动物模型,动脉粥样硬化 z-bo 2020-08-07 906

　　As early as 1908, Ignatowski and others recognized that a high-cholesterol diet may be related to the formation of experimental atherosclerosis. In 1913, Anichkov and Chalatov established the first rabbit model of atherosclerosis with a high-cholesterol diet (i.e., the bait method) and has been used today, becoming the most commonly used and traditional experimental model in atherosclerosis research. With the development of interventional medicine, immunology and molecular biology, researchers in recent years have gradually developed atherosclerosis models assisted by mechanical injury methods, atherosclerosis models established by immune methods, and natural mutations and genetic engineering. A mouse model of atherosclerosis.

　　One, prey-induced atherosclerosis animal model

　　prey-induced As animal model refers to an animal model induced by feeding a high-fat diet. Most copies of this model follow Ignatowski's method, adding cholesterol to the feed to induce pathology. Usually within a certain range, the higher the cholesterol content in the feed, the more obvious the increase in plasma cholesterol content, and the faster the occurrence of As lesions. However, the plasma cholesterol level is not positively correlated with the formation of As and the degree of disease. Excessive plasma cholesterol levels often cause animal poisoning and even death. It is generally believed that the plasma cholesterol level should be controlled at 15.6-20.8mmol/L (600-800mg/dl).

　　(1) Rabbit Atherosclerosis Model

　　1. Animals are usually 3 to 4 months old New Zealand white rabbits or Japanese big-eared white rabbits with a weight of 2.0 to 2.5 kg. Generally male animals are more sensitive than female animals, and the choice of sex depends on the purpose of the experiment.

　　2. Feed cholesterol 0.5%, lard 5%, basic feed 94.5%, feed for 6-8 weeks. Serum cholesterol can be increased to 39-52mmol/L (1500-2000mg/dl), and the incidence of aortic As lesions can reach 80%-100%. If 15% egg yolk powder is added to the feed, the cholesterol in the feed is subtracted after 3 weeks of feeding, and the serum cholesterol can be increased to about 52mmol/L (2000mg/dl) after feeding for 3 weeks, and the incidence of aortic disease Up to 100%.

　　Others add lard and egg yolk powder to the basic feed, that is, each rabbit is fed with 30-50g of basic feed, 1.5g lard and 5g egg yolk powder every day, and the lard and egg yolk powder are subtracted for the 8th week. At the 14th week, the rabbit aortic lesion area reached 92%, and the lipid precipitation and liver damage of the mononuclear phagocyte system were lighter. The animal was in stable health and the mortality rate was low in the experiment.

　　3. Features Rabbits are the most commonly used animals to replicate hyperlipidemia and As models. Rabbits and humans have some similarities in lipoprotein metabolism. For example, the plasma of rabbits and humans have CETP activity; the coding site of ApoB is in the liver, and ApoB48 is only present in chylomicrons; the lipoprotein composition is mainly LDL. The expression of LDL receptors in the liver is low, and the ability to clear blood lipids is low. After intravenous injection of cholesterol floatation, hyperlipidemia can last for 3 to 4 days. Rabbits have a high absorption rate of exogenous cholesterol, up to 75% to 90%. Therefore, rabbits are very sensitive to high-fat feed, and hyperlipidemia causes As lesions to occur quickly and in a wide range. Under certain conditions, such as intermittent feeding of cholesterol or extending the experimental period to more than 1 year, As lesions similar to humans can be replicated, including atheroma and compound lesions. But rabbits lack liver lipase, nor do they have human ApoA analogs. The disadvantage is that serum cholesterol must reach a high level to form plaques. At this time, the internal organs are prone to lipid deposition, the animal's lifespan is short, the resistance is poor, and it is easy to die from secondary infection. Rabbits are herbivores, and their lipid metabolism is quite different from that of humans. Rabbit coronary artery disease mostly occurs in the small arteries of the heart, and mainly occurs in the large branches of the coronary arteries.

　　Another scholar reported that cystine can also induce As in rabbits. When cystine 0.3g/d was added to the basal feed, 3 months later, the rabbit aortic wall showed obvious atherosclerotic lesions, the cholesterol content in the aortic tissue was also significantly increased, and the accumulation of foam cells was observed under microscope.

　　Some scholars reported that rabbits were injected subcutaneously with homocysteine thiolactone at 20-25 mg/kg daily (with a concentration of 1 mg/ml in a 5% glucose solution) for 20-25 days. Adult rabbits and The typical lesions of As can appear in young rabbits. If homocysteine thiolactone is injected and 2% cholesterol is added to the feed, all animals will have significant As lesions.

　　Note: The fat content and fatty acid properties in the feed can affect the formation of As lesions. If the high-fat feed contains a large amount of saturated fatty acids, even a small amount of cholesterol can cause serious As disease: On the contrary, if the feed contains more polyvalent unsaturated fatty acids, it can reduce the degree of As disease. The amount of sugars in the feed can also affect the replication of the As experimental model, especially sucrose can promote the increase of serum cholesterol levels and accelerate the formation of lesions. This may be caused by the massive loss of trace element chromium in the refining process of sucrose. There are data indicating that chromium deficiency is related to As. Excessive protein content in feed can also promote the formation of animal As disease, especially the content of sulfur-containing amino acid-methionine.

　　(2) Rat model

　　1. Animals Wisar or SD rats aged 6-8 weeks, weighing 160-190g. In order to avoid the influence of sexual cycle on blood lipid metabolism, it is advisable to use male animals.

　　2. There are many formulas currently used in feed, such as: cholesterol 4%, bile salt 1%, basic feed 95%, feeding for 2 to 4 weeks; cholesterol 1% to 4%, lard 10%, methionine 0.2% , Basic feed 86%-89%, feeding for 1 to 2 weeks; egg yolk powder 10%, lard 5%, bile salt 0.5%, basic feed 84.5%, feeding for 1 to 2 weeks.

　　Although high-fat diet alone can increase the serum cholesterol level of rats, it is not prone to As lesions, inhibiting thyroid function or destroying rat thyroid with radioactive iodine can accelerate the induction of As lesions. Patek et al. found that rats after pituitary removal can also achieve the purpose of causing early As by inhibiting thyroid function. Jofis et al. added high-fat diet to the anti-thyroid drug thiouracil, which can induce atherosclerotic plaques in Wistar rats.

　　It has been reported in the literature that the calcium content in the walls of healthy coronary arteries is at a low level, while the calcium content in the lipid streaks of the atherosclerosis can be increased by 13 times, the calcium content in fibrous plaques increased by 24 times compared with normal, and the calcium content in late composite plaques can reach It is 80 times that of healthy coronary arteries, so some people consider establishing an As model with calcium overload. Domestic scholars reported that vitamin D was used to induce As in rats, and a one-time intraperitoneal injection of 600,000 U/kg of vitamin D into Wistar male rats. On the 18th day, aortic wall smooth muscle hyperplasia, endothelium and subendothelial edema and degeneration were observed. In the first day, the vascular smooth muscle proliferation is obvious, the arrangement of muscle cells is disordered, the structure of the elastic fiber layer is unclear, foam cells are formed, and atheromas protrude into the lumen. Wilgram also applied vitamin D2 to rats by gavage for 4 consecutive days, and fed a diet containing 1.5% cholesterol, 0.5% cholic acid, and 0.2% propylthiouracil. After 6 weeks, obvious As lesions appeared. Jellinek et al. believe that fat emulsions containing soybean extract, medium-chain triglycerides, phospholipids, and glycerin are easier to form an As model than cholesterol. The former only takes 8 days to produce As lesions in blood vessels, while the latter takes several months.

　　3. Features Rats are rarely used to replicate As models because of their small aorta and difficulty in producing As lesions. However, rats are convenient to feed, strong in resistance, and similar in eating habits to humans. They are still one of the main animal models for pharmacological research on blood lipids.

　　(3) Mini pig model

　　1. Animals Mini-swine (mini-swine) refers to pig breeds whose maximum weight is 1/8 to 1/6 of the maximum weight of ordinary domestic pigs. At present, the small pigs commonly used abroad include Landrace, Yuctan, Hanfold, Nebraska, Yorkshire and other lines. Some breeds of miniature pigs have also been developed in China, but they are not very mature, and there are not many miniature pigs available for biomedical research. At present, the most commonly used miniature pig is Guizhou Xiangzhu, whose maximum weight is 35kg, which is about half of the maximum weight of foreign miniature pigs of 75kg.

　　2. Feed cholesterol 1%, egg yolk powder 12.5%, basic feed 86.5%, feeding for 6 to 12 months, serum cholesterol significantly increased and the formation of As lesions.

　　3. Characteristics Pigs and humans have many things in common in physiology, biochemistry and anatomy. Bustad reported that the organ coefficients of mini-pigs are roughly similar to those of humans, and the biochemical properties, LDL structure and apolipoproteins of porcine plasma lipoproteins are also similar to humans. Due to this similarity, minipigs have a good effect of replicating As, and their lesion distribution and pathological characteristics are similar to those of humans. As lesions in the coronary arteries of miniature pigs are heavier than the aortic lesions, and the left coronary artery is more prone than the right coronary artery. Feeding with high cholesterol (4%) feed can induce progressive As lesions, and severe atheromas appear in both coronary arteries and aorta.

　　It is currently believed that miniature pigs are the preferred mammals besides primates in the As study.

　　(four) quail model

　　1. Animals Japanese or Korean male quails aged 5-6 weeks should weigh 90-110g.

　　(7) Pigeon model Pigeon is an omnivorous animal, and its food variety is close to human. Normal pigeons have higher plasma cholesterol levels, which can be said to be natural hypercholesterolemia animals. 70% of their plasma cholesterol is distributed in HDL, and lipoproteins lack ApoE and ApoB48.

　　pigeons are more sensitive to high-fat diets and are likely to induce As disease. After feeding the feed containing 0.5% cholesterol and 10% lard, the plasma total cholesterol level can reach as high as 52mmol/L (2000mg/dl), and As lesions appear. As lesions of WC (white carneau) pigeons usually occur in the thoracic aorta, abdominal aorta, iliac artery, carotid artery, renal artery and coronary artery. The lesions include foam cells, lipid nuclei, extracellular matrix, etc.; progressive plaques The mass may have calcification and angiogenesis, and eventually ulcers, bleeding, thrombosis and myocardial infarction may occur. In addition, there are some pathological manifestations similar to humans, including enhanced platelet adhesion, thrombosis, weakened endothelial function and vascular smooth muscle cell function. Therefore, the WC pigeon is one of the best animal models for studying human As. It should be noted that different breeds of pigeons have very different sensitivity to hypercholesterolemia and As induced by diet.

　　(8) Dog model Dogs rarely show spontaneous As, and are resistant to As induced by high-cholesterol diet. It is difficult to successfully replicate the prey-induced As model. Someone fed a feed containing 5% cholesterol and 16% hydrogenated coconut oil for 1 year, plasma cholesterol increased by about 8 times, coronary artery, abdominal aorta, iliac artery and other severe As disease.

　　2. Artificially bred animals with natural defects

　　In the research of hyperlipidemia and atherosclerosis, some animals with natural defects are often selected. These animals with certain genetic defects have been artificially selected and cultivated to form experimental animals with specific pathological characteristics or disease tendencies. The following are often used.

　　1. WHHL rabbit and STH rabbit Watanabe heritable hyperlipidemic rabbit (Watanabe heritable hyperlipidemic rabbit, WHHL rabbit) is a spontaneous hyperlipidemic Japanese white rabbit discovered by Watanabe in 1974. After 5 years of selection and passage in 1979 Nurturing success. WHHL rabbits have congenital defects in LDL receptors. The cells of their skin, liver, adrenal glands and other organs lack LDL receptors. They are hypercholesterolemia at birth. Serum total cholesterol, triglycerides and phospholipids are abnormally elevated. Spontaneous formation of atherosclerosis, myocardial infarction, xanthoma, etc. at age, which is very similar to human patients with familial hypercholesterolemia. Therefore, WHHL rabbit is a rare animal model in lipid metabolism and atherosclerosis research. However, its reproductive capacity is low, its body resistance is poor, and its susceptibility to coccidiosis is high, making it difficult to raise.

　　In 1987, Ville of St. Thomas's hospital in London discovered a New Zealand white rabbit with hereditary lipid metabolism, named "St. Thomas's hospital rabbit", or STH rabbit. The liver of STH rabbits is hyperactive in synthesizing VLDL. Feeding normal feed can cause the concentration of LDL, IDL and VLDL in the blood to increase. It has spontaneous hypercholesterolemia and hypertriglyceridemia, and its performance is similar to human high triglycerides. Esteremia and compound hyperlipidemia, the As lesions are also very similar to humans.

　　2. Spontaneous hypertensive rats (spontaneous hypertensive rats, SHR) were screened and bred from Wistar rats by Okamoto and Aoki of Japan in 1963. They are characterized by the continuous increase of spontaneous blood pressure, male SHR Also accompanied by extensive nodular periarteritis. SHR is often used as an experimental animal model for hypertensive diseases and nodular periarteritis, and is widely used in medical research. In addition to hypertension research, SHR is often used in As research topics such as exploring the relationship between blood pressure and arterial intima. What’s more valuable is that two subtypes have been selected and cultivated from SHR: one is SHR-ALR (arteriolipidos-sprone rats), which is prone to arterial lipid deposition. The reaction is strong. Serum cholesterol can rise to 13～20.8mmol/L (500～800mg/dl) after feeding high-fat diet for 1 week. Lipid deposits appear in small arteries such as mesenteric artery and cerebral artery, which can be seen after a few weeks Obvious atherosclerotic plaques are especially suitable for the study of cerebral arteriosclerosis; the other is myocardial-ischemic rats with a high incidence of ischemic heart disease, which can spontaneously congestive heart failure As well as extensive myocardial ischemic lesions, HDL levels decrease and the LDL/HDL ratio rises after feeding high-fat diets. As lesions can occur in a short period of time, which manifests as intimal thickening, lipid deposition, and can cause coronary artery stenosis And thrombosis and other manifestations, can be used for coronary heart disease, heart failure and other research.

　　2. The feed consists of 69% flour, 14% lard, 6% bone meal, 6% peanut oil, 4% bran, 1% cholesterol and sand (150g per 5kg feed). Blood lipids increased significantly in the second week of feeding, and As-like lesions appeared in the 6th to 8th weeks, with an incidence of 80% to 90%.

　　3. Features The application of high-fat feed can quickly induce quail As, and the coronary arteries and aorta can be affected. There is no obvious relationship between the degree of aortic disease and gender, but male quail coronary As disease is more serious than female, and can cause myocardial infarction. Arterial lesions in quail are similar to early human lipid streaks, mainly confined to the intima, and more severe at the bifurcation of the artery. The quail is small in size, consumes less drugs in experiments, is convenient for blood collection and administration, is cheap, easy to raise, and has sufficient sources for some experiments.

　　(5) Non-human primate model

　　1. Animals Non-human primates used to replicate As models include squirrel monkeys, baboons, curly monkeys, and spider monkeys. The blood lipids of monkeys and the nature and distribution of As lesions are very similar to humans. However, studies have shown that monkeys of different species have great differences in sensitivity to As, and it is generally believed that rhesus monkeys (or rhesus monkeys) are the most ideal.

　　2. The feed consists of 86.2% basic feed, 0.6% cholesterol, 6.6% egg yolk powder, and 6.6% lard. The serum cholesterol level can be as high as 7.8-15.6mmol/L (300-600mg/dl) after feeding the above-mentioned feed for 1 to 3 months. As lesions appear, severe cases can cause myocardial infarction.

　　3. Characteristics Monkeys have different sensitivity to As lesions, and the distribution of As lesions is also different. As lesions in male rhesus monkeys are mainly distributed in the anterior descending and circumflex branches of the left coronary artery; As lesions in cebus monkeys mainly occur in the carotid branches; lesions in cynomolgus monkeys are usually in the coronary arteries but the aorta is not affected; Africa The abdominal aorta of green monkeys has more lesions than the thoracic aorta. Due to the uncertainty of the lesion site of monkey As, the price is expensive, the animal source is limited, it is difficult to handle or capture, and it is more harmful, which limits the use of monkeys.

　　(Six) mouse model

　　1. Animals C57BL/6J strain mice, 9 weeks old, weighing about 20g are suitable.

　　2. Feed Add palm oil 10%, milk powder 4%, cholesterol 2%, and sodium cholate 0.4% to the basic feed, mix and grind to make pellets, dry in the sun, and autoclave for later use. The concentration of total cholesterol, free cholesterol and triglyceride in serum can be doubled after 16 weeks of feeding the above feed, and typical As lesions containing a lot of foam cells are formed in the aortic valve and coronary artery.

　　3. Features The use of mice to replicate the prey-induced As model has the advantages of small individual, less feed and medicine consumption, convenient feeding and management, and simple operation. However, because the individual is too small, the available samples are limited, sampling is difficult, and some experiments are not suitable. It should be noted that many other species of mice are resistant to As, and it is difficult to replicate successfully.

　　3. Natural mutant pig Prescott et al. (1991) reported a natural mutant pig with three allelic mutations related to ApoB and ApoR (Lpb5, Lpr1, Lpu1), which can occur under low-fat and cholesterol-free feed conditions Obvious hypercholesterolemia and As lesions. At 7 months of age, lipid streaks appeared in coronary arteries, iliac arteries, and femoral arteries. Progressive lesions could be observed within 2 years, including pathological changes such as extracellular cholesterol crystals, foam cells, necrotic lipid nuclei and fibrous caps. Interestingly, this mutant pig, unlike patients with familial hypercholesterolemia and WHHL rabbits, has normal LDL receptor activity.

　　4. LDL receptor-deficient monkeys Kusumi (1993) and Scanu (1988) have reported that familial LDL receptor-deficient monkeys were found in rhesus monkeys, their LDL cholesterol and Lp(a) levels were elevated, and the aorta developed progressive As Lesions, while the coronary artery lesions are smaller.