动物造模,转基因,动脉粥样硬化 z-bio 2022-01-25 295

　　A transgenic animal refers to a type of animal that uses experimental introduction methods to stably integrate foreign genes in the animal chromosomal genome and can be passed on to offspring. At present, most of the commonly used animals are transgenic animals and gene knockout animals. The research of genetically modified animals is based on classical genetics, molecular genetics, structural genetics and DNA recombination technology. As a kind of biological high-tech, the research of genetically modified animals has far-reaching theoretical value and significant application value. . The foreign gene integrated into the animal genome is called a transgene. Since the foreign gene may only be integrated into the genome of part of the animal’s tissue cells when establishing a transgenic animal, it may also be integrated into the genome of all tissue cells of the animal. Therefore, an animal with a foreign gene integrated in the genome of only part of the tissue is called Chimera (chimera, mosaic animals). Such animals can only inherit the foreign gene they carry to their offspring when the "part of the tissue cells" into which the foreign gene is integrated are just germ cells; otherwise, the foreign gene can be passed on to the offspring. The gene will not be passed on to offspring. Generally, the embryonic stem cell method and the first generation transgenic animals produced by the retroviral vector method are all chimeric animals, and about 20% of the first generation transgenic animals obtained by the microinjection method are such animals. If all cells of an animal are integrated with foreign genes, they have the ability to inherit the foreign genes to their offspring. Such animals are usually called transgenic animals. When a foreign gene is expressed in a transgenic animal and an animal model with a phenotype similar to human disease symptoms is cultivated, it is called a transgenic animal model. Currently, there are two main strategies for establishing transgenic animals. One is to allow the transgene to be overexpressed in animals, and the most commonly used method is microinjection. Transgene can use the promoter of the gene itself, or the exogenous promoter of splicing tissue-specific expression: it can be transferred into a single gene, or it can be transferred into a double gene. Another method is to inactivate the gene in the body and lose its function, that is, gene knockout technology. This is the technology of gene targeting using embryonic stem cells developed in recent years to produce genetically defective transgenic animals. In the medical field, genetically modified animals have been used in the research of genetic diseases, tumors, cardiovascular diseases, and immune diseases, including establishing pathological models of diseases, exploring their etiology, pathogenesis and gene therapy methods, studying gene expression regulation, and conducting drug interventions and interventions. Development and testing of new drugs, etc. The establishment of genetically modified animal models of diseases is an important research content in the application of genetically modified animal technology to medicine. Transgenic technology can build animal models of human diseases by changing the expression level of certain genes on the basis of the original genetic background of animals. The cause of the disease in this model is clear (caused by the transferred foreign gene), and the symptoms of the model animals are single, close to those of the patient. Transgenic mice are excellent animal models for studying the interaction between genetic and environmental factors in As. Mouse ApoE gene was the first gene to be successfully knocked out (Zhang et al., 1992), and then LDL receptor gene knockout mice (Veniant et al., 1998) and liver lipase gene knockout mice (Mezdour et al., 1997), human ApoB100 transgenic mice (Purcell-Huynh et al., 1995) and human CETP transgenic mice (Marotti et al., 1993) and so on. The results of the study showed that the entire arterial tree of ApoE knockout mice, LDL receptor gene knockout mice and ApoB100 transgenic mice can have obvious As lesions, and they have the typical characteristics of human atheroma. Therefore, transgenic mice are the most important transgenic animal models in As research. The preparation technology of transgenic animals is briefly introduced as follows.

　　1. Transgenic and knockout mice

　　(1) ApoE knockout mice ApoE is a glycoprotein with a molecular weight of approximately 34kDa, which is mainly synthesized in the liver, brain and other tissues of humans and mice. ApoE is the structural component of all lipoprotein molecules except LDL. ApoE is the most important apolipoprotein in the blood. It is a ligand for the receptors of LDL, VLDL, CM and CM residues, and is closely related to the metabolism of many lipoproteins. ApoE can promote the outflow of cholesterol from peripheral cells and the clearance of plasma cholesterol esters, and can also inhibit lipoprotein oxidation, reduce the formation of oxidized low-density lipoprotein and oxidized very low-density lipoprotein, thereby inhibiting the formation of foam cells. Piedrahita et al. (1992) used gene targeting technology to successfully replicate ApoE knockout mice, which can spontaneously develop atherosclerosis and are very similar to human atherosclerotic plaques. Most mice have natural resistance to As, therefore, the establishment of transgenic or gene knockout animals usually select C57BL/6J, CBA/J, SJLF2, 129Sv and other strains that are more sensitive to As.

　　Jorge et al. used homologous recombination to produce ApoE knockout mice. The production process is roughly as follows: cloning ApoE gene → constructing plasmid → cell culture and electrotransfer → screening homologous recombinants → producing blastodermal chimera animals.

　　ApoE gene knockout mice are unable to recognize and bind LDL, VLDL/IDL, etc. due to the failure of ApoE, so that the clearance of these lipoproteins is delayed and hyperlipidemia occurs. Feed low-cholesterol and low-fat diets and plasma cholesterol levels are sufficient. Up to 10.4～15.6mmol/L (400～600mg/dl). Hyperlipidemia induces the oxidative modification of lipoproteins and promotes the formation of As lesions. 5-6 weeks old mice can see that monocytes adhere to the vascular endothelium and migrate through the endothelial cells to the endothelium. Lipid streaks can be seen after 10 weeks. Fibrous plaques similar to human As can appear after 20 weeks. The As lesions in ApoE knockout mice are extensive, with large and middle arteries throughout the body such as the aortic root, thoracic aorta, and carotid artery. Renal artery, coronary artery, femoral artery, etc. can occur. If you eat high-fat and high-cholesterol feed, plasma cholesterol levels will increase and As lesions will form faster and more serious.

　　ApoE knockout mouse is the first mouse model that can spontaneously develop As under normal diet conditions, and is currently the most widely used ideal animal model in the field of As research. However, the detailed mechanism of spontaneous As lesions after ApoE knockout is not yet fully understood. Hakamata used VLDL/IDL and LDL isolated from the plasma of ApoE knockout mice to compare with the mouse's macrophages. The results showed that VLDL/IDL The increase in cholesterol ester content in macrophages is 7 times higher than that caused by LDL. It is speculated that in addition to the effect of raising LDL, high levels of VLDL/IDL in plasma also play a role in the formation of foam cells.

　　"(2) LDL-R knockout mice LDL-R mainly recognizes ApoB on the surface of LDL particles and ApoE on the surface of IDL particles, and mediates the internalization of related lipoproteins and their further metabolism in the cell. Therefore, these As-causing lipoproteins in the plasma of LDL-R knockout mice increased, and the plasma cholesterol level increased by 2 times. Among them, VLDL and LDL cholesterol increased significantly, and HDL cholesterol content decreased. If fed with high cholesterol feed containing cholic acid, the plasma cholesterol level can exceed 39mmol/L (1500mg/dl) and form a large number of As lesions such as lipid streaks, but no fibrous plaque formation is seen.

　　The method of producing LDL-R knockout mice is basically similar to that of ApoE knockout mice. The technical route is roughly as follows: Amplify LDL-R cDNA by PCR → construct vector → cultivate embryonic stem cells, and transfer the recombinant vector into embryo by electrotransfer Stem cells → Inject the stem cell clone containing the LDL-R mutant gene into the blastocysts of C57BL/6J mice.

　　LDL-R gene knockout mouse model was prepared by Ishibashi et al. using gene targeting technology in 1993. LDL-R -/- mice are the best model for studying human familial hypercholesterolemia. Similar to untreated familial hypercholesterolemia, these mice developed extensive atheroma and atherosclerotic lesions in the thoracic aorta and other blood vessels. Interestingly, the offspring of LDL-R -/- mice crossed with ApoBEC -/- mice or crossed with human ApoB100 transgenic mice can significantly increase plasma LDL levels and develop arteries under a low-cholesterol diet. Atherosclerosis. The pathological characteristics of LDL-R -/- mice are similar to those of ApoE -/- mice.

　　Sakaguchi et al. found that VLDL isolated from the plasma of LDL-R knockout mice can induce foaming of macrophages, indicating that the As lesions in LDL-R knockout mice may be related to the concentration of VLDL in plasma in addition to the accumulation and retention of LDL. Elevated related. Hasty et al. reported that the plasma triglycerides and cholesterol in the offspring of LDLR -/- mice and ob/ob mice were significantly increased. The offspring of homozygous offspring fed a low-cholesterol diet for 6 months can produce extensive atherosclerotic lesions.

　　(3) Human ApoB100 transgenic mice The basic method of ApoB100 transgenic mice replication is as follows: using the human genomic DNA library constructed on phage P1 as a template, using PCR to amplify human ApoB100 gene → target gene (human ApoB100 gene) screening and Identification→Isolate and purify the target gene from the recombinant vector→Adjust the concentration of the purified target fragment with microinjection buffer, and microinject it into the mouse fertilized egg cell→Insert the transgenic fertilized egg from the back into the fallopian tube of the pseudopregnant female mouse Inside, let the embryo mature in the foster mother → extract DNA from the tail of the mouse, and use the 32P-labeled human ApoB probe for Southern blot analysis to screen and identify transgenic mice with foreign gene integration.

　　The apolipoprotein ApoB on the surface of LDL is the ligand of the LDL receptor. Like LDL receptor knockout mice, ApoB100 transgenic mice will not have As lesions when fed with low-fat diet, and their plasma cholesterol levels are 2.6-5.2mmol/L (100-200mg/dl). After being fed high-cholesterol diet for 18 weeks, plasma cholesterol increased as high as 7.8-13mmol/L (300-500mg/dl), hypercholesterolemia and severe As lesions appeared, and progressive lesions formed after 6 months, with larger Necrotic lipid core and fibrous cap.

　　(4) ApoE3-Leiden (E3L) mutant gene The mouse ApoE3-Leiden mutation is a rare dominant-negative mutation of the human Apoe3 gene, which is characterized by repeated codons in series between the 120-126 bases. Sequence, which is associated with human familial β-lipoprotein abnormalities. ApoE3-Leiden mutant gene mouse (E3L) is produced by introducing human Apo-E3-Leiden gene into C57BL/6 mice. In addition to the introduction of the ApoE3-Leiden gene, it also includes the ApoC1 gene and a promoter element that regulates the expression of Apo-E and Apo-C1 genes. ApoE3-Leiden(E3L) mutant mice are highly sensitive to diets containing fat, sugar and cholesterol. When fed a high-cholesterol and high-fat diet, plasma cholesterol and triglyceride levels are significantly increased, accompanied by obvious VLDL and LDL Lipoprotein is elevated. Increasing the level of cholesterol and sugar in the diet can make the plasma lipid level rise rapidly to 41.6-52mmol/L (1600-2000mg/dl), which is easy to cause prey-induced As lesions, forming lipid streaks and fibrous plaque lesions. Compared with ApoE -/- and LDLR -/- transgenic mice, the E3L mouse model is a model of atherosclerosis with moderately elevated blood lipids (the plasma cholesterol level of ordinary diet is about 2mmol/L; the plasma cholesterol level of high-fat diet does not exceed 25mmol /L). In addition, plasma cholesterol and triglyceride levels change with the production of VLDL in the liver. E3L mice can have all the characteristics of human vascular disease, from lipid streaks to severe plaques. The lesion starts at the root of the aorta and progresses to the entire artery branch in a time-dependent manner. E3L mice are an ideal animal model for studying the effects of genetic and environmental factors on the disease.

　　(5) Human scavenger receptor AI (hSR-AI) transgenic mice Human scavenger receptor AI transgenic mice The replication method is as follows: Construction of mouse tie-1 promoter and human scavenger receptor AI cDNA expression Vector→Identified by restriction enzyme digestion and sequencing→Inject the prepared tie-1-hSR-A-BGH poly A fragment into the fertilized egg by microinjection method→Transfer the surviving fertilized egg into the pseudo-pregnant female mouse→offspring After polymerase chain reaction and Southern blot analysis, the positive transgenic mice integrated with the foreign target gene were screened out → Tissue RNA reverse transcription polymerase chain reaction and immunohistochemical staining to detect the expression level and expression site of human scavenger receptor AI .

　　Human scavenger receptor AI transgenic mice have human scavenger receptor AI expression in the aorta, kidney, liver and other tissues, and they are mainly concentrated on vascular endothelial cells. The aortic endothelial cells of the transgenic mice were obviously edema, with polycystic and insect-eating changes on the surface, more blisters in the cytoplasm, and plasma triglyceride levels were significantly higher than those of the control mice. Transgenic mice showed early pathological changes of As in blood vessels and were more sensitive to As.

　　(6) Other genetically engineered mouse atherosclerosis models

　　1. Related to inflammation Macrophages infiltrate the blood vessel wall after endothelial injury in the early stage of atherosclerosis. Therefore, only blocking the infiltration of macrophages can further clarify the role of macrophages. Since macrophage colony stimulating factor (MCSF) can affect the development of monocytes and macrophages, apolipoprotein E -/- mice were cross-bred with OP mice with mutations in the MCSF gene. The blood monocytes and tissue macrophages in the hybrid mice are reduced, and the As lesions are only 1/7 of those in the simple apolipoprotein E-deficient mice, and they do not develop to the fibrotic stage, indicating the "clean" of macrophages. The role is to promote the development of As. The As lesions in MCPI-1 -/- mice were milder; the As lesions in mice overexpressing MCPI-1 gene were aggravated.

　　2. Related to protease and extracellular matrix Vascular smooth muscle cells (VSMC) and collagen that are vulnerable to plaque are significantly reduced, and the fiber cap is easy to rupture, which leads to various complications. Recent studies have revealed that TGF-β negative-dominant mutant mice have increased vascular inflammation, reduced VSMC and collagen components, and formed a vulnerable plaque phenotype. This indicates that the anti-inflammatory factor TGF-β stabilizes As plaques and inhibits the development of As. Mice with matrix GLA protein deficiency (Mgp knockout) spontaneously calcified arterial wall elastic tissue and muscular tissue, and died of aneurysm rupture within 2 months, but no plaque formation. After plasminogen is activated into plasmin, it participates in the degradation of extracellular matrix. Apolipoprotein E and plasminogen double gene knockout mice As lesions expand and foam

　　The cell infiltration is obvious. Plasminogen activator inhibitor 1 (PAI-1) is another pathway of fibrin/fibrinogen lysis and inhibits plasminogen activation. However, regardless of PAI-1 gene knockout or overexpression, it does not affect the As process, which may be because the mouse has other substances that selectively inhibit the activation of plasminogen. Matrix metalloproteinases (MMP) degrade the extracellular matrix, balance the processes of promoting proteolysis and resisting proteolysis, and play an important role in the As process. The arterial lesions of MMP23 knockout mice progressed, and a large amount of collagen accumulated in the plaques, and the incidence of aneurysms was very low. Conversely, overexpression of MMP21 by gene transfection technology can promote the degradation of neointimal extracellular matrix and reduce As disease.

　　3. Related to immune regulation Atherosclerotic lesions are mainly infiltration of macrophages and T cells, indicating that the immune system is involved in the formation of As lesions. On this basis, an As mouse model related to immune regulation was established. In recent years, the role of T cells in the formation of As has been basically clear. The study found that T cells promoted the formation of lipid streaks, and the lipid streaks of C57BL/6 mice lacking CD4+ and CD8+ cells were significantly reduced. After immunodeficient apolipoprotein E-/- mice obtain CD4+ cells from immunocompetent mice, As lesions are significantly worsened, indicating that CD4+ cells promote the progression of the lesions. Cytotoxic CD8+ cells induce apoptosis of apolipoprotein E-/- mouse VSMC and promote the formation of lesions. Compared with wild-type mice, mice with MHC-Ⅰ and MHC-Ⅱ gene deletions are more ill. Recombinase activator gene 2 (RAG2) deletion mice established in 1997 lacked B cells and T cells. Compared with the normal group, the lack of autoantibodies and T cells did not affect As lesions.

　　4. Related to glucose metabolism Diabetes is a risk factor for cardiovascular disease, but its mechanism is unclear. The spontaneous diabetic mice induced by streptozotocin (STZ) was used as a model to clarify the damage effect of high glucose on blood vessels. Advanced glycation end product (AGE), a by-product of hyperglycemia, combines with AGE receptor (advanced glycation end product receptor, RAGE) to induce the production of inflammatory markers and aggravate As lesions. After the apolipoprotein E-/- mice were injected with STZ to develop diabetes, daily injection of RAGE could alleviate As lesions. These results prove that there is a close relationship between AGE and diabetes-related As lesions. However, it has recently been reported that diabetes is a factor in the resistance to As. Injecting apolipoprotein E-/- mice with glucosamine gold to destroy the hypothalamic satiety center, induce obesity and diabetes in the mice, and the non-diabetic apolipoprotein E- /-Compared with mice, As develops slowly, which raises questions about the role of diabetes in promoting As. Therefore, further research is particularly important. Clinically, type 1 and type 2 diabetes have different pathophysiological characteristics and cardiovascular complications, and the exact mechanism is still unclear. Therefore, the use of mouse models to explore the relationship between type 1 and type 2 diabetes and As is still limited, which will be a future research area.

　　5. Related to Hypertension Hypertension is closely related to As. Endothelial → nitric oxide synthase (endothelial nitric oxide synthase, eNOS) regulates vasomotion. eNOS-/- mice developed hypertension, and after mating with apolipoprotein E-/- mice, the offspring had both hypertension and As. However, there are reports that eNOS and NO produce superoxide anions, oxidize low-density lipoproteins, and accelerate the pathological process of As. In 2002, some scholars reported that the offspring of eNOS-/- mice mated with apolipoprotein E-/- mice had less pathological damage than the control group apolipoprotein E-/- mice, indicating that eNOS, NO and As The relationship is still unclear. In recent years, it has been discovered that angiotensin II stimulates the proliferation of vascular smooth muscle cells and the accumulation of cholesterol in macrophages, which accelerates the pathological process of As. Compared with apolipoprotein E-/-PACE+/+ mice, the offspring of apolipoprotein E-/- mice mated with ACE-/- mice have significantly reduced As lesions and oxidative stress, indicating that ACE is involved in As form.

　　6. Other hyperhomocysteinemia is also an important risk factor for As. The pathological damage area of Apolipoprotein E-/- mice with homocysteinemia was enlarged, the vascular inflammation was aggravated, and multiple complications occurred. Vitamin C can add hydroxide ions at both ends of proline and lysine to stabilize collagen. The Gulo-/- mice and apolipoprotein E-/- mice with the deletion of the vitamin C synthase gene had significantly reduced collagen content in the heterozygous lesions.

　　Two, genetically modified rabbits

　　Although transgenic mice are the most widely used in transgenic animals, there have been reports on the application of transgenic rabbits to study As and lipid metabolism in recent years. The first transgenic rabbit animal model was obtained in 1985 by Hammer et al. by microinjection. Rabbits are the standard animal model for biomedical research and transgenic rabbits are widely used in the research of various human diseases. Most of the rabbits that make genetically modified rabbits use special pathogen free (SPF) New Zealand rabbits and Japanese big-eared white rabbits. The production method mainly uses the direct injection of the constructed foreign gene into the male pronucleus of the fertilized egg, which is similar to the process of making transgenic mice by this method. Specifically, rabbits aged 5 to 7 months are selected, and the rabbits are superovulated by injection of PMSG (pregnant mare's serum gonadotropin) or FSH (follicular stimulating hormone). Then, the exogenous gene is injected into the male pronucleus of the fertilized egg under a microscope, cultured in vitro for 2 to 3 hours, and the surviving fertilized egg is selected for transplantation into the oviducts on both sides of the recipient rabbit. Four weeks after the birth of the newborn rabbits, ear tissues were taken, and Southern blot was used to detect whether the foreign gene was integrated into the rabbit's genome. In addition, transgenic rabbits can also be obtained through techniques such as sperm and oocyte injection, ES cell-mediated methods, and somatic cell nuclear transfer, but these methods are still immature and not widely used.

　　(1) ApoAI transgenic rabbits In 1993, Perevozchikov and others successfully bred transgenic rabbits with human ApoAI cDNA. Three years later, Duverger et al. introduced 11 kbp DNA containing the human ApoAI gene into the NZW rabbit genome and expressed human ApoAI in the liver. After 14 weeks of feeding with 0.4% cholesterol, the plasma HDL cholesterol content of ApoAI transgenic rabbits was twice that of the control group. In the later stage of the experiment, the lipid accumulation on the surface of the aorta of the transgenic rabbits was significantly reduced compared with the control group, and the total cholesterol was 46% of the control group. Studies have shown that overexpression of human ApoAI seems to have a tendency to inhibit this development.

　　(2) ApoB transgenic rabbit Fan Jianglin et al. injected human ApoB genomic DNA with a full length of 80kb into the male pronucleus of NZW rabbit fertilized eggs, and bred transgenic rabbits carrying the human ApoB100 gene. Only human ApoB100 is expressed in the plasma of transgenic rabbits, but ApoB48 is not expressed, because the liver of rabbits lacks the function of encoding the corresponding ApoB mRNA. Compared with non-transgenic rabbits of the same sex, the same age, and the same conditions, the plasma cholesterol and triglyceride content of the transgenic rabbits is 2 to 3 times that of the control group, and the HDL cholesterol is significantly reduced. Almost all cholesterol and human ApoB100 in transgenic rabbit plasma are in the form of LDL particles rich in triglycerides. Through gradient polyacrylamide gel analysis, the diameter of the transgenic rabbit LDL particles was significantly reduced compared with the control group. In this study, whether transgenic rabbits carrying the human ApoB100 gene are susceptible to As has not been determined.

　　(3) Apo(a) genetically modified rabbits. Apo(a) is only found in humans, old world monkeys and hedgehogs in nature. Apo(a), which is the main component of lipoprotein (a), and human coronary heart disease, Stroke is closely related to restenosis after coronary artery dilation, but there has been a lack of suitable animal models to study human Apo(a). Human Apo(a) expressed by transgenic mice cannot combine with rodent ApoB to form LP(a) particles, so mice are not an ideal animal model. Rouy et al. established a transgenic rabbit model expressing human Apo(a). Studies have shown that these transgenic rabbits expressing human Apo(a) can form human-like Lp(a)-like particles, suggesting that such transgenic rabbits are useful models for studying Lp(a). In order to determine the relationship between Apo(a) and As, the transgenic rabbits were fed normal and high-cholesterol diets. It was found that the transgenic rabbits fed normal diets did not develop As lesions, which showed that low levels of Apo(a) could not lead to the formation of As. After feeding rabbits with feed containing 0.3% cholesterol for 16 weeks, the plasma cholesterol content of transgenic rabbits and non-transgenic rabbits are similar, but the As disease of transgenic rabbits is much more serious. Transgenic rabbits have atherosclerotic lesions in the aorta, iliac artery and carotid artery. Regional expansion.

　　(4) ApoE2 transgenic rabbits Transgenic rabbits expressing human ApoE2 (Cys112, Cys158) at high concentrations were successfully bred by Huang et al. Type I hyperlipoproteinemia is a manifestation of homozygous ApoE2 gene. Such patients are susceptible to As before they reach adulthood. Huang’s study found that the plasma levels of total cholesterol and HDL cholesterol in the transgenic rabbits overexpressing human ApoE2 were higher than those in the control group, and the total cholesterol and HDL cholesterol concentrations of the transgenic rabbits were significantly different between males and females. Analyzing the plasma lipoproteins of transgenic rabbits, it was found that it was consistent with the characteristics of type I hyperlipoproteinemia, β-VLDL aggregation, VLDL, IDL concentration increased significantly, and gender differences were shown. Feeding these rabbits with normal diet for 11 months spontaneously formed As lesions in the aortic arch and abdominal aorta, showing that the overexpression of human ApoE2 in transgenic rabbits can promote the spontaneous formation of As. The study also found that male rabbits have more extensive lesions than female rabbits, suggesting that sex hormones play an important role in type I hyperlipoproteinemia.

　　(5) ApoE3 transgenic rabbit Research reported that human ApoE3 genomic DNA was linked to the liver regulatory region and introduced into the rabbit genome. The transgenic rabbit expressed human ApoE3 (Cys112, Arg158). Compared with non-transgenic rabbits, transgenic rabbits have lower VLDL and higher LDL. There are almost no large VLDL particles (>36nm) in the plasma of transgenic rabbits, and about 20% of such particles in non-transgenic rabbits. The difference is very significant. It may be that ApoE3 increases the receptor-mediated clearance of VLDL. Research by Huang et al. showed that overexpression of ApoE3 in transgenic rabbits stimulates the liver to produce VLDL, enhances VLDL clearance, inhibits VLDL lipolysis, and causes hyperlipidemia. The difference in the expression of different levels of human ApoE in a narrow range plays an important critical role in adjusting plasma cholesterol and triglyceride concentrations, and may play a decisive role in the type of hyperlipoproteinemia.

　　(6) Transgenic rabbits with liver lipase Some researchers have introduced the regulatory region of human ApoE/C Ⅰ expression in the liver and liver lipase cDNA into the rabbit genome. Six rabbits expressed human liver lipase in the liver. Liver lipase plays a pivotal role in the process of lipoprotein metabolism. The activity of endogenous liver lipase in the rabbit itself is very low. Transgenic rabbits expressing human liver lipase have an important effect on the metabolism of lipids and lipoproteins in the blood. Total RNA was extracted from lo tissues, and it was found that the human liver lipase gene was only expressed in the liver of rabbits. Overexpression of liver lipase caused a significant decrease in HDL, VLDL, and IDL, and total cholesterol and triglycerides in transgenic rabbits decreased by 42% and 58%. After feeding the liver lipase transgenic rabbits with 0.3% cholesterol and 3% soybean oil for 5 weeks, the blood cholesterol content of the transgenic rabbits was only 1/3 of the control group, and the trend of hypercholesterolemia in the transgenic rabbits was weakened. Preliminary studies have shown that reducing the blood cholesterol concentration of transgenic rabbits with liver lipase is related to the reduction of the aortic atherosclerotic area.

　　(7) Lipoprotein lipase transgenic rabbits In order to study the role of lipoprotein lipase (LPL) in lipid metabolism and the relationship with As, Araki et al. injected human LPL cDNA with chicken β-actin promoter into rabbits for fertilization Among the eggs, a transgenic rabbit expressing human LPL was obtained. Northern blot showed that human LPL is expressed in heart, kidney, adrenal gland, and intestinal tissues. In transgenic rabbits, plasma triglycerides decreased by 80%, and HDL decreased by 59%. Analysis of lipoprotein density particles shows that increasing the expression of LPL in transgenic rabbits can significantly reduce the levels of VLDL and IDL, and the level of LDL cholesterol is significantly increased. When the transgenic rabbits were fed cholesterol feed, the transgenic rabbits significantly inhibited the formation and development of hypercholesterolemia and As lesions, suggesting that over-expression of LPL has an inhibitory effect on feed-induced hypercholesterolemia and As.

　　(八) LCAT transgenic rabbit Human phosphatidylcholine cholesterol acyltransferase (LCAT) is a key enzyme in cholesterol metabolism. In 1996, Hoeg and his colleagues reported that the genomic DNA covering the human LCAT gene was introduced into NZW rabbits to cultivate transgenic rabbits that successfully carried human LCAT to study the effects of overexpression of LCAT on plasma lipid and lipoprotein metabolism. . Northern blot showed that LCAT is mainly expressed in the liver, but also expressed in the brain, heart and muscle. The plasma lipid and lipoprotein content of transgenic rabbits changed significantly, and the concentration of total cholesterol, free cholesterol and esterified cholesterol increased significantly. The increase in cholesterol is mainly due to a significant increase in the concentration of HDL cholesterol. After feeding the rabbits with 0.3% cholesterol feed for 17 weeks, the HDL cholesterol content of the transgenic rabbits increased by 20% compared with the control group. The arterial surface lesions in transgenic rabbits were only 5%, compared with 35% in the control rabbits, suggesting that LCAT has an inhibitory effect on feed-induced As. However, the study by Mehlum et al. found that the LCAT activity of homozygous transgenic mice carrying human LCAT was 14-27 times higher than that of the control group, and the HDL cholesterol concentration was 2 times higher, but there was no obvious inhibitory effect on feed-induced As.

　　(9) 15-lipoxygenase transgenic rabbit 15-lipoxygenase (15-LO) is expressed in foamy macrophages in As lesions, which may be related to the oxidation of LDL during the occurrence of As. Shen et al. used lysozyme macrophage-specific promoter to link human 15-LO gene