本文關(guān)鍵詞：豚鼠動脈粥樣硬化模型的建立及機理探討　出處：《中國協(xié)和醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 動脈粥樣硬化 豚鼠 CETP LCAT PPARα LDL-R SR-BI CD36 Ox-LDL ABCA1

【摘要】： 背景 動脈粥樣硬化及其并發(fā)癥嚴重危害人類健康。動脈粥樣硬化的發(fā)病機制復(fù)雜,是多種因素長期共同作用的結(jié)果。探明動脈粥樣硬化發(fā)病機理、篩選研究有效治療藥物是國內(nèi)外研究的重點和難點,而建立一種與人類動脈粥樣硬化發(fā)病機制、臨床表現(xiàn)相一致或接近的整體動物模型則是進行上述研究的關(guān)鍵。近年來,國內(nèi)外學(xué)者使用鳥類、鼠類、兔、豬、非人靈長類動物和轉(zhuǎn)基因動物等用于實驗性動脈粥樣硬化的研究,在取得諸多進展的同時,也逐步發(fā)現(xiàn)模型的缺陷和不足,限制了其應(yīng)用。研究資料提示,豚鼠是極少數(shù)通過LDL攜帶和轉(zhuǎn)運大部分膽固醇的動物之一,這點與人類極為相似。其次,豚鼠血漿脂質(zhì)構(gòu)成、膽固醇和脂蛋白代謝方式上也與人類存在諸多相似之處,對藥物治療的反應(yīng)性與臨床研究結(jié)果一致。提示將豚鼠作為由脂代謝紊亂造成的動脈粥樣硬化的動物模型,可能具有一定的優(yōu)勢。 目的 探索建立豚鼠動脈粥樣硬化模型的方法,使模型具有典型的動脈粥樣硬化病變特征。從整體、細胞和分子水平探討豚鼠動脈粥樣硬化的發(fā)病機制,并與另一種嚙齒類動物大鼠模型進行平行比較,闡明豚鼠作為一種新型動脈粥樣硬化動物模型的特點及優(yōu)勢所在。 方法 將豚鼠和大鼠分別隨機分為正常、模型組1組和模型2組。正常對照組飼常規(guī)飼料,模型1組飼含0.5%膽固醇、10%豬油的高脂飼料,模型2組飼含1%膽固醇、10%豬油的高脂飼料,連續(xù)10周的方法建立動脈粥樣硬化模型。觀測動物體重、攝食量和狀態(tài)變化。誘導(dǎo)結(jié)束后首先檢測血清脂質(zhì)和肝臟脂質(zhì)的變化；酶聯(lián)免疫吸附法測定血清CETP、LCAT、hs-CRP和Ox-LDL濃度；取主動脈弓至胸主動脈下端進行蘇Ⅳ染色,觀察主動脈壁內(nèi)脂質(zhì)沉積、動脈粥樣硬化病灶面積百分比；取主動脈根部至主動脈弓部的動脈進行石蠟切片,Masson染色法觀察動脈內(nèi)膜膠原纖維的變化,HE染色法分析內(nèi)膜面積／中膜面積、內(nèi)膜炎性細胞浸潤和內(nèi)膜表層斑塊的形成情況；免疫組化檢測血管內(nèi)膜ABCA1、CD36、ICAM-1蛋白表達的變化；實時熒光定量法檢測肝臟PPARa、LCAT、LDL-R、SR-BI mRNA相對表達的變化,對豚鼠SR-BI基因進行克隆測序。 結(jié)果 1.血脂和肝脂的變化 高脂飼料誘導(dǎo)10周后,與正常組相比,豚鼠兩個模型組血漿TC、TG、LDL-C水平均顯著升高,形成了典型的高脂血癥。而大鼠只有模型1組LDL-C和模型2組TC、LDL-C出現(xiàn)顯著升高,且升高幅度明顯低于相同條件誘導(dǎo)的豚鼠。豚鼠和大鼠各模型組肝臟TC和TG均顯著性升高,且升高幅度近似。 豚鼠模型1組和模型2組的血清HDL-C水平顯著升高了9.9和10.8倍,HDL的兩個主要亞型HDL3/ HDL2比值也都顯著升高,HDL組成和分布發(fā)生變化,小顆粒的HDL3堆積。大鼠兩個模型組的血清HDL-C水平和HDL3/ HDL2比值與正常組相比都未發(fā)生變化。 2.動脈粥樣硬化病變 正常豚鼠和大鼠主動脈壁光滑、平坦,內(nèi)皮細胞完整、連接緊密、緊貼于平直的內(nèi)彈力板上,平滑肌細胞呈長梭形、排列整齊,無炎癥細胞聚集,無斑塊形成。豚鼠經(jīng)高脂飼料誘導(dǎo)10周后,主動脈壁大體染色出現(xiàn)斑塊(脂質(zhì)斑塊)；高倍鏡下觀察(×400),內(nèi)膜明顯增厚(表現(xiàn)為大量單核／巨噬細胞來源的泡沫細胞堆積于內(nèi)膜表層)；內(nèi)膜單核細胞、巨噬細胞等炎性細胞浸潤與聚集增加,大量的泡沫細胞聚集形成斑塊；泡沫細胞內(nèi)富含脂質(zhì),細胞外形成脂滴,破壞內(nèi)膜平滑肌細胞之間的正常連接,使平滑肌細胞被這些脂質(zhì)包繞分割。少數(shù)病變嚴重的豚鼠主動脈出現(xiàn)細胞外脂質(zhì)融合和纖維帽,形成纖維斑塊。但病灶內(nèi)未發(fā)現(xiàn)明顯組織壞死、鈣化、斑塊破裂出血和血栓形成。按AHA的動脈粥樣硬化分期標(biāo)準,豚鼠模型組形成了Ⅱ-Va型動脈粥樣硬化病變,介于動脈粥樣硬化的早期和晚期之間。對豚鼠動脈粥樣硬化病理變化進行半定量分析,模型1和模型2組斑塊的發(fā)生率分別為70%(7／10)和75%(6／8),主動脈斑塊面積%、纖維面積%、內(nèi)膜面積%,內(nèi)膜面積／中膜面積比值較正常對照組都明顯升高。而相同條件誘導(dǎo)下大鼠動脈管壁未形成明顯的動脈粥樣硬化病變,半定量分析也無顯著變化。 3.分子機制研究 與正常組相比,豚鼠模型組肝臟LDL-R mRNA表達下調(diào),血漿Ox-LDL水平顯著升高,說明豚鼠正常的LDL-C受體代謝途徑受阻,大量LDL被氧化修飾成Ox-LDL。同時豚鼠模型組主動脈CD36蛋白表達上調(diào),說明大量的Ox-LDL經(jīng)巨噬細胞膜CD36受體攝入細胞內(nèi),形成泡沫細胞。豚鼠模型組血漿CETP水平顯著升高,一方面促進小而致密的LDL形成并沉積于動脈,另一方面促進小顆粒HDL3水平相對升高。此外,豚鼠模型組血漿LCAT水平和肝臟LCAT mRNA表達都顯著下降,表明LCAT催化HDL3向HDL2的酯化轉(zhuǎn)變減少,HDL成熟過程受阻,也是HDL3/HDL2比值升高的原因之一。豚鼠攝入高脂飲食后,主動脈粘附分子ICAM-1蛋白表達增加,促進了炎性細胞的粘附與聚集；血漿hs-CRP水平升高,提示豚鼠出現(xiàn)血管炎癥反應(yīng),促進了粥樣硬化的發(fā)生發(fā)展。而相同高脂飲食誘導(dǎo)的大鼠只有模型2組血漿Ox-LDL、hs-CRP水平和主動脈CD36蛋白表達升高,且變化幅度不如豚鼠明顯。 此外,高脂飲食的攝入導(dǎo)致豚鼠兩個模型組肝臟SR-BI mRNA和主動脈ABCA1蛋白表達代償性升高,而大鼠未出現(xiàn)此變化。 結(jié)論 1.采用0.5%膽固醇和10%豬油的高脂膳食誘導(dǎo)豚鼠10周可建立典型的動脈粥樣硬化模型。 2.與大鼠相比較,豚鼠對飲食性膽固醇更敏感,通過高脂飲食誘導(dǎo)更易于形成動脈粥樣硬化病變。 3.豚鼠容易形成動脈粥樣硬化病變的原因和機制與血漿脂蛋白的代謝、動脈粥樣硬化形成過程中關(guān)鍵酶、蛋白、細胞因子和受體(包括CETP、LCAT、CD36、LDL-R、Ox-LDL、ICAM-1等)的變化有關(guān)。 4.此法建立的豚鼠動脈粥樣硬化模型具有造模周期較短、指標(biāo)穩(wěn)定、操作簡便、具有典型動脈粥樣硬化病變特征的特點,為脂代謝紊亂及動脈粥樣硬化治療藥物篩選和評價提供了一種較為合適的動物模型。
[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.

【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R-332;R543

【參考文獻】

相關(guān)期刊論文 前10條

1 吳新偉,傅明德,劉秉文;Ⅳ型高脂血癥患者血清HDL亞類組成的初步研究[J];華西醫(yī)科大學(xué)學(xué)報;1999年03期

2 徐燕華,傅明德,徐勇霞,劉秉文;肥胖者血清高密度脂蛋白亞類組成的研究[J];華西醫(yī)科大學(xué)學(xué)報;2001年04期

3 徐勇霞,傅明德,徐燕華,于倩;冠心病患者血清HDL亞類組成的研究[J];華西醫(yī)科大學(xué)學(xué)報;2002年03期

4 勾藍圖,傅明德,徐燕華,顏丙玉,楊雨葉,劉宇;血清脂蛋白膽固醇水平與HDL亞類組成的關(guān)系[J];四川大學(xué)學(xué)報(醫(yī)學(xué)版);2005年01期

5 馬建e，

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