JPWO2013183725A1 - Non-human model animal, method for producing non-human model animal, and screening method for antithrombotic drug - Google Patents
Non-human model animal, method for producing non-human model animal, and screening method for antithrombotic drug Download PDFInfo
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Abstract
高コレステロール食を摂餌した非ヒト動物の動脈の内皮の一部を傷害する工程と、当該非ヒト動物にアンギオテンシンIIを持続投与する工程とによって得られる非ヒトモデル動物は、プラークびらんに起因する血栓症を発症する。当該非ヒトモデル動物における血栓症は、血管内腔を閉塞する閉塞性である。また、当該非ヒトモデル動物における血栓症は、血小板血栓及びフィブリン血栓の少なくとも一方が血管内腔に形成される病態を示す。The non-human model animal obtained by damaging a part of the endothelium of an artery of a non-human animal fed with a high cholesterol diet and the step of continuously administering angiotensin II to the non-human animal is caused by plaque erosion. Develops thrombosis. Thrombosis in the non-human model animal is occlusive, which occludes the blood vessel lumen. The thrombosis in the non-human animal model indicates a pathological condition in which at least one of platelet thrombus and fibrin thrombus is formed in the blood vessel lumen.
Description
本発明は、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に関する。 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.
日本では、心筋梗塞、脳卒中、末梢動脈疾患等の動脈硬化性疾患の罹患率が増加している。これらの疾患の多くは、動脈硬化巣（プラーク）を素地とした血栓症により発症し、患者の生活の質の低下をもたらし、死因及び寝たきりの主たる原因となっている。 In Japan, the prevalence of arteriosclerotic diseases such as myocardial infarction, stroke and peripheral arterial disease is increasing. 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.
動脈硬化性疾患の中でも、急性心筋梗塞症は、患者の生命予後を脅かす最も重篤な疾患である。世界中で年間７００万人が急性心筋梗塞症を含む冠動脈疾患で死亡しており、死因の第１位である（非特許文献１参照）。急性心筋梗塞症の超急性期（発症６−８時間以内）に対する再灌流法の普及及び内科的治療（β遮断薬、ＡＣＥ阻害剤、スタチン等）の標準化により、急性心筋梗塞症の急性期（発症１ヶ月以内）の生命予後は改善している。 Among arteriosclerotic diseases, acute myocardial infarction is the most serious disease that threatens the patient's prognosis. Around the world, 7 million people die from coronary artery disease including acute myocardial infarction annually, and is the leading cause of death (see Non-Patent Document 1). With the spread of reperfusion for the hyperacute phase (within 6-8 hours of onset) of acute myocardial infarction and the standardization of medical treatment (beta blockers, ACE inhibitors, statins, etc.), the acute phase of acute myocardial infarction ( Life prognosis within 1 month of onset has improved.
急性心筋梗塞症を発症した後の生存者が増加する一方で、急性心筋梗塞症を発症した後の心不全等に対する慢性期診療体制の整備が必要となっている。例えば、長期間でみたときの急性心筋梗塞症による死亡率は依然として高いままであり、現行の医療で最善を尽くしても急性心筋梗塞症の患者の長期予後は改善されないという問題がある。急性心筋梗塞症は、冠動脈の不安定プラークに形成される閉塞性血栓に起因することが知られており（非特許文献２参照）、急性心筋梗塞症の発生機序をさらに解明することは、適切な診断、予防又は治療等の対策となりうる。 While the number of survivors after developing acute myocardial infarction is increasing, it is necessary to develop a chronic care system for heart failure after developing acute myocardial infarction. For example, the mortality rate due to acute myocardial infarction when viewed over a long period of time remains high, and the long-term prognosis of patients with acute myocardial infarction is not improved even if the best is done with current medical care. 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.
ここに、不安定プラークは、プラークの破綻及びプラークびらんの２つのタイプに分類される。プラークの破綻は、薄い線維性被膜、強い炎症細胞の浸潤、脂質コアを特徴とする。プラークの破綻では、線維性被膜の破綻が血管内膜の剥離、血管内膜下のコラーゲンや組織因子による血中の凝固因子の活性化及び血小板凝集を惹起し、閉塞性血栓が形成されると考えられている（非特許文献３参照）。また、プラークびらんは、血管内膜の剥離を特徴とする。プラークびらんの病理学的特徴は、線維性被膜の破綻がない、新生内膜が主に平滑筋細胞やプロテオグリカンによって形成されている、炎症細胞の浸潤が上記プラークの破綻に比べて少ない等の点でプラークの破綻と相異する（非特許文献４参照）。 Here, unstable plaque is classified into two types: plaque failure and plaque erosion. Plaque failure is characterized by a thin fibrous cap, strong inflammatory cell infiltration, and a lipid core. In 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).
病態の発生機序を解明する方法として動物モデルを使用する方法がある。最近、ＡｐｏＥ欠損マウス等の高コレステロール血症マウスの腕頭動脈において、血栓は形成されないものの、薄い線維性被膜、炎症細胞の浸潤、脂質コア等のヒトの不安定プラーク及びその破綻に類似した病理像が得られたことが報告された（非特許文献５参照）。また、高コレステロール食を負荷したウサギの大動脈をバルーンカテーテルによって傷害し、自己赤血球を投与すると脂質コアと炎症が惹起され、不安定プラークが生じるとする報告もある（非特許文献６参照）。また、血栓が形成されるモデル動物として、Ｎｉｅｍａｎｎ−Ｐｉｃｋ Ｃ１及びＡｐｏＥの双方を欠損したマウスが知られている。このマウスでは、大動脈弁付近の上行大動脈に中膜の変性及び破壊と外膜の炎症とを伴う非閉塞性血栓が形成されることが報告されている（非特許文献７参照）。さらに、機械的刺激や薬物負荷によって急性血栓が誘発される動物モデルも報告されている（特許文献１及び非特許文献８参照）。 There is a method of using an animal model as a method for elucidating the pathogenesis of a disease state. Recently, in the brachiocephalic artery of hypercholesterolemic mice such as ApoE-deficient mice, although a thrombus is not formed, pathology similar to human unstable plaque such as thin fibrous capsule, infiltration of inflammatory cells, lipid core, and its failure It was reported that an image was obtained (see Non-Patent Document 5). In addition, there is a report that when a rabbit's aorta loaded with a high cholesterol diet is injured with a balloon catheter and administered with autologous red blood cells, lipid cores and inflammation are induced, resulting in unstable plaque (see Non-Patent Document 6). As a model animal in which a thrombus is formed, a mouse deficient in both Niemann-Pick C1 and ApoE is known. In this mouse, it has been reported that 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). Furthermore, 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).
しかしながら、不安定プラークの表面に血栓が形成されるモデル動物は未だ存在しない。不安定プラークのうち、プラークびらんは、急性心筋梗塞患者の２０〜２５％、心臓突然死患者の３０〜３５％の責任病変であるとされている。このため、プラークびらんに起因する血栓の形成機序の解明に有用な非ヒトモデル動物が臨床的にも求められている。 However, there is still no model animal in which a thrombus is formed on the surface of unstable plaque. Of vulnerable plaque, plaque erosion is said to be responsible for 20-25% of patients with acute myocardial infarction and 30-35% of patients with sudden cardiac death. For this reason, a nonhuman model animal useful for elucidating the formation mechanism of a thrombus caused by plaque erosion is also clinically required.
本発明は、上記実情に鑑みてなされたものであり、プラークびらんに起因する血栓症のモデルとして有用な非ヒトモデル動物、当該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を提供することを目的とする。 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.
本発明の第１の観点に係る非ヒトモデル動物は、
プラークびらんに起因する血栓症を発症する。The non-human model animal according to the first aspect of the present invention is:
It develops thrombosis due to plaque erosion.
この場合、前記血栓症は、
血管内腔を閉塞する閉塞性である、
こととしてもよい。In this case, the thrombosis is
Is occlusive to occlude the lumen of the blood vessel,
It is good as well.
また、前記血栓症は、
血小板血栓及びフィブリン血栓の少なくとも一方が血管内腔に形成される、
こととしてもよい。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.
また、上記非ヒトモデル動物は、
ウサギである、
こととしてもよい。The non-human model animal is
A rabbit,
It is good as well.
本発明の第２の観点に係るプラークびらんに起因する血栓症を発症する非ヒトモデル動物の作製方法は、
高コレステロール食を摂餌した非ヒト動物の動脈の内皮の一部を傷害する工程と、
前記非ヒト動物に昇圧剤を持続投与する工程と、
を含む。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.
この場合、前記昇圧剤は、
アンギオテンシンIIである、
こととしてもよい。In this case, the pressor is
Angiotensin II,
It is good as well.
また、上記非ヒト動物は、
ウサギである、
こととしてもよい。The non-human animal is
A rabbit,
It is good as well.
本発明の第３の観点に係る抗血栓薬のスクリーニング方法は、
上記本発明の第１の観点に係る非ヒトモデル動物を用いる。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.
この場合、抗血栓症薬のスクリーニング方法は、
前記非ヒトモデル動物に被験物質を投与する工程と、
前記被験物質を投与された前記非ヒトモデル動物と前記被験物質を投与されなかった前記非ヒトモデル動物との間で、血栓症に関連するマーカーを比較する工程と、
を含む、
こととしてもよい。In this case, 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.
本発明によれば、プラークびらんに特徴的な病変を呈する血栓が非ヒト動物に形成される。このため、プラークびらんに起因する血栓症のモデルとして有用な非ヒトモデル動物が得られる。 According to the present invention, 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.
本発明の実施の形態に係る非ヒトモデル動物は、プラークびらんに起因する血栓症を発症する。プラークとは、動脈における肥厚し硬化した部位、いわゆる動脈硬化巣に存在する内膜の斑状肥厚性病変である。プラークは、中心に粥腫を含み、線維性被膜によって被覆されている。プラークは、その内部にコレステロールエステルからなる脂質コアを有することが多い。プラークを覆う線維性被膜が、脂質コアに達しない傷害によって破綻する病変を、プラークびらんという。プラークびらんには、明らかな脂質コアや薄い線維性被膜がほとんどなく、炎症細胞の浸潤が乏しい。また、プラークびらんは、平滑筋細胞とプロテオグリカンに富むプラークに多く認められる。これに対し、プラークの破綻には、明らかな脂質コア、炎症細胞の浸潤が強い、薄い線維性被膜がある、線維性被膜内の平滑筋細胞が少ない等の特徴がある。 The non-human model animal according to the embodiment of the present invention 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.
血栓症は、血管内腔に血栓が形成される病態であるが、広くは、形成された血栓に関連する病態をも含む。血栓に関連する病態は、例えば、血栓が形成されたことによる虚血や梗塞、動脈硬化、心筋梗塞、狭心症、脳血栓等である。 Thrombosis is a condition in which a thrombus is formed in a blood vessel lumen, 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.
本実施の形態に係る非ヒトモデル動物における血栓症は、血管内腔を閉塞する閉塞性であってもよい。閉塞性の血栓症の場合、例えば、上記非ヒトモデル動物の血管内腔の面積の７５％以上、より好ましくは９０％以上、さらに好ましくは９５％以上が血栓で占められている。血管内腔の面積に占める血栓の割合は、体表管エコーにより得られる血管切断面の画像に基づいて算出することもできる。また、血管サンプルの病理学的解析により、血管内腔の面積に占める血栓の割合を把握することもできる。 The thrombosis in the non-human model animal according to the present embodiment may be occlusive that obstructs the blood vessel lumen. In the case of 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. Moreover, 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.
本実施の形態に係る非ヒトモデル動物における血栓症は、血小板血栓及びフィブリン血栓の少なくとも一方が血管内腔に形成される。血小板血栓は、白色血栓ともいい、その形成機序に血小板が大きく関与している。フィブリン血栓は、赤色血栓ともいい、主に血液凝固反応によって形成される。ヒトにおける閉塞性血栓症では、血小板血栓が最初に生じて、引き続き、フィブリン血栓によって血管が閉塞されることが知られている。下記実施例において、上記非ヒトモデル動物における血栓症では、血小板血栓が生じた後にフィブリン血栓によって血管が閉塞されることが示唆されている。このため、上記非ヒトモデル動物は、ヒトにおける閉塞性血栓症の経緯を再現している点で、ヒトで自然発生する閉塞性血栓症のモデルとしても有用である。 In the thrombosis in the non-human model animal according to the present embodiment, 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. In occlusive thrombosis in humans, it is known that platelet thrombosis occurs first, followed by occlusion of blood vessels by fibrin thrombosis. In the following examples, it is suggested that in the thrombosis in the non-human model animal, blood vessels are occluded by fibrin thrombus after platelet thrombus occurs. For this reason, 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.
次に、本実施の形態に係る非ヒトモデル動物の作製方法を説明する。上記非ヒトモデル動物の作製方法は、高コレステロール食を摂餌した非ヒト動物の動脈の内皮の一部を傷害する工程と、当該非ヒト動物に昇圧剤を持続投与する工程とを含む。 Next, a method for producing a non-human model animal according to the present embodiment will be described. 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.
非ヒト動物は、ヒトを除く動物であれば特に限定されず、実験動物として用いられる哺乳類動物、鳥類等を用いてもよい。例えば、非ヒト動物として、マウス、ラット、ハムスター、ウサギ、イヌ、ブタ、サル、ニワトリ等を用いることができる。ヒトにおけるプラークびらんに起因する血栓症により近い病態を再現するために、ゲノムがヒトに近いサル等を非ヒト動物として用いるのが好ましい。また、脂質代謝系及び凝固線溶系がヒトに近い非ヒト動物を用いるのがより好ましい。脂質代謝系及び凝固線溶系がヒトに近い非ヒト動物は、例えば、ウサギである。ウサギは、飼育が比較的容易であるという点で非ヒト動物として適している。ウサギの種類は、特に限定されないが、例えば、日本白色種ウサギが挙げられる。 The non-human animal is not particularly limited as long as it is an animal excluding humans, and mammals and birds used as laboratory animals may be used. For example, mice, rats, hamsters, rabbits, dogs, pigs, monkeys, chickens and the like can be used as non-human animals. In order to reproduce a pathological condition closer to thrombosis caused by plaque erosion in humans, it is preferable to use monkeys and the like whose genome is close to that of humans as non-human animals. It is more preferable to use 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.
非ヒト動物としてマウス、ラット、ハムスター等を用いる場合は、凝固線溶系において凝固系が線溶系に対して相対的に強くなるように、例えば、凝固系に関連する因子の機能を強めたり、線溶系に関連する因子の機能を弱めたりしたマウス、ラット、ハムスター等を用いればよい。例えば、凝固系に関連する因子の機能を強めるためには、凝固系に関連する因子の遺伝子の発現量が増加するようにした遺伝子改変動物を用いることができる。凝固系に関連する因子の遺伝子は、凝固系を促進するフィブリン、フィブリノーゲン、プロトロンビン、トロンビン、トロンボプラスチン、第VI因子、第VII因子、第VIII因子、第IX因子、第X因子、第XI因子、第XII因子、第XIII因子の凝固因子等の遺伝子である。このような遺伝子改変動物は、遺伝子ノックイン等の公知の遺伝子操作技術によって、あらかじめ作製することができる。このとき、単一の凝固因子等の遺伝子の発現量が増加するようにしてもよいが、複数の凝固因子等の遺伝子の発現量が増加するようにすれば、凝固系が線溶系に対して相対的により強くすることができる。 When 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. For example, in order to strengthen the function of a factor related to the coagulation system, 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.
線溶系に関連する因子の機能を弱めるためには、例えば、線溶系に関連する因子の遺伝子を欠損した、又は線溶系に関連する因子の遺伝子の発現量が低下するようにした遺伝子改変動物を用いることができる。線溶系に関連する因子の遺伝子は、プラスミン、プラスミノゲン、組織型プラスミノゲン活性化因子（ｔ‐ＰＡ）及びウロキナーゼ（ｕ‐ＰＡ）等の遺伝子である。このような遺伝子改変動物は、遺伝子ノックアウト等の公知の遺伝子操作技術によって、作製することができる。なお、線溶系に関連する因子の遺伝子の発現量を低下させるために、ｓｉＲＮＡ、ｍｉＲＮＡ、アンチセンス核酸等を用いてもよい。また、線溶系に関連する因子の機能を弱めるために、線溶系に関連する因子の機能を阻害する阻害剤、抗体、ＤＮＡアプタマー又はＲＮＡアプタマー等をマウス、ラット、ハムスター等に投与してもよい。 In order to weaken the function of a factor related to the fibrinolytic system, for example, 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. Can be used. Factor genes related to the fibrinolytic system are genes such as plasmin, plasminogen, tissue-type plasminogen activator (t-PA) and urokinase (u-PA). Such a genetically modified animal can be produced by a known gene manipulation technique such as gene knockout. It should be noted that 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. In addition, in order to weaken the function of a factor related to 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. .
次に、高コレステロール食を摂餌した非ヒト動物の動脈の内皮の一部を傷害する工程について説明する。高コレステロール食は、例えば、用いる非ヒト動物に対する一般的な飼料に、コレステロールを更に添加した飼料である。高コレステロール食のコレステロール含有量は、０．５〜４％、好ましくは１〜３％、より好ましくは１．５％〜２．５％である。 Next, the process of damaging a part of the endothelium of an artery of a non-human animal that has been fed a high cholesterol diet will be described. 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%.
高コレステロール食を非ヒト動物に摂餌させる期間は、特に限定されず、例えば、使用する非ヒト動物の種類、又は高コレステロール食のコレステロール含有量に応じて、適宜選択することができる。例えば、非ヒト動物としてウサギを用いる場合には、コレステロール含有量が好ましくは０．５〜４％、好ましくは１〜３％、より好ましくは１．５％〜２．５％の高コレステロール食を１〜１０週間摂餌させることが好ましく、２〜８週間摂餌させることがより好ましい。高コレステロール食を非ヒト動物に摂餌させることによって、当該非ヒト動物における血液中の総コレステロールの値が１００〜１０００ｍｇ／ｄｌになることが好ましく、３００〜８００ｍｇ／ｄｌになることがより好ましい。なお、当該非ヒト動物における血液中の総コレステロールの値は、１０００ｍｇ／ｄｌ以上になってもよい。 The period during which a non-human animal is fed a high-cholesterol diet 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. For example, when a rabbit is used as a non-human animal, a high cholesterol diet with a cholesterol content of preferably 0.5-4%, preferably 1-3%, more preferably 1.5% -2.5%. It is preferable to feed for 1 to 10 weeks, and it is more preferable to feed for 2 to 8 weeks. By feeding a high-cholesterol diet to a non-human animal, the value of total cholesterol in the blood in the non-human animal is preferably 100 to 1000 mg / dl, and more preferably 300 to 800 mg / dl. In addition, the value of the total cholesterol in the blood in the said non-human animal may be 1000 mg / dl or more.
内皮を傷害する動脈の種類は特に限定されない。動脈の種類は、例えば、大動脈、心房動脈、肝動脈、下行動脈等である。ウサギ等の比較的大きな動物では、四肢の動脈等でもよい。より具体的には、大腿動脈末端から総腸骨動脈起始部までの内皮を傷害してもよい。 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.
内皮の傷害は、種々の方法で実施することができる。例えば、内皮を傷害する動脈の部位にバルーンカテーテルを挿入し、バルーンカテーテルに加圧することで内皮を傷害することができる。このとき、バルーンカテーテルの大きさは、傷害する部位の大きさを考慮して、適宜決定すればよい。バルーンカテーテルへの加圧は、用いるバルーンカテーテルの仕様にもよるが、例えば、１〜４ａｔｍ、より好ましくは、１〜２ａｔｍである。 Endothelial injury can be performed in various ways. For example, the endothelium can be injured by inserting a balloon catheter into an arterial site that damages the endothelium and pressurizing the balloon catheter. At this time, the size of the balloon catheter may be appropriately determined in consideration of the size of the injured site. The pressure applied to the balloon catheter is, for example, 1 to 4 atm, more preferably 1 to 2 atm, although it depends on the specifications of the balloon catheter to be used.
この他、内皮の傷害は、動脈の内皮に対して一定の物理的な負荷を加える手段を適宜選択できる。例えば、縫合糸を用いて動脈を結搾する方法等がある。 In addition, for the injury of the endothelium, means for applying a certain physical load to the endothelium of the artery can be appropriately selected. For example, there is a method of ligating an artery using a suture thread.
次に、上記非ヒト動物に昇圧剤を持続投与する工程について説明する。昇圧剤は、血圧を上昇させる作用を有する限り、化合物、抗体、ペプチド、ＤＮＡアプタマー、ＲＮＡアプタマー、ｓｉＲＮＡ、ｍｉＲＮＡ、アンチセンス核酸等のいずれでもよい。具体的には、例えば、昇圧剤としては、ノルエピネフリン、ドブタミン等が好ましく、アンギオテンシンIIがより好ましい。アンギオテンシンIIは、昇圧作用を有するポリペプチドである。昇圧剤を持続投与する方法は、特に限定されない。例えば、昇圧剤としてアンギオテンシンIIを用いる場合、アンギオテンシンII溶液を充填した浸透圧ポンプを、上記非ヒト動物の皮下に埋め込むことでアンギオテンシンIIを持続投与することができる。持続投与するアンギオテンシンIIの濃度は、用いる非ヒト動物の種類に応じて、適宜決定される。例えば、非ヒト動物としてウサギを用いた場合、アンギオテンシンIIを毎分１０〜１００ｎｇ／ｋｇ、好ましくは毎分３０〜７０ｎｇ／ｋｇ、より好ましくは毎分４０〜６０ｎｇ／ｋｇ、特に好ましくは毎分４５〜５５ｎｇ／ｋｇで投与すればよい。 Next, the process of continuously administering a pressor to the non-human animal will be described. 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. Specifically, for example, as 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. For example, when angiotensin II is 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. The concentration of angiotensin II to be continuously administered is appropriately determined according to the type of non-human animal used. For example, when a rabbit is used as the 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.
なお、上記非ヒト動物の静脈等にアンギオテンシンII溶液を定期的に静脈等に注射することにより、アンギオテンシンIIを持続投与してもよい。 Angiotensin II may be continuously administered by periodically injecting an angiotensin II solution into the vein or the like of the non-human animal.
次に、上記非ヒトモデル動物の使用方法を説明する。例えば、上記非ヒトモデル動物を用いて、抗血栓症薬のスクリーニングが可能である。例えば、当該抗血栓症薬のスクリーニング方法は、上記非ヒトモデル動物に被験物質を投与する工程と、被験物質を投与された上記非ヒトモデル動物と被験物質を投与されなかった上記非ヒトモデル動物との間で、血栓症に関連するマーカーを比較する工程とを含む。 Next, a method for using the non-human model animal will be described. For example, an antithrombotic drug can be screened using the non-human model animal. For example, 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.
上記非ヒトモデル動物への被験物質の投与では、化合物、抗体、ペプチド、ＤＮＡアプタマー、ＲＮＡアプタマー、ｓｉＲＮＡ、ｍｉＲＮＡ、アンチセンス核酸等の被験物質を、上記非ヒトモデル動物に投与する。投与の方法は、特に限定されないが、被験物質に適切な方法を選択するのが好ましい。例えば、被験物質が化合物の場合には、経口投与であってもよいし、腹腔内投与であってもよい。また、被験物質が抗体、ペプチド、ＤＮＡアプタマー、ＲＮＡアプタマー、ｓｉＲＮＡ、ｍｉＲＮＡ、アンチセンス核酸等の場合は、静脈注射、皮下注射等であってもよい。被験物質の投与は、単回でもよいし、複数回であってもよい。被験物質の濃度は、被験物質の種類によって適宜決定すればよい。また、被験物質の濃度を数段階希釈した複数の濃度の被験物質を投与してもよい。 In the administration of the test substance to the non-human model animal, 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. For example, when the test substance is a compound, it may be administered orally or intraperitoneally. Further, when 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 | concentration of a test substance suitably with the kind of test substance. Moreover, you may administer the test substance of the several density | concentration which diluted the density | concentration of the test substance several steps.
血栓症に関連するマーカーは、例えば、血管内腔に形成された血栓の有無であってもよいし、形成された血栓の大きさ等の値であってもよい。血栓の有無は、血管造影、体表管エコー、組織染色等の病理学的解析等を用いて判断できる。また、血栓の大きさ等の値は、血管造影、体表管エコー、組織染色等の病理学的解析において、画像内の血栓に相当する領域の大きさ等から算出できる。この他、血栓症に関連するマーカーは、組織中酸素分圧から得られる組織中酸素飽和度、第X因子等の凝固系に関連する因子の活性、プラスミン等の線溶系に関連する因子の活性等であってもよい。 The marker related to thrombosis may be, for example, the presence or absence of a thrombus formed in a blood vessel lumen, 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. In addition, 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. In addition, 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.
この他、上記非ヒトモデル動物の使用方法としては、血栓症、特にプラークびらんに起因する血栓症の病態観察、血栓症の診断用の各種マーカーの探索等が挙げられる。なお、上記抗血栓症薬は、血栓症を治療する薬剤、血栓症を予防する薬剤等を含む。 In addition, examples of the method of 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.
以上詳細に説明したように、本実施の形態に係る非ヒトモデル動物では、下記実施例に示すように、明らかな脂質コアや薄い線維性被膜の所見がなく、血小板血栓が生じて、それに続いてフィブリン血栓が形成される。このように、上記非ヒトモデル動物は、プラークびらんに特徴的な病変を呈する血栓症を発症しており、プラークびらんにおける血栓の形成機序を再現する。このため、本実施の形態に係る非ヒトモデル動物は、プラークびらんに起因する血栓症のモデルとして有用である。 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.
また、本実施の形態に係る非ヒトモデル動物が発症する血栓症は、血管内腔を閉塞する閉塞性であってもよい。冠動脈の不安定プラークに形成される閉塞性血栓症は、動脈硬化性疾患の中でも生命予後を脅かす最も重篤な疾患である急性心筋梗塞症の原因である。このため、本実施の形態に係る非ヒトモデル動物は、急性心筋梗塞症の発症機序の解明、治療薬及び予防薬の探索、予防法の開発に有用である。 In addition, the thrombosis that develops in the non-human model animal according to the present embodiment may be obstructive that blocks the blood vessel lumen. 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.
また、本実施の形態に係る非ヒトモデル動物が発症する血栓症は、血小板血栓及びフィブリン血栓の少なくとも一方が血管内腔に形成される。これにより、血小板血栓が先行して形成され、フィブリン血栓が続いて形成されるヒトでの閉塞性血栓症と極めて類似した病態を、当該非ヒトモデル動物において再現できる。このため、ヒトでの自然発生に近い閉塞性血栓のモデルが得られる。 In the thrombosis that develops in the non-human model animal according to the present embodiment, at least one of platelet thrombus and fibrin thrombus is formed in the blood vessel lumen. Thereby, 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.
また、本実施の形態では、非ヒトモデル動物は、ウサギであってもよいこととした。ウサギは、脂質代謝系及び凝固線溶系がヒトに近いため、遺伝子改変等を加えなくてもプラークびらんに起因する血栓症のモデルが得られる。また、ウサギは、サル等に比べて安価で、かつ飼育が容易なので、被験物質のスクリーニング等に非ヒトモデル動物を利用しやすい。 In the present embodiment, 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.
なお、本実施の形態における非ヒトモデル動物の作製方法によれば、下記実施例に示すように、高い確率で非ヒト動物にプラークびらんに起因する血栓症を発症させることができる。 In addition, according to the method for producing a non-human model animal in the present embodiment, as shown in the following examples, thrombosis caused by plaque erosion can be caused in a non-human animal with high probability.
また、本実施の形態では、昇圧剤としてアンギオテンシンIIを用いるようにした。アンギオテンシンIIは、生体由来の生理活性物質であって生体との適合性が高いため、副作用を回避しつつ、プラークびらんに起因する血栓症を発症させることができる。 In the present embodiment, angiotensin II is used as the pressor. 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.
また、本実施の形態における非ヒトモデル動物の作製方法では、非ヒト動物は、ウサギであってもよいとした。ウサギは、上述のように脂質代謝系及び凝固線溶系がヒトに近いため、遺伝子改変等を行わずに、プラークびらんに起因する血栓症を発症させることができる。このようにすることで、プラークびらんに起因する血栓症を発症する非ヒトモデル動物を容易に早く確立することができる。 In the method for producing a non-human model animal in the present embodiment, 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.
また、本実施の形態における抗血栓薬のスクリーニング方法によれば、プラークびらんに起因する血栓症に対する被験物質の治療効果を確かめられるので、ヒトの血栓症に対して高い治療効果を有する被験物質をより確実に選択することができる。 Further, according to 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.
以下の実施例により、本発明をさらに具体的に説明するが、本発明は実施例によって限定されるものではない。 The following examples further illustrate the present invention, but the present invention is not limited to the examples.
（実施例１：閉塞性血栓症モデルの作製１）
体重２．５〜３．０ｋｇの雄の９匹の日本白色種ウサギに、９２ｇのＬＲＣ４粉末と、２ｇのコレステロールと、６ｇの落花生油とを配合した高コレステロール食（ＬＲＣ４＋２％コレステロール＋６％ピーナッツ油添加飼料、オリエンタル酵母工業株式会社）を８週間に渡って投与した。高コレステロール食の投与開始２週間後に、拡張時の直径が３×１５ｍｍのバルーンカテーテルを用いて、１〜２ａｔｍの加圧によって両側大腿動脈末端から総腸骨動脈起始部に対して内皮傷害を実施した。これと同時にアンギオテンシンIIの投与を開始した。アンギオテンシンII（Ａｎｇｉｏｔｅｎｓｉｎ II ｈｕｍａｎ；シグマ‐アルドリッチ社製）は、ウサギ背面皮下に埋め込んだ浸透圧ポンプ（Ａｌｚｅｔ（登録商標） ｏｓｍｏｔｉｃ ｍｉｎｉ ｐｕｍｐ）によって５０ｎｇ／ｋｇ／ｍｉｎの濃度で投与した。アンギオテンシンIIの溶媒には、０．９％生理食塩水を用いた。アンギオテンシンIIの投与は、バルーンカテーテルによる内皮傷害から４週間継続した。
バルーンカテーテルによる内皮傷害の６週間後、ウサギにヘパリンを静脈注射した後にウサギを安楽死させた。安楽死させたウサギの両側腸骨動脈から大腿動脈までの血管サンプルを採取した。血管サンプルを３ｍｍ間隔で輪状に切断し、病理学的解析（ヘナトキシリン‐エオジン染色；ＨＥ染色及びエラスチカ‐ワンギーソン染色；ＥＶＧ染色）を行った。(Example 1: Production of obstructive thrombosis model 1)
A high-cholesterol diet (LRC4 + 2% cholesterol + 6% peanut oil) containing nine Japanese white rabbits weighing 2.5-3.0 kg and 92 g of LRC4 powder, 2 g of cholesterol, and 6 g of peanut oil Additive feed, Oriental Yeast Co., Ltd.) was administered over 8 weeks. Two weeks after the start of administration of the high-cholesterol diet, endothelial injury was caused from the bilateral femoral artery to the common iliac artery origin by pressurizing 1 to 2 atm using a balloon catheter with a diameter of 3 × 15 mm when dilated. Carried out. At the same time, administration of angiotensin II was started. Angiotensin II (manufactured by Sigma-Aldrich) 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.
（結果）
安楽死前の血管造影において、実験に用いた９匹のウサギのうち７匹、のべ下肢１８本中９本に大腿動脈の閉塞を認めた。図１は、安楽死前のウサギの血管造影画像の図である。図中の矢印Ａから末端側（矢印Ｂ）に向かって、本来造影される血管が造影されていない。このことから、この部位における血管の閉塞を確認した。(result)
In angiography before euthanasia, femoral artery occlusion was observed in 7 out of 9 rabbits used in the experiment and 9 out of 18 lower limbs. 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.
図２は、閉塞を有する大腿動脈を輪状に切断した血管サンプルの写真である。図２中の矢印Ａ及び矢印Ｂは、図１中の矢印Ａ及び矢印Ｂにそれぞれ対応する。血管造影によって造影されなかった部位が実際に黒っぽい血栓で閉塞されていることを確認した。 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.
図３は、前記切断した血管サンプルに対するＨＥ染色及びＥＶＧ染色の結果を示す。図３では、上から下に向かって中枢端側から末端側の血管の染色画像が配置されている。ＨＥ染色では、鮮明に血管内が赤色に染まった。また、ＥＶＧ染色では、鮮明に血管内が黄色に染まった。これにより、病理所見としてフィブリン血栓による血管閉塞を確認した。このような結果は、大腿動脈の閉塞を認めた７匹のうち２匹において得られた。 FIG. 3 shows the results of HE staining and EVG staining of the cut blood vessel sample. In FIG. 3, stained images of blood vessels from the central end side to the distal end side are arranged from top to bottom. In the HE staining, the blood vessel was vividly stained red. Further, in 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.
図４は、図１と異なるウサギの安楽死前の血管造影図である。このウサギにおいても、図中の矢印Ａから末端側（矢印Ｂ）に向かって、血管の一部における閉塞を確認した。図４のウサギから採取した血管サンプルを輪状に切断した写真を図５に示す。図５中の矢印Ａ及び矢印Ｂは、図４中の矢印Ａ及び矢印Ｂにそれぞれ対応する。血管造影によって造影されなかった部位が実際に白っぽい血栓で閉塞されていることを確認した。 FIG. 4 is an angiogram of the rabbit before euthanasia 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. The photograph which cut | disconnected the blood vessel sample extract | 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.
図６は、前記切断した血管サンプルに対するＨＥ染色及びＥＶＧ染色の結果を示す。ＨＥ染色では、血管内が赤色に染まり、青紫色に染まった領域が散見された。また、ＥＶＧ染色においても、血管内が赤色に染まり、青紫色に染まった領域が散見された。これにより、病理所見として器質化した血小板血栓による血管閉塞を確認した。このような結果は、大腿動脈の閉塞を認めた７匹のうち５匹において得られた。 FIG. 6 shows the results of HE staining and EVG staining of the cut blood vessel sample. In the HE staining, blood vessels were stained red and blue-violet areas were scattered. Also in EVG staining, blood vessels were stained red and blue-violet areas were scattered. Thereby, 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.
本実験において閉塞した血管の病理所見では、血管壁の新生内膜が著明に肥厚していた。また、炎症細胞の浸潤が新生内膜、中膜に認められた。さらに、外弾性板の変性が認められた。一方、一部に脂質の沈着やコレステロールクリスタルの沈着を認めたが、プラークの破綻の特徴である明らかな脂質コア及び薄い線維性被膜は認められなかった。このことから、本実験で得られたウサギの血管に形成された閉塞性血栓は、プラークの破綻に起因するものではなく、プラークびらんに起因するものであると考えられる。 In the pathological findings of the obstructed blood vessels in this experiment, 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.
（実施例２：実行可能性試験）
血栓性閉塞の発生率を評価するために、上記実施例１と同様の実験をウサギの匹数ｎ＝８（１６本の大腿動脈を採取）で行った。なお、本実験では、アンギオテンシンIIを投与しない点のみが異なるアンギオテンシンII非投与群（ｎ＝４、８本の大腿動脈を採取）をさらに加えた。(Example 2: Feasibility test)
In order to evaluate the incidence of thrombotic occlusion, the same experiment as in Example 1 was performed with the number of rabbits n = 8 (16 femoral arteries collected). In this experiment, an angiotensin II non-administration group (n = 4, 8 femoral arteries were collected), which differed only in that angiotensin II was not administered, was further added.
（結果）
図７は、ウサギの大腿動脈における血栓性閉塞の発生率を示す。アンギオテンシンII非投与群のウサギでは、血栓性閉塞が発生した大腿動脈はなかった。一方、アンギオテンシンII投与群のウサギでは、３１％の大腿動脈（ｎ＝５）でフィブリン血栓（赤色血栓）と血小板血栓（白色血栓）が混在した新鮮な血栓がみられた。また、アンギオテンシンII投与群のウサギでは、３１％の大腿動脈（ｎ＝５）で器質化した血栓がみられた。すなわち、アンギオテンシンII投与群のウサギでは、６２％の大腿動脈で血栓性閉塞が発生していた。両群の間には、有意な差が認められた（ｐ＝０．００２９）。
この結果から、昇圧剤アンギオテンシンIIを持続投与することで、プラークびらんに起因する血栓性閉塞の発生が促されることが示された。(result)
FIG. 7 shows the incidence of thrombotic occlusions in rabbit femoral arteries. None of the rabbits in the angiotensin II non-administered group had a femoral artery with thrombotic occlusion. On the other hand, in the rabbits in the angiotensin II administration group, 31% of femoral arteries (n = 5) showed fresh thrombus mixed with fibrin thrombus (red thrombus) and platelet thrombus (white thrombus). In rabbits in the angiotensin II administration group, 31% of femoral arteries (n = 5) showed organized thrombus. That is, thrombotic occlusion occurred in 62% of femoral arteries in rabbits in the angiotensin II administration group. There was a significant difference between the two groups (p = 0.629).
From these results, it was shown that the continuous administration of the pressor angiotensin II promotes the occurrence of thrombotic occlusion caused by plaque erosion.
（実施例３：閉塞性血栓症モデルの作製２）
体重２．５〜３．０ｋｇの雄の４匹の日本白色種ウサギに高コレステロール食（ＬＲＣ４＋２％コレステロール＋６％ピーナッツ油添加飼料、オリエンタル酵母工業株式会社）を１０週間に渡って投与した。以下、上記実施例１と同様に、内皮傷害を実施し、アンギオテンシンIIの投与を開始した。アンギオテンシンIIの投与は８週間継続した。なお、アンギオテンシンIIの投与においては、バルーンカテーテルによる内皮傷害から４週間後に浸透圧ポンプを入れ替えた。
バルーンカテーテルによる内皮傷害後より、体表管エコーを用いて大腿動脈における血管の閉塞を週に３回観察した。体表管エコーは、Ｖｉｓｕａｌ ｓｏｎｉｃｓ社のＶｅｖｏ２１００を使用した。体表管エコーにおけるプローブはＭＳ５５０Ｄで、４０ｍＨｚを条件とした。
また、パルスドップラー法（２５Ｍｈｚ）を用いて大腿動脈末梢側、大腿動脈狭窄部、伏在動脈、膝下動脈の収縮期最高血流速度（ＰＳＶ）及び波形変化を観察した。パルスドップラー法における観察において、血流の消失を認めた場合、０．１ｍｇ／ｍｌの濃度で２ｍｌの硝酸イソソルビドを静脈に注射することによって血管攣縮を抑制した。さらに、最初に観察された血流の消失後２４時間以内に再度、同じ部位において血流の消失を認めた場合、血管閉塞と判断した。血管閉塞を発症したウサギについては、Ｏｘｙｌａｂ ｐＯ２（登録商標、Ｏｘｆｏｒｄ Ｏｐｔｒｏｎｉｘ社）を用いて、両下肢の組織中酸素分圧を測定した。さらに血管閉塞を発症したウサギについては、０．１ｍｇ／ｍｌ硝酸イソソルビド添加生理食塩水１００ｍｌ及び固定液（９５％エタノールと５％酢酸）１００ｍｌを１００ｍｍＨｇで還流し、腸骨動脈から大腿動脈までの血管サンプルを採取した。採取した血管サンプルに対して、長軸像の病理学的解析を行った。(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.
After endothelium injury by the balloon catheter, blood vessel occlusion in the femoral artery was observed three times a week using body surface tube echo. The 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.
Moreover, 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). In the observation by the pulse Doppler method, when disappearance of blood flow was observed, vasospasm was suppressed by injecting 2 ml of isosorbide nitrate at a concentration of 0.1 mg / ml into the vein. Furthermore, when the disappearance of the blood flow was observed again at the same site within 24 hours after the disappearance of the first observed blood flow, it was determined that the blood vessel was occluded. For rabbits that developed vascular occlusion, the partial pressure of oxygen in the tissues of both lower limbs was measured using Oxylab pO2 (registered trademark, Oxford Optronix). Furthermore, for rabbits that developed vascular occlusion, 100 ml of 0.1 mg / ml physiological saline supplemented with isosorbide nitrate and 100 ml of fixative (95% ethanol and 5% acetic acid) was refluxed at 100 mmHg, and blood vessels from the iliac artery to the femoral artery A sample was taken. A pathological analysis of the long axis image was performed on the collected blood vessel sample.
（結果）
バルーンカテーテルによる内皮傷害後、実験に用いた４匹のウサギ全てについて、７週目までに体表管エコーによる観察において大腿動脈末梢部付近で血管の閉塞を認めた。図８は、体表管エコーにより得られた大腿動脈末梢部付近の血管内腔の画像を示す。画像中の波線で示す動脈において、血管内腔の閉塞を認めた。(result)
After endothelium injury by the balloon catheter, all four rabbits used in the experiment had occlusion of blood vessels in the vicinity of the peripheral part of the femoral artery as observed by body surface echo by week 7. 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.
図９は、体表管エコーにより血管内腔の閉塞を認めたウサギの血管造影図である。図中の矢印Ａから矢印Ｂまでの間に、本来造影される血管が造影されていない。このことから、この部位における血管の閉塞を確認した。 FIG. 9 is an angiogram of a rabbit whose occlusion of the blood vessel lumen was recognized 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.
図１０は、血管サンプルに対するＨＥ染色及びＥＶＧ染色の結果を示す。ＨＥ染色では、血管内の大部分が赤色に染まったが、青紫色に染まった領域の散在を認めた。また、ＥＶＧ染色では、血管内の大部分が黄色に染まったが、赤色に染まった領域の散在を認めた。病理所見としては、新生内膜の肥厚、散在性の炎症細胞の浸潤を認めた。コレステロールクリスタルの沈着はなく、脂質コア及び薄い線維性被膜は認められなかった。また、外弾性板の変性はほとんど認められなかった。血管の閉塞部分は、中枢端から末梢端まで血栓により閉塞していたが、血栓と血管壁の脂質との連続性は認められなかった。このことから、本実験で得られたウサギの血管に形成された閉塞性血栓は、プラークの破綻に起因するものではなく、プラークびらんに起因するものであると考えられる。 FIG. 10 shows the results of HE staining and EVG staining for a blood vessel sample. In the HE staining, most of the blood vessels were stained red, but scattered regions of blue-violet were observed. In the EVG staining, most of the blood vessels were stained yellow, but scattered regions of red were observed. As pathological findings, thickening of the neointima and infiltration of scattered inflammatory cells were observed. There was no deposition of cholesterol crystals and no lipid core or thin fibrous capsule was observed. Further, 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.
上記実施例１及び実施例３において、病理学的解析における病理所見では、いずれの血管サンプルにおいても明らかな脂質コア及び薄い線維性被膜は認められなかった。明らかな脂質コア及び薄い線維性被膜は、プラークの破綻による閉塞性血栓の特徴であって、プラークびらんでは、ほとんどみられない。また、内皮傷害の６週間後にほとんどのウサギにおいて血小板血栓による血管閉塞を確認した。さらに、内皮傷害の７〜８週間後に全てのウサギにおいてフィブリン血栓による血管閉塞を確認した。このように、上記各実施例で得られた非ヒトモデル動物は、ヒトで報告されている閉塞性血栓症のプロセスである血小板血栓が最初に形成され、引き続いてフィブリン血栓が形成される病態を再現している。これらのことは、上記各実施例で得られた非ヒトモデル動物がプラークびらんに起因する血栓症のモデルとして妥当であることを示唆している。 In the above Example 1 and Example 3, no clear lipid core and thin fibrous capsule were observed in any blood vessel sample in the pathological findings in the 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. Furthermore, vascular occlusion due to platelet thrombus was confirmed in most rabbits 6 weeks after endothelial injury. Furthermore, vascular occlusion due to fibrin thrombus was confirmed in all rabbits 7-8 weeks after endothelial injury. Thus, 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. These facts suggest that the non-human animal model obtained in each of the above examples is valid as a model of thrombosis caused by plaque erosion.
（実施例４：アテローム血栓性閉塞の発生率の評価）
アテローム血栓性閉塞の発生について評価するために、上記実施例３と同様の実験をウサギの匹数ｎ＝６で行った。本実験では、アンギオテンシンIIを投与しない点のみが異なるアンギオテンシンII非投与群（ｎ＝５）をさらに加えた。また、バルーンカテーテルによる内皮傷害そのものによる血栓の形成を抑制するために、内皮傷害の前後２週間に渡って、アセチルサリチル酸を２０ｍｇ／日で経口投与した。(Example 4: Evaluation of incidence of atherothrombotic occlusion)
In order to evaluate the occurrence of atherothrombotic occlusion, the same experiment as in Example 3 was performed with the number of rabbits n = 6. In this experiment, an angiotensin II non-administered group (n = 5), which was different only in that no angiotensin II was administered, was further added. In addition, in order to suppress thrombus formation due to the endothelial injury itself by the balloon catheter, acetylsalicylic acid was orally administered at 20 mg / day for 2 weeks before and after the endothelial injury.
（結果）
図１１は、閉塞が発生せずに生存したウサギの割合の経時変化を示す。アンギオテンシンII非投与群のウサギでは、１０週間に渡って１匹を除いて閉塞を認めなかった。一方、アンギオテンシンII投与群では、４週目から閉塞を認めるウサギが現れ、９週までにすべてのウサギで閉塞が認められた。(result)
FIG. 11 shows the change over time in the proportion of rabbits that survived without occlusion. In rabbits in the angiotensin II non-administered group, no obstruction was observed except for one animal over 10 weeks. On the other hand, in the angiotensin II administration group, rabbits with obstruction appeared from the 4th week, and obstruction was observed in all rabbits by 9 weeks.
図１２は、採取した血管サンプルに対するＨＥ染色の結果を示す。血管内腔が閉塞していないアンギオテンシンII非投与群に対して、アンギオテンシンII投与群では、血管内腔が血栓で閉塞していた。なお、血圧及び心拍数に関して両群に有意な差はなかったため、アテローム血栓性閉塞の形成は、血圧又は心拍数の変化に起因するものではない。
この結果から、昇圧剤アンギオテンシンIIを持続投与することで、アテローム血栓性閉塞を自然発生させることができることが示された。FIG. 12 shows the result of HE staining for the collected blood vessel sample. In contrast to the angiotensin II non-administered group in which the vascular lumen was not occluded, 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.
図１３は、アンギオテンシンII投与群から採取したアテローム血栓性閉塞を伴う大腿動脈に対するＨＥ染色及びＥＶＧ染色の結果を示す。血管内には、白色血栓の領域と赤色血栓の領域が観察された。 FIG. 13 shows the results of HE staining and EVG staining for 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.
図１４乃至図１６は、上記アテローム血栓性閉塞を伴う大腿動脈に対する免疫組織化学解析の結果を示す。免疫組織化学解析は、ＰＥＣＡＭ−１、α−ｓｍｏｏｔｈ ｍｕｓｃｌｅ ａｃｔｉｎ（αＳＭＡ）、Ｒａｂｂｉｔ ｓｍｏｏｔｈ ｍｕｓｃｌｅ ｍｙｏｓｉｎ ｈｅａｖｙ ｃｈａｉｎ（ＳＭ１）、ｓｍｏｏｔｈ ｍｕｓｃｌｅ ｍｙｏｓｉｎ ｈｅａｖｙ ｃｈａｉｎ（ＳＭ２）、Ｒａｂｂｉｔ ｔｉｓｓｕｅ ｆａｃｔｏｒ（組織因子）、ｎｏｎｍｕｓｃｌｅ ｍｙｏｓｉｎ ｈｅａｖｙ ｃｈａｉｎ（ＳＭｅｍｂ）、Ｈｕｍａｎ ｃａｌｐｏｎｉｎ（カルポニン）、及びｒａｂｂｉｔ ｍａｃｒｏｐｈａｇｅ（ＲＡＭ−１１）について行った。免疫組織化学解析では、固定した腸骨大腿動脈をパラフィンで包埋し、５ｍｍ毎に５μｍの厚さの連続切片サンプルを作製した。サンプルは抗原賦活化のため、クエン酸バッファーで２０分間煮沸し、３％スキムミルクでブロッキングした後、一次抗体を４℃で一晩かけて付着させた。使用した一次抗体は、ＰＥＣＡＭ−１（サンタクルズ社、ｓｃ−１５０６、希釈倍率は１：５０）、αＳＭＡ（シグマ社、Ａ２５４７、希釈倍率は１：１００）、ＳＭ１（ヤマサ醤油社、７６００、希釈倍率は１：１００）、ＳＭ２（ヤマサ醤油社、７６０１、希釈倍率は１：１００）、組織因子（アメリカンダイアグノスティカ社、４５１３、希釈倍率は１：１００）、ＳＭｅｍｂ（ヤマサ醤油社、７６０２、希釈倍率は１：２００）、カルポニン（ダコ社、Ｍ３５５６、希釈倍率は１：５０）、及びＲＡＭ−１１（ダコ社、Ｍ０６３３、希釈倍率は１：１００）である。 14 to 16 show the results of immunohistochemical analysis for the femoral artery with the above-mentioned atherothrombotic occlusion. Immunohistochemical analysis was performed using PECAM-1, α-smooth muscle actin (αSMA), Rabbit smooth muscle heavy chain (SM1), smooth muscle myskin heavier tissue (SM2). (SMemb), Human calponin (calponin), and rabbit macrophage (RAM-11). In the immunohistochemical analysis, the fixed iliac femoral artery was embedded in paraffin, and serial section samples having a thickness of 5 μm were prepared every 5 mm. 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), αSMA (Sigma, A2547, dilution factor 1: 100), SM1 (Yamasa Shoyu Co., Ltd., 7600, dilution factor). 1: 100), SM2 (Yamasa Soy Sauce, 7601, dilution factor 1: 100), tissue factor (American Diagnostics, 4513, dilution factor 1: 100), SMemb (Yamasa Soy Sauce, 7602, dilution) The magnification is 1: 200), calponin (Dako, M3556, dilution factor is 1:50), and RAM-11 (Dako, M0633, dilution factor is 1: 100).
上記一次抗体に対する陰性対照として、ノーマルマウスＩｇＧ（サンタクルズ社、ｓｃ−２０２５）及びノーマルヒツジＩｇＧ（サンタクルズ社、ｓｃ−２７１７）を使用した。二次抗体としてヒストファインＳＡＢ−ＰＯ（Ｍ）キット（ニチレイバイオサイエンス社、４２４０２２）及びウサギ抗ヒツジＩｇＧ（Ｚｙｍｅｄ（商標）、インビトロジェン社、６１−８６４０）を使用した。なお、発色にはＤＡＢバッファータブレット（メルク社、１０２９２４）を使用した。 Normal mouse IgG (Santa Cruz, sc-2025) and normal sheep IgG (Santa Cruz, sc-2717) were used as negative controls for the primary antibody. As a secondary antibody, a Histofine SAB-PO (M) kit (Nichirei Bioscience, 422402) and a rabbit anti-sheep IgG (Zymed ™, Invitrogen, 61-8640) were used. A DAB buffer tablet (Merck, 102924) was used for color development.
図１４を参照して、アテローム血栓性閉塞を伴う大腿動脈には、（１）プラークの破綻及び脂質コアはみられず、（２）ＰＥＣＡＭ−１陽性の内皮層はみられず、（３）内膜／血栓の界面に未成熟な平滑筋様の細胞（αＳＭＡ陽性、ＳＭ１陰性、ＳＭ２陰性）が多く存在した。 Referring to FIG. 14, in the femoral artery with atherothrombotic occlusion, (1) no plaque rupture and lipid core are observed, (2) no PECAM-1-positive endothelial layer is observed, (3) There were many immature smooth muscle-like cells (αSMA positive, SM1 negative, SM2 negative) at the intima / thrombus interface.
図１５は、閉塞のある動脈及び閉塞のない動脈に対する免疫組織化学解析の結果を比較したものである。αＳＭＡ陽性、ＳＭ１陰性、ＳＭ２陰性の平滑筋様の細胞は、閉塞のある動脈に特異的にみられた。
また、内膜／血栓の界面に見られるαＳＭＡ陽性、ＳＭ１陰性、ＳＭ２陰性の平滑筋様の細胞では、脱分化型平滑筋細胞の分化マーカーであるＳＭｅｍｂ及びカルポニンは発現していなかった（図１６）。
上記のように、内皮層が見られず、血管内膜／血栓の界面にαＳＭＡ陽性、ＳＭ１陰性、ＳＭ２陰性の平滑筋様の細胞が見られるため、当該モデル動物におけるアテローム血栓性閉塞は、プラークびらんに起因して形成されたものであることが示唆された。FIG. 15 compares the results of immunohistochemical analysis for an artery with occlusion and an artery without occlusion. αSMA positive, SM1 negative, SM2 negative smooth muscle-like cells were found specifically in occluded arteries.
In addition, SMEMA and calponin, which are differentiation markers for dedifferentiated smooth muscle cells, were not expressed in αSMA-positive, SM1-negative, and SM2-negative smooth muscle-like cells found at the intima / thrombus interface (FIG. 16). ).
As described above, since the endothelial layer is not seen and αSMA positive, SM1 negative, SM2 negative smooth muscle-like cells are seen at the intima / thrombus interface, atherothrombotic occlusion in the model animal It was suggested that it was formed due to erosion.
（実施例５：閉塞性血栓症モデルでの薬効評価）
上記実施例３で作製した閉塞性血栓症モデルを用いて、薬物の抗血栓作用を評価した。上記実施例３と同じ処理を行ったウサギに対して、薬物投与群として内皮傷害の１週前からアセチルサリチル酸を２０ｍｇ／日で経口投与し、内皮傷害時からダビガトランを１０ｍｇ／ｋｇ／日で経口投与した。(Example 5: Efficacy evaluation in obstructive thrombosis model)
Using the occlusive thrombosis model prepared in Example 3 above, the antithrombotic action of the drug was evaluated. For the rabbits treated in the same manner as in Example 3, acetylsalicylic acid was orally administered at 20 mg / day 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.
（結果）
図１７は、閉塞が発生せずに生存したウサギの割合の経時変化を示す。薬物投与群のウサギでは、１０週間に渡って閉塞を認めなかった。一方、薬物非投与群では、３週目から閉塞を認めるウサギが現れ、９週までにすべてのウサギで閉塞が認められ、生存しなかった。両群の間には、有意な差が認められた（ｐ＝０．００３９）。組織染色でも、アセチルサリチル酸とダビガトランの投与によって、アテローム血栓性閉塞の発生が抑制されたことが示された（図１８）。(result)
FIG. 17 shows the change over time in the proportion of rabbits that survived without occlusion. In the drug administration group, no obstruction was observed for 10 weeks. On the other hand, in the drug non-administered group, rabbits with obstruction appeared from the 3rd week, and obstruction was observed in all rabbits by 9 weeks and did not survive. There was a significant difference between the two groups (p = 0.039). Tissue staining also showed that the administration of acetylsalicylic acid and dabigatran suppressed the occurrence of atherothrombotic occlusion (FIG. 18).
これまで、非ヒトモデル動物として汎用されるマウス、ラット等では、プラークびらんに起因する血栓症を発症する非ヒトモデル動物は、全く得られなかった。上記実施例では、ヒトと脂質代謝系及び凝固線溶系が近いウサギを採用することによって、遺伝子改変等を行わずにプラークびらんに起因する血栓症を発症させることができた。上記実施例で得られたウサギは、遺伝子改変等を加えておらず、内皮傷害をきっかけとして発生するプラークびらん及び閉塞性血栓の病態を再現するため、自然発症に近いプラークびらんに起因する血栓症のモデルとして特に有用である。 Until now, no mouse, rat, etc., which are widely used as non-human model animals, have obtained no non-human model animals that develop thrombosis caused by plaque erosion. In the above example, by employing a rabbit that is close to the human lipid metabolism system and coagulation / fibrinolysis system, it was possible to develop thrombosis caused by plaque erosion without genetic modification or the like. Rabbits obtained in the above examples were not genetically modified, etc., and to reproduce the pathology of plaque erosion and obstructive thrombosis caused by endothelial injury, thrombosis caused by plaque erosion close to spontaneous occurrence It is particularly useful as a model.
上記のように、自然発症に近いプラークびらんに起因する血栓症のモデルを用いて、血栓症及びこれに関連する心筋梗塞、脳卒中、末梢動脈疾患等の動脈硬化性疾患の治療又は予防のための薬物のスクリーニングが可能になる。上記モデル動物を用いた薬物のスクリーニングは、当該モデルがプラークびらん及び閉塞性血栓の病態を再現するため、有効性の高い薬物の探索に好適である。 As described above, for the treatment or prevention of arteriosclerotic diseases such as thrombosis and related myocardial infarction, stroke, peripheral arterial disease using a model of thrombosis caused by plaque erosion close to spontaneous occurrence Drug screening becomes possible. Drug screening using the above model animals is suitable for searching for highly effective drugs because the model reproduces the pathology of plaque erosion and obstructive thrombosis.
本発明は、本発明の広義の精神と範囲を逸脱することなく、様々な実施の形態及び変形が可能とされるものである。また、上述した実施の形態は、本発明を説明するためのものであり、本発明の範囲を限定するものではない。すなわち、本発明の範囲は、実施の形態ではなく、特許請求の範囲によって示される。そして、特許請求の範囲内及びそれと同等の発明の意義の範囲内で施される様々な変形が、本発明の範囲内とみなされる。 Various embodiments and modifications can be made to the present invention without departing from the broad spirit and scope of the present invention. The above-described embodiments are for explaining the present invention and do not limit the scope of the present invention. In other words, the scope of the present invention is shown not by the embodiments but by the claims. Various modifications within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.
本出願は、２０１２年６月６日に出願された日本国特許出願特願２０１２−１２９３４０号に基づく。本明細書中に日本国特許出願特願２０１２−１２９３４０号の明細書、特許請求の範囲、図面全体を参照として取り込むものとする。 This application is based on Japanese Patent Application No. 2012-129340 filed on June 6, 2012. The specification, claims, and entire drawing of Japanese Patent Application No. 2012-129340 are incorporated herein by reference.
本発明は、プラークびらんに起因する血栓症の非ヒトモデル動物に好適である。本発明を適用することにより、プラークびらんに起因する血栓症の病態の解明及び血栓症の治療薬等の研究開発が促進される。 The present invention is suitable for a non-human animal model of thrombosis caused by plaque erosion. By applying the present invention, elucidation of the pathological condition of thrombosis caused by plaque erosion and research and development of a therapeutic agent for thrombosis, etc. are promoted.
Claims (9)
非ヒトモデル動物。Develop thrombosis due to plaque erosion,
Non-human model animal.
血管内腔を閉塞する閉塞性である、
ことを特徴とする請求項１に記載の非ヒトモデル動物。The thrombosis is
Is occlusive to occlude the lumen of the blood vessel,
The non-human model animal according to claim 1.
血小板血栓及びフィブリン血栓の少なくとも一方が血管内腔に形成される、
ことを特徴とする請求項１又は２に記載の非ヒトモデル動物。The thrombosis is
At least one of a platelet thrombus and a fibrin thrombus is formed in the blood vessel lumen;
The non-human model animal according to claim 1 or 2.
ウサギである、
ことを特徴とする請求項１乃至３のいずれか一項に記載の非ヒトモデル動物。The non-human model animal is
A rabbit,
The non-human model animal according to any one of claims 1 to 3, wherein:
前記非ヒト動物に昇圧剤を持続投与する工程と、
を含む、
プラークびらんに起因する血栓症を発症する非ヒトモデル動物の作製方法。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,
A method for producing a non-human model animal that develops thrombosis caused by plaque erosion.
アンギオテンシンIIである、
ことを特徴とする請求項５に記載の非ヒトモデル動物の作製方法。The vasopressor is
Angiotensin II,
The method for producing a non-human model animal according to claim 5.
ウサギである、
ことを特徴とする請求項５又は６に記載の非ヒトモデル動物の作製方法。The non-human animal is
A rabbit,
The method for producing a non-human model animal according to claim 5 or 6.
抗血栓症薬のスクリーニング方法。Using the non-human model animal according to any one of claims 1 to 4,
Screening method for antithrombotic drugs.
前記被験物質を投与された前記非ヒトモデル動物と前記被験物質を投与されなかった前記非ヒトモデル動物との間で、血栓症に関連するマーカーを比較する工程と、
を含む、
ことを特徴とする請求項８に記載の抗血栓症薬のスクリーニング方法。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,
The method for screening an antithrombotic drug according to claim 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014520051A JPWO2013183725A1 (en) | 2012-06-06 | 2013-06-06 | Non-human model animal, method for producing non-human model animal, and screening method for antithrombotic drug |
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| JP2014520051A JPWO2013183725A1 (en) | 2012-06-06 | 2013-06-06 | Non-human model animal, method for producing non-human model animal, and screening method for antithrombotic drug |
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