WO2013183725A1 - Animal modèle non humain, procédé de création d'un animal modèle non humain et procédé de criblage d'un médicament antithrombogénique - Google Patents

Animal modèle non humain, procédé de création d'un animal modèle non humain et procédé de criblage d'un médicament antithrombogénique Download PDF

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WO2013183725A1
WO2013183725A1 PCT/JP2013/065741 JP2013065741W WO2013183725A1 WO 2013183725 A1 WO2013183725 A1 WO 2013183725A1 JP 2013065741 W JP2013065741 W JP 2013065741W WO 2013183725 A1 WO2013183725 A1 WO 2013183725A1
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model animal
human model
human
thrombosis
animal
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健輔 江頭
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国立大学法人九州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/25—Animals on a special diet
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0375—Animal model for cardiovascular diseases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis

Definitions

  • the present invention relates to a non-human model animal, a method for producing a non-human model animal, and a method for screening an antithrombotic drug.
  • arteriosclerotic diseases such as myocardial infarction, stroke and peripheral arterial disease
  • Many of these diseases are caused by thrombosis based on arteriosclerotic lesions (plaque), resulting in a decrease in the quality of life of the patient and the main cause of death and bedriddenness.
  • Non-Patent Document 1 The acute phase of acute myocardial infarction (within 6-8 hours of onset of acute myocardial infarction) through the spread of reperfusion and standardization of medical treatment ( ⁇ -blockers, ACE inhibitors, statins, etc.) Life prognosis within 1 month of onset has improved.
  • Acute myocardial infarction is known to be caused by an occlusive thrombus formed in unstable plaque of coronary arteries (see Non-Patent Document 2), and further elucidating the mechanism of acute myocardial infarction, It can be an appropriate diagnosis, prevention or treatment.
  • Plaque failure is characterized by a thin fibrous cap, strong inflammatory cell infiltration, and a lipid core.
  • plaque rupture the rupture of the fibrous cap causes detachment of the intima, activation of coagulation factors in the blood and platelet aggregation by collagen and tissue factor under the intima, and formation of an occlusive thrombus It is considered (see Non-Patent Document 3).
  • Plaque erosion is also characterized by vascular intimal detachment.
  • the pathological features of plaque erosion include the absence of fibrous capsule failure, the neointimal is formed mainly by smooth muscle cells and proteoglycans, and the infiltration of inflammatory cells is less than the above-mentioned plaque failure. This is different from the failure of plaque (see Non-Patent Document 4).
  • mice lacking both Niemann-Pick C1 and ApoE are known as model animals in which thrombus is formed.
  • a non-occlusive thrombus accompanied by degeneration and destruction of the media and inflammation of the adventitia is formed in the ascending aorta near the aortic valve (see Non-Patent Document 7).
  • an animal model in which acute thrombus is induced by mechanical stimulation or drug loading has been reported (see Patent Document 1 and Non-Patent Document 8).
  • the present invention has been made in view of the above circumstances, and provides a non-human model animal useful as a model of thrombosis caused by plaque erosion, a method for producing the non-human model animal, and a screening method for an antithrombotic drug. For the purpose.
  • the non-human model animal according to the first aspect of the present invention is: It develops thrombosis due to plaque erosion.
  • the thrombosis is Is occlusive to occlude the lumen of the blood vessel, It is good as well.
  • the thrombosis is At least one of a platelet thrombus and a fibrin thrombus is formed in the blood vessel lumen; It is good as well.
  • the non-human model animal is A rabbit, It is good as well.
  • a method for producing a non-human model animal that develops thrombosis caused by plaque erosion according to the second aspect of the present invention, Damaging a portion of the endothelium of an artery of a non-human animal fed a high cholesterol diet; Continuously administering a pressor to the non-human animal; including.
  • the pressor is Angiotensin II, It is good as well.
  • the non-human animal is A rabbit, It is good as well.
  • the antithrombotic drug screening method according to the third aspect of the present invention comprises: The non-human model animal according to the first aspect of the present invention is used.
  • the screening method for antithrombotic drugs is: Administering a test substance to the non-human model animal; Comparing a marker associated with thrombosis between the non-human model animal to which the test substance has been administered and the non-human model animal to which the test substance has not been administered; including, It is good as well.
  • a thrombus exhibiting a lesion characteristic of plaque erosion is formed in a non-human animal. For this reason, a non-human model animal useful as a model of thrombosis caused by plaque erosion can be obtained.
  • FIG. 3 is a first diagram showing an angiographic image of a rabbit in Example 1. It is a figure which shows the blood-vessel sample extract
  • FIG. 6 is a (second) diagram illustrating an angiographic image of a rabbit in Example 1. It is a figure which shows the blood-vessel sample extract
  • FIG. 4 It is a figure which shows the image of the body surface tube echo with respect to the rabbit in Example 3.
  • FIG. It is a figure which shows the angiographic image of the rabbit shown in FIG.
  • FIG. It is a figure which shows the result of the tissue dyeing
  • Example 4 it is a figure which shows the time-dependent change of the ratio of the rabbit which survived without obstruction
  • Example 6 is a graph showing the results of tissue staining of the femoral artery of rabbits in the angiotensin II administration group in Example 4. It is a figure which shows the result of the immunohistochemical analysis regarding the various markers with respect to the femoral artery of FIG. It is a figure which shows the result of the immunohistochemical analysis with respect to the artery with obstruction
  • Example 5 it is a figure which shows the time-dependent change of the ratio of the rabbit which survived without occlusion. It is a figure which shows the result of the tissue dyeing
  • the non-human model animal develops thrombosis caused by plaque erosion.
  • Plaque is a thickened lesion of the intima present in a thickened and hardened site in the artery, the so-called arteriosclerotic lesion.
  • the plaque contains an atheroma in the center and is covered by a fibrous capsule.
  • Plaques often have a lipid core made of cholesterol ester inside.
  • Plaque erosion is a lesion in which the fibrous capsule covering the plaque fails due to injury that does not reach the lipid core. Plaque erosion has few apparent lipid cores and thin fibrous capsules and poor inflammatory cell infiltration. Plaque erosion is often observed in plaques rich in smooth muscle cells and proteoglycans. In contrast, plaque failure is characterized by a clear lipid core, strong infiltration of inflammatory cells, a thin fibrous capsule, and a few smooth muscle cells in the fibrous capsule.
  • Thrombosis is a condition in which a thrombus is formed in the lumen of a blood vessel, but broadly includes a condition related to the formed thrombus.
  • the pathological condition related to the thrombus is, for example, ischemia or infarction due to the formation of a thrombus, arteriosclerosis, myocardial infarction, angina pectoris, cerebral thrombus, or the like.
  • the thrombosis in the non-human model animal may be occlusive that obstructs the blood vessel lumen.
  • obstructive thrombosis for example, 75% or more, more preferably 90% or more, and still more preferably 95% or more of the area of the blood vessel lumen of the non-human model animal is occupied by thrombus.
  • the ratio of the thrombus occupying the area of the blood vessel lumen can also be calculated based on the image of the blood vessel cut surface obtained by body surface tube echo.
  • the ratio of the thrombus in the area of the blood vessel lumen can be grasped by the pathological analysis of the blood vessel sample.
  • the thrombosis in the non-human model animal at least one of platelet thrombus and fibrin thrombus is formed in the blood vessel lumen.
  • Platelet thrombus is also called white thrombus, and platelets are greatly involved in the formation mechanism.
  • Fibrin thrombus is also called red thrombus and is mainly formed by blood coagulation reaction.
  • occlusive thrombosis in humans it is known that platelet thrombosis occurs first, followed by occlusion of blood vessels by fibrin thrombosis.
  • fibrin thrombosis it is suggested that in the thrombosis in the non-human model animal, blood vessels are occluded by fibrin thrombus after platelet thrombus occurs.
  • the said non-human model animal is useful also as a model of the obstructive thrombosis which occurs naturally in the human in the point which reproduces the history of the obstructive thrombosis in human.
  • the method for producing a non-human model animal includes a step of damaging a part of the arterial endothelium of a non-human animal fed with a high cholesterol diet and a step of continuously administering a pressor to the non-human animal.
  • Non-human animals are not particularly limited as long as they are animals other than humans, and mammals and birds used as experimental animals may be used. For example, mice, rats, hamsters, rabbits, dogs, pigs, monkeys, chickens and the like can be used as non-human animals.
  • monkeys and the like whose genome is close to that of humans as non-human animals.
  • a non-human animal whose lipid metabolism system and coagulation / fibrinolysis system are close to humans.
  • the non-human animal whose lipid metabolism system and coagulation / fibrinolysis system are close to humans is, for example, a rabbit.
  • Rabbits are suitable as non-human animals because they are relatively easy to breed. Although the kind of rabbit is not specifically limited, For example, a Japanese white breed rabbit is mentioned.
  • mice, rats, hamsters, etc. are used as non-human animals, for example, the function of factors related to the coagulation system is strengthened or A mouse, rat, hamster or the like that has weakened the function of factors related to the dissolution system may be used.
  • a genetically modified animal in which the expression level of the gene of the factor related to the coagulation system is increased can be used.
  • Coagulation-related factor genes include fibrin, fibrinogen, prothrombin, thrombin, thromboplastin, factor VI, factor VII, factor VIII, factor IX, factor X, factor XI, factor These are genes such as factor XII and factor XIII coagulation factor.
  • Such a genetically modified animal can be prepared in advance by a known gene manipulation technique such as gene knock-in. At this time, the expression level of a gene such as a single coagulation factor may be increased. However, if the expression level of a gene such as a plurality of coagulation factors is increased, the coagulation system is compared to the fibrinolytic system. Can be relatively stronger.
  • a genetically modified animal in which the gene of the factor related to the fibrinolytic system is deleted or the expression level of the gene of the factor related to the fibrinolytic system is decreased.
  • Factor genes related to the fibrinolytic system are genes such as plasmin, plasminogen, tissue-type plasminogen activator (t-PA) and urokinase (u-PA).
  • t-PA tissue-type plasminogen activator
  • u-PA urokinase
  • Such a genetically modified animal can be produced by a known gene manipulation technique such as gene knockout.
  • siRNA, miRNA, antisense nucleic acid, etc. may be used in order to reduce the expression level of the factor gene associated with the fibrinolytic system.
  • an inhibitor, antibody, DNA aptamer or RNA aptamer etc. that inhibits the function of the factor related to the fibrinolytic system may be administered to mice, rats, hamsters, etc. .
  • a high-cholesterol diet is, for example, a diet obtained by further adding cholesterol to a general diet for non-human animals to be used.
  • the cholesterol content of the high cholesterol diet is 0.5-4%, preferably 1-3%, more preferably 1.5% -2.5%.
  • the period of feeding the high-cholesterol diet to the non-human animal is not particularly limited, and can be appropriately selected according to, for example, the type of non-human animal used or the cholesterol content of the high-cholesterol diet.
  • a high-cholesterol diet with a cholesterol content of preferably 0.5 to 4%, preferably 1 to 3%, more preferably 1.5% to 2.5%. Feeding for 1 to 10 weeks is preferable, and feeding for 2 to 8 weeks is more preferable.
  • the total cholesterol level in the non-human animal is preferably 100 to 1000 mg / dl, and more preferably 300 to 800 mg / dl.
  • the value of the total cholesterol in the blood in the said non-human animal may be 1000 mg / dl or more.
  • the type of artery that damages the endothelium is not particularly limited.
  • the types of arteries are, for example, the aorta, atrial artery, hepatic artery, descending artery and the like. In a relatively large animal such as a rabbit, the limb artery may be used. More specifically, the endothelium from the end of the femoral artery to the origin of the common iliac artery may be damaged.
  • Endothelial injury can be performed by various methods.
  • the endothelium can be injured by inserting a balloon catheter into an arterial site that damages the endothelium and pressurizing the balloon catheter.
  • the size of the balloon catheter may be appropriately determined in consideration of the size of the injured site.
  • the pressurization to the balloon catheter is, for example, 1 to 4 atm, more preferably 1 to 2 atm, depending on the specification of the balloon catheter used.
  • means for applying a certain physical load to the endothelium of the artery can be appropriately selected.
  • a method of ligating an artery using a suture thread there is a method of ligating an artery using a suture thread.
  • the vasopressor may be any compound, antibody, peptide, DNA aptamer, RNA aptamer, siRNA, miRNA, antisense nucleic acid, etc., as long as it has an action of increasing blood pressure.
  • the pressor norepinephrine and dobutamine are preferable, and angiotensin II is more preferable.
  • Angiotensin II is a polypeptide having a pressor action.
  • the method of continuously administering the vasopressor is not particularly limited.
  • angiotensin II when used as a pressor, angiotensin II can be continuously administered by implanting an osmotic pump filled with an angiotensin II solution under the skin of the non-human animal.
  • concentration of angiotensin II to be continuously administered is appropriately determined according to the type of non-human animal used.
  • angiotensin II when a rabbit is used as a non-human animal, angiotensin II is 10 to 100 ng / kg per minute, preferably 30 to 70 ng / kg per minute, more preferably 40 to 60 ng / kg per minute, particularly preferably 45 per minute. It may be administered at ⁇ 55 ng / kg.
  • angiotensin II may be continuously administered by periodically injecting the angiotensin II solution into the vein of the non-human animal.
  • an antithrombotic drug can be screened using the non-human model animal.
  • the antithrombotic drug screening method includes the steps of administering a test substance to the non-human model animal, the non-human model animal administered with the test substance, and the non-human model animal not administered with the test substance. And comparing markers associated with thrombosis.
  • a test substance such as a compound, antibody, peptide, DNA aptamer, RNA aptamer, siRNA, miRNA, antisense nucleic acid or the like is administered to the non-human model animal.
  • the method of administration is not particularly limited, but it is preferable to select an appropriate method for the test substance.
  • the test substance when the test substance is a compound, it may be administered orally or intraperitoneally.
  • the test substance is an antibody, peptide, DNA aptamer, RNA aptamer, siRNA, miRNA, antisense nucleic acid or the like, intravenous injection, subcutaneous injection or the like may be used.
  • the test substance may be administered once or multiple times. What is necessary is just to determine the density
  • the marker related to thrombosis may be, for example, the presence or absence of a thrombus formed in the lumen of a blood vessel, or a value such as the size of the formed thrombus.
  • the presence or absence of a thrombus can be determined using pathological analysis such as angiography, body surface tube echo, and tissue staining.
  • values such as the size of the thrombus can be calculated from the size of the region corresponding to the thrombus in the image in pathological analysis such as angiography, body surface tube echo, and tissue staining.
  • markers related to thrombosis include tissue oxygen saturation obtained from tissue oxygen partial pressure, activity of factors related to coagulation such as factor X, activity of factors related to fibrinolytic systems such as plasmin Etc.
  • examples of the method for using the non-human model animal include observation of thrombosis, particularly thrombosis caused by plaque erosion, search for various markers for thrombosis diagnosis, and the like.
  • the antithrombotic drug includes a drug for treating thrombosis, a drug for preventing thrombosis, and the like.
  • the non-human model animal according to the present embodiment As described in detail above, in the non-human model animal according to the present embodiment, as shown in the following examples, there is no apparent lipid core or thin fibrous capsule, and platelet thrombus occurs, followed by Fibrin thrombus is formed. Thus, the non-human model animal has developed thrombosis exhibiting a characteristic lesion in plaque erosion, and reproduces the formation mechanism of thrombus in plaque erosion. For this reason, the non-human model animal according to the present embodiment is useful as a model of thrombosis caused by plaque erosion.
  • the thrombosis that occurs in the non-human model animal according to the present embodiment may be occlusive that obstructs the lumen of the blood vessel.
  • Obstructive thrombosis formed in vulnerable plaque of coronary arteries is the cause of acute myocardial infarction, which is the most serious disease that threatens life prognosis among arteriosclerotic diseases. Therefore, the non-human model animal according to the present embodiment is useful for elucidating the onset mechanism of acute myocardial infarction, searching for therapeutic drugs and preventive drugs, and developing preventive methods.
  • the thrombosis that develops in the non-human model animal at least one of platelet thrombus and fibrin thrombus is formed in the blood vessel lumen.
  • a pathological condition very similar to obstructive thrombosis in humans in which a platelet thrombus is formed in advance and a fibrin thrombus is subsequently formed can be reproduced in the non-human model animal. For this reason, a model of an occlusive thrombus that is close to the natural occurrence in humans is obtained.
  • the non-human model animal may be a rabbit. Since rabbits have a lipid metabolism system and a coagulation / fibrinolysis system similar to those of humans, a model of thrombosis caused by plaque erosion can be obtained without genetic modification. In addition, since rabbits are cheaper and easier to breed than monkeys and the like, it is easy to use non-human model animals for screening of test substances.
  • thrombosis caused by plaque erosion can be caused in a non-human animal with high probability.
  • angiotensin II is used as a pressurizing agent.
  • Angiotensin II is a biologically active substance derived from a living body and has high compatibility with the living body, and therefore can cause thrombosis caused by plaque erosion while avoiding side effects.
  • the non-human animal may be a rabbit. Rabbits can develop thrombosis caused by plaque erosion without genetic modification or the like because the lipid metabolism system and coagulation / fibrinolysis system are close to those of humans as described above. By doing in this way, the non-human model animal which develops the thrombosis resulting from plaque erosion can be established easily and quickly.
  • the screening method for an antithrombotic drug in the present embodiment since the therapeutic effect of a test substance against thrombosis caused by plaque erosion can be confirmed, a test substance having a high therapeutic effect against human thrombosis is obtained. You can select more reliably.
  • Example 1 Production of obstructive thrombosis model 1
  • a high-cholesterol diet (LRC4 + 2% cholesterol + 6% peanut oil) containing 9 g Japanese white rabbits weighing 2.5-3.0 kg and 92 g LRC4 powder, 2 g cholesterol, and 6 g peanut oil Additive feed, Oriental Yeast Co., Ltd.) was administered over 8 weeks.
  • angiotensin II administration of angiotensin II was started.
  • Angiotensin II Angiotensin II human; manufactured by Sigma-Aldrich
  • Angiotensin II human was administered at a concentration of 50 ng / kg / min by an osmotic pump (Alzet® osmotic mini pump) implanted subcutaneously in the back of the rabbit. 0.9% physiological saline was used as a solvent for angiotensin II.
  • Angiotensin II administration was continued for 4 weeks after the endothelial injury by the balloon catheter. Six weeks after endothelial injury with the balloon catheter, the rabbits were euthanized after intravenous injection of heparin.
  • Blood samples from bilateral iliac arteries to femoral arteries of euthanized rabbits were collected.
  • the blood vessel sample was cut into a ring shape at intervals of 3 mm, and pathological analysis (henatoxylin-eosin staining; HE staining and elastica-Wangieson staining; EVG staining) was performed.
  • FIG. 1 is an angiographic image of a rabbit before euthanasia. From the arrow A in the figure toward the end side (arrow B), the blood vessel originally contrasted is not contrasted. This confirmed the occlusion of the blood vessel at this site.
  • FIG. 2 is a photograph of a blood vessel sample obtained by cutting a femoral artery having an obstruction into a ring shape.
  • An arrow A and an arrow B in FIG. 2 correspond to the arrow A and the arrow B in FIG. 1, respectively. It was confirmed that the part not imaged by angiography was actually occluded by a dark thrombus.
  • FIG. 3 shows the results of HE staining and EVG staining of the cut blood vessel sample.
  • stained images of blood vessels from the central end side to the distal end side are arranged from top to bottom.
  • the blood vessel was vividly stained red.
  • the EVG staining the inside of the blood vessel was stained yellow. Thereby, vascular occlusion due to fibrin thrombus was confirmed as a pathological finding. Such a result was obtained in 2 out of 7 animals with femoral artery occlusion.
  • FIG. 4 is an angiogram of the rabbit before euthanasia which is different from FIG. Also in this rabbit, occlusion in a part of the blood vessel was confirmed from the arrow A to the terminal side (arrow B) in the figure.
  • collected from the rabbit of FIG. 4 in ring shape is shown in FIG. Arrows A and B in FIG. 5 correspond to arrows A and B in FIG. 4, respectively. It was confirmed that the part not imaged by angiography was actually occluded by a whitish thrombus.
  • FIG. 6 shows the results of HE staining and EVG staining of the cut blood vessel sample.
  • HE staining blood vessels were stained red and blue-violet areas were scattered.
  • EVG staining blood vessels were stained red and blue-violet areas were scattered.
  • vascular occlusion due to organized platelet thrombus was confirmed as a pathological finding.
  • Such a result was obtained in 5 out of 7 animals with femoral artery occlusion.
  • the neointima of the blood vessel wall was markedly thickened. Inflammatory cell infiltration was observed in the neointima and media. Furthermore, modification of the outer elastic plate was observed. On the other hand, some lipid deposits and cholesterol crystal deposits were observed, but no obvious lipid core and thin fibrous capsule, which are characteristic of plaque failure, were observed. From this, it is considered that the obstructive thrombus formed in the blood vessel of the rabbit obtained in this experiment is not caused by the breakdown of the plaque but is caused by the plaque erosion.
  • Example 2 Feasibility test
  • n 8 (16 femoral arteries collected).
  • Example 3 Production 2 of obstructive thrombosis model
  • a high-cholesterol diet (LRC 4 + 2% cholesterol + 6% peanut oil-added feed, Oriental Yeast Co., Ltd.) was administered to four Japanese white rabbits weighing 2.5-3.0 kg over 10 weeks. Thereafter, endothelial injury was performed in the same manner as in Example 1, and administration of angiotensin II was started. Administration of angiotensin II continued for 8 weeks. In the administration of angiotensin II, the osmotic pump was replaced 4 weeks after the endothelial injury by the balloon catheter.
  • body surface tube echo used Vevo 2100 of Visual sonics.
  • the probe in the body surface echo was MS550D, and the condition was 40 mHz.
  • the systolic blood flow velocity (PSV) and waveform change of the femoral artery peripheral side, the femoral artery stenosis, the saphenous artery, and the inferior arteries were observed using the pulse Doppler method (25 Mhz).
  • FIG. 8 shows an image of the blood vessel lumen in the vicinity of the peripheral part of the femoral artery obtained by body surface tube echo. In the artery indicated by the wavy line in the image, occlusion of the vascular lumen was observed.
  • FIG. 9 is an angiogram of a rabbit whose occlusion of the blood vessel lumen was confirmed by body surface tube echo. Between the arrow A and the arrow B in the figure, the blood vessel originally contrasted is not imaged. This confirmed the occlusion of the blood vessel at this site.
  • FIG. 10 shows the results of HE staining and EVG staining of a blood vessel sample.
  • HE staining most of the blood vessels were stained red, but scattered regions of blue-violet were observed.
  • EVG staining most of the blood vessels were stained yellow, but scattered regions of red were observed.
  • thickening of the neointima and infiltration of scattered inflammatory cells were observed.
  • almost no modification of the outer elastic plate was observed.
  • the occluded portion of the blood vessel was blocked by a thrombus from the central end to the peripheral end, but continuity between the thrombus and the lipid on the blood vessel wall was not observed. From this, it is considered that the obstructive thrombus formed in the blood vessel of the rabbit obtained in this experiment is not caused by the breakdown of the plaque but is caused by the plaque erosion.
  • Example 1 and Example 3 above no obvious lipid core and thin fibrous capsule were observed in any blood vessel sample in pathological findings in pathological analysis.
  • Obvious lipid cores and thin fibrous caps are characteristic of obstructive thrombi due to plaque rupture and are rarely seen in plaque erosion.
  • vascular occlusion due to platelet thrombus was confirmed in most rabbits 6 weeks after endothelial injury.
  • vascular occlusion due to fibrin thrombus was confirmed in all rabbits 7-8 weeks after endothelial injury.
  • the non-human animal model obtained in each of the above examples has a pathological condition in which platelet thrombosis, which is a process of obstructive thrombosis reported in humans, is first formed, followed by fibrin thrombosis. It is reproduced.
  • Example 4 Evaluation of incidence of atherothrombotic occlusion
  • FIG. 11 shows the change over time in the proportion of rabbits that survived without occlusion.
  • rabbits in the angiotensin II non-administered group no obstruction was observed except for one animal over 10 weeks.
  • rabbits with obstruction appeared from the 4th week, and obstruction was observed in all rabbits by 9 weeks.
  • FIG. 12 shows the result of HE staining for the collected blood vessel sample.
  • the vascular lumen was occluded by a thrombus in the angiotensin II administered group. Since there was no significant difference between the two groups regarding blood pressure and heart rate, the formation of atherothrombotic occlusion was not due to changes in blood pressure or heart rate. From this result, it was shown that atherothrombotic occlusion can be spontaneously generated by continuously administering the pressor angiotensin II.
  • FIG. 13 shows the results of HE staining and EVG staining of the femoral artery with atherothrombotic occlusion collected from the angiotensin II administration group. A white thrombus region and a red thrombus region were observed in the blood vessel.
  • the sample was boiled with citrate buffer for 20 minutes for antigen activation, blocked with 3% skim milk, and then the primary antibody was allowed to adhere overnight at 4 ° C.
  • the primary antibodies used were PECAM-1 (Santa Cruz, sc-1506, dilution factor 1:50), ⁇ SMA (Sigma, A2547, dilution factor 1: 100), SM1 (Yamasa Shoyu Co., Ltd., 7600, dilution factor).
  • Normal mouse IgG (Santa Cruz, sc-2025) and normal sheep IgG (Santa Cruz, sc-2717) were used as negative controls for the primary antibody.
  • a Histofine SAB-PO (M) kit (Nichirei Bioscience, 422402) and rabbit anti-sheep IgG (Zymed TM, Invitrogen, 61-8640) were used.
  • a DAB buffer tablet (Merck, 102924) was used for color development.
  • FIG. 15 compares the results of immunohistochemical analysis for an artery with occlusion and an artery without occlusion.
  • ⁇ SMA positive, SM1 negative, SM2 negative smooth muscle-like cells were found specifically in occluded arteries.
  • SMEMA and calponin which are differentiation markers for dedifferentiated smooth muscle cells, were not expressed in ⁇ SMA-positive, SM1-negative, SM2-negative smooth muscle-like cells found at the intima / thrombosis interface (FIG. 16). ).
  • Example 5 Efficacy evaluation in obstructive thrombosis model
  • Rabbits treated in the same manner as in Example 3 were administered orally with 20 mg / day of acetylsalicylic acid from the week before endothelial injury as a drug administration group, and dabigatran was orally administered at 10 mg / kg / day from the time of endothelial injury. Administered.
  • FIG. 17 shows the change over time in the proportion of rabbits that survived without occlusion.
  • the drug administration group no obstruction was observed for 10 weeks.
  • Tissue staining also showed that the administration of acetylsalicylic acid and dabigatran suppressed the occurrence of atherothrombotic occlusion (FIG. 18).
  • the present invention is suitable for a non-human animal model of thrombosis caused by plaque erosion.
  • elucidation of the pathological condition of thrombosis caused by plaque erosion and research and development of a therapeutic agent for thrombosis, etc. are promoted.

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Abstract

La présente invention concerne un animal modèle non humain obtenu par une étape destinée à léser une portion de l'enthothélium d'une artère d'un animal non humain qui a ingéré un aliment à haute teneur en cholestérol et une étape d'administration continue à l'animal non humain d'angiotensine II, l'animal développant une thrombose provoquée par l'érosion de plaque. La thrombose dans l'animal modèle non humain est occlusive, occluant l'espace intravasculaire. En outre, la thrombose dans l'animal modèle non humain présente un état pathologique dans lequel une thrombose plaquettaire et/ou un thrombus de fibrine se forme dans l'espace intravasculaire.
PCT/JP2013/065741 2012-06-06 2013-06-06 Animal modèle non humain, procédé de création d'un animal modèle non humain et procédé de criblage d'un médicament antithrombogénique WO2013183725A1 (fr)

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JP2014520051A JPWO2013183725A1 (ja) 2012-06-06 2013-06-06 非ヒトモデル動物、非ヒトモデル動物の作製方法及び抗血栓薬のスクリーニング方法

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CN110313445A (zh) * 2019-08-11 2019-10-11 江苏珂玛麒生物科技有限公司 一种冠状动脉硬化性心脏病的动物模型、构建方法、及应用
CN115362987A (zh) * 2022-10-26 2022-11-22 首都医科大学宣武医院 血栓制备方法及其应用

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Publication number Priority date Publication date Assignee Title
CN110313445A (zh) * 2019-08-11 2019-10-11 江苏珂玛麒生物科技有限公司 一种冠状动脉硬化性心脏病的动物模型、构建方法、及应用
CN115362987A (zh) * 2022-10-26 2022-11-22 首都医科大学宣武医院 血栓制备方法及其应用

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