL水平显著升高,说明豚鼠正常的LDL-C受体代谢途径受阻,大量LDL被氧化修饰成Ox-LDL。同时豚鼠模型组主动脉CD36蛋白表达上调,说明大量的Ox-LDL经巨噬细胞膜CD36受体摄入细胞内,形成泡沫细胞。豚鼠模型组血浆CETP水平显著升高,一方面促进小而致密的LDL形成并沉积于动脉,另一方面促进小颗粒HDL3水平相对升高。此外,豚鼠模型组血浆LCAT水平和肝脏LCAT mRNA表达都显著下降,表明LCAT催化HDL3向HDL2的酯化转变减少,HDL成熟过程受阻,也是HDL3/HDL2比值升高的原因之一。豚鼠摄入高脂饮食后,主动脉粘附分子ICAM-1蛋白表达增加,促进了炎性细胞的粘附与聚集；血浆hs-CRP水平升高,提示豚鼠出现血管炎症反应,促进了粥样硬化的发生发展。而相同高脂饮食诱导的大鼠只有模型2组血浆Ox-LDL、hs-CRP水平和主动脉CD36蛋白表达升高,且变化幅度不如豚鼠明显。此外,高脂饮食的摄入导致豚鼠两个模型组肝脏SR-BI mRNA和主动脉ABCA1蛋白表达代偿性升高,而大鼠未出现此变化。结论 1.采用0.5%胆固醇和10%猪油的高脂膳食诱导豚鼠10周可建立典型的动脉粥样硬化模型。 2.与大鼠相比较,豚鼠对饮食性胆固醇更敏感,通过高脂饮食诱导更易于形成动脉粥样硬化病变。 3.豚鼠容易形成动脉粥样硬化病变的原因和机制与血浆脂蛋白的代谢、动脉粥样硬化形成过程中关键酶、蛋白、细胞因子和受体(包括CETP、LCAT、CD36、LDL-R、Ox-LDL、ICAM-1等)的变化有关。 4.此法建立的豚鼠动脉粥样硬化模型具有造模周期较短、指标稳定、操作简便、具有典型动脉粥样硬化病变特征的特点,为脂代谢紊乱及动脉粥样硬化治疗药物筛选和评价提供了一种较为合适的动物模型。
[Abstract]:background
Atherosclerosis and its complications serious harm to human health. The pathogenesis of atherosclerosis is complex, the interaction of many factors. Results proved the pathogenesis of atherosclerosis, effects of drug therapy is the focus and difficulty of research at home and abroad, and the establishment of a human and animal model of atherosclerosis, the overall clinical manifestation is consistent with or close to the key of the research. In recent years, domestic and foreign scholars use birds, rodents, rabbits, pigs, for non-human primate animal and transgenic animal research laboratory in atherosclerosis, made a lot of progress at the same time, also gradually found model flaws and shortcomings, limiting its application. The research data suggest that guinea pigs are one of the few carried by LDL and most of the cholesterol transport of animal and human, which is extremely similar. Secondly, guinea pig The way of plasma lipid composition, cholesterol and lipoprotein metabolism also has many similarities with humans, response to therapy and clinical research results. Guinea pigs were used as animal model caused by the disorder of lipid metabolism in atherosclerosis, may have certain advantages.
objective
Explore the establishment of atherosclerosis in guinea pig model, the model has the characteristics of typical atherosclerotic lesions. On the whole, the cellular and molecular level to explore the pathogenesis of atherosclerosis in guinea pig, and the model with another rodent animal rat parallel comparison, clarify the guinea pig as a new animal model of atherosclerosis characteristics and advantages.
Method
The guinea pigs and rats were randomly divided into normal group, model group 1 and model group 2. Control group fed with normal forage, the model 1 group were fed with high fat diet containing 0.5% cholesterol, 10% lard, 2 model group were fed with high fat diet containing 1% cholesterol, 10% lard, for 10 consecutive weeks of establishment of artery atherosclerosis model. Observation of animal weight, food intake and status change. After the first detection of induced changes in lipid and liver lipid serum; serum CETP was measured by enzyme-linked immunosorbent assay, LCAT, hs-CRP and Ox-LDL concentration; at the lower end of the aortic arch to the thoracic aorta of Su IV staining, observe the lipid deposition in aortic atherosclerosis. The lesion area percentage; aorta root to the aortic arch artery for paraffin sections, observe the changes of intimal collagen Masson staining method, analysis of the intimal area / medial area HE staining, endometrial inflammatory cells The changes of ABCA1, CD36 and ICAM-1 protein expression in the intima were detected by immunohistochemistry, and the relative expression of PPARa, LCAT, LDL-R and SR-BI mRNA in liver was detected by real-time immunohistochemistry. The SR-BI gene of guinea pig was cloned and sequenced.
Result
1. changes in blood lipid and liver fat
High fat diet after 10 weeks of induction, compared with the normal group, the guinea pig two model group plasma TC, TG, LDL-C levels were significantly increased, forming a typical hyperlipidemia rats model. Only 1 groups of LDL-C and TC in model group 2, LDL-C increased significantly, and the increase was significantly lower than that induced by the same conditions the guinea pigs in guinea pig and rat liver TC and TG of the model group were significantly increased, and the increased range of approximation.
The guinea pig model group 1 and model 2 group serum HDL-C level significantly increased 9.9 and 10.8 times, the two main subtypes of HDL3/ HDL2 ratio of HDL also increased significantly, HDL composition and distribution changes, small particles of HDL3 accumulation. Two rats in model group serum HDL-C level and HDL3/ ratio compared to HDL2 and the normal group did not change.
2. atherosclerotic lesions
The normal guinea pig and rat aortic endothelial cell wall is smooth, flat, complete, tight, elastic plate close to the flat, smooth muscle cells were fusiform, arranged neatly, no accumulation of inflammatory cells, plaque formation in guinea pigs. The high-fat diet for 10 weeks, the aortic wall plaque (lipid staining appeared in general plaque); observed at high magnification (* 400), endometrial thickening (represented by a large number of monocyte / macrophage derived foam cell accumulation in the endometrial surface); endometrial monocytes, macrophages and other inflammatory cell infiltration and aggregation increased, a large number of foam cells and plaque formation; foam cells in lipid rich cells. The outer lipid droplet formation, damage between intimal smooth muscle cells in the normal connection, so that the smooth muscle cells by the lipid wrapping segmentation. Some lesions serious appearance of extracellular lipid in guinea pig aorta and fusion of fibrous cap, fiber forming Dimensional plaque. But the lesions in no obvious tissue necrosis, calcification, plaque rupture bleeding and thrombosis. According to AHA staging criteria of atherosclerosis model group, guinea pig form II -Va type of atherosclerotic lesions, between early and late stages of atherosclerosis. A semi quantitative analysis of pathological changes of atherosclerosis in guinea pig, model 1 and model group 2 plaque the incidence rate was 70% (7 / 10) and 75% (6 / 8), aortic plaque area%, fiber area%, intimal area%, neointimal area / media area ratio compared with the normal control group were significantly increased. The same conditions did not form obvious atherosclerotic lesions induced by rat arterial wall. Semi quantitative analysis also showed no significant changes.
Study on 3. molecular mechanism
Compared with the normal group, model group of guinea pig liver LDL-R mRNA expression, the level of plasma Ox-LDL increased significantly, that blocked the pathway of LDL-C receptor metabolism in normal guinea pig, a large amount of LDL was oxidized into Ox-LDL. and CD36 expression in aorta was increased, indicating the large number of Ox-LDL by macrophage membrane CD36 receptors into the cell, the formation of foam cells. The guinea pig model group plasma CETP levels were significantly increased, small and dense LDL formation and deposition in the artery to promote hand, small particles of HDL3 level is relatively increased. On the other hand, in addition, the guinea pig model group plasma LCAT levels and liver LCAT mRNA expression decreased significantly, showed that the LCAT catalyzed HDL3 to HDL2 transformation to reduce HDL esterification, mature the process is blocked, but also one of the reasons for the ratio of HDL3/HDL2 increased. The guinea pigs eat a high-fat diet, the expression of ICAM-1 protein increased in aortas, promote inflammatory Cell adhesion and aggregation; plasma levels of hs-CRP, suggesting that guinea vascular inflammation, promoting the development of atherosclerosis. And induced by the same diet rat model only 2 groups of plasma Ox-LDL, the expression level of hs-CRP and aortic CD36 protein increased, and the amplitude of variation as guinea pigs significantly.
In addition, the intake of high fat diet led to a compensatory increase in the expression of SR-BI mRNA and ABCA1 protein in the liver of the two model groups of guinea pigs, while the rats did not appear to be changed.
conclusion
1. a high fat diet of 0.5% cholesterol and 10% lard could be used to induce a typical atherosclerotic model in guinea pigs for 10 weeks.
2. compared with rats, guinea pigs were more sensitive to dietary cholesterol and were more likely to form atherosclerotic lesions through a high fat diet.
3., the causes and mechanisms of the formation of atherosclerotic lesions in guinea pigs are related to the metabolism of plasma lipoproteins, the changes of key enzymes, proteins, cytokines and receptors (including CETP, LCAT, CD36, LDL-R, Ox-LDL, ICAM-1, etc.) in the process of atherosclerosis.
4. the establishment of atherosclerosis in guinea pig model with short cycle modeling, stability index, simple operation, has the characteristics of typical features of atherosclerotic lesions, provide a suitable animal model for lipid metabolism and atherosclerosis drug screening and evaluation.

【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R-332;R543

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