KR102088556B1 - Ischemic injury stroke resistance animal model and method for producing the same - Google Patents
Ischemic injury stroke resistance animal model and method for producing the same Download PDFInfo
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Abstract
본 발명은 허혈성 뇌질환 동물 모델 및 이의 제조 방법에 관한 것이다. 본 발명의 허혈성 뇌질환 저항성 동물 모델은 DAPK1 및 Pin1 유전자가 이중 결손되어 저항성을 나타내므로, 종래 허혈성 뇌질환 감수성 동물 모델들과 비교하여 허혈성 뇌질환과 관련된 시험 물질의 스크리닝에 유용하게 이용될 수 있고, 허혈성 뇌질환과 관련된 유전자 발현 연구에 참고용으로 이용될 수 있어, 관련된 약학 분야에서 유용하게 이용될 수 있다.The present invention relates to an ischemic brain disease animal model and a method for manufacturing the same. The ischemic brain disease resistant animal model of the present invention can be usefully used for screening test substances related to ischemic brain disease compared to conventional ischemic brain disease susceptible animal models because the DAPK1 and Pin1 genes are double-deficient and exhibit resistance. , May be used as a reference in the study of gene expression related to ischemic brain disease, it may be useful in the related pharmaceutical field.
Description
본 발명은 허혈성 뇌질환 저항성 동물 모델 및 이의 제조 방법에 관한 것이다. The present invention relates to an ischemic brain disease resistant animal model and a method for manufacturing the same.
2001년 우리나라 사망원인 통계조사에 따르면, 뇌혈관 질환에 의한 사망률(인구 십만 명당 사망자수)이 암 다음으로 2위인 73.8명에 이르며, 이에 따른 사회, 경제적 비용은 2조 3,138억 원으로 추정되었다. 뇌혈관 질환 치료제에 대한 수요가 매우 높으므로, 뇌혈관 질환 치료를 통한 사회적 경제적 손실을 줄이기 위한 치료제 개발연구가 중요하다. 뇌혈관 질환은 특히 노령 연령층에 발병률이 높으며, 일단 발생하면 사망률이 매우 높다. 통계청에서 2003년 10월에 발표한 우리나라 노령 인구의 비율을 살펴보면, 우리나라는 지난 2000년 65세 이상 인구가 총인구에서 차지하는 비중이 7.2%에 이르러 고령화 사회에 들어섰으며, 오는 2019년에는 이 비율이 14%를 넘어 고령사회에 진입할 것으로 전망되고 있다.According to a statistical survey on the causes of death in Korea in 2001, the mortality rate due to cerebrovascular disease (the number of deaths per 100,000 people) reached the second place after cancer, 73.8, and the social and economic costs were estimated at 2,3,138 billion won. Since the demand for the treatment of cerebrovascular disease is very high, it is important to research and develop therapeutic agents to reduce social and economic losses through the treatment of cerebrovascular disease. Cerebrovascular disease has a high incidence, especially in the elderly, and once it occurs, the mortality rate is very high. Looking at the proportion of the elderly population in Korea announced by the National Statistical Office in October 2003, Korea entered an aging society in 2000, when the population aged 65 and over accounted for 7.2% of the total population. It is expected to enter the aging society by more than%.
뇌혈관 질환은 크게 2가지 형태로 분류될 수 있다. 하나는 뇌출혈 등에서 볼 수있는 출혈성 뇌질환이고, 다른 하나는 뇌혈관의 폐쇄 등에 의해 나타나는 허혈성 뇌질환이다. 출혈성 뇌질환은 교통사고 등에 의해서 주로 나타나며, 허혈성 뇌질환은 주로 노령의 사람들에서 자주 나타나는 질환이다. 허혈성 뇌질환은 혈전증(thrombosis), 색전증(em-bolism), 일과성 허혈 발작(transient ischemic attack) 및 소경색(lacune)등으로 세분할 수 있는데, 허혈성 뇌혈관질환은 주로 혈전과 색전 등에 의하여 뇌에 혈류를 공급하는 혈관에 병리학적 이상이 생긴 것이다.Cerebrovascular disease can be broadly classified into two types. One is hemorrhagic brain disease, which can be seen in brain hemorrhage, and the other ischemic brain disease caused by occlusion of cerebral vessels. Hemorrhagic brain disease is mainly caused by traffic accidents, etc., and ischemic brain disease is a disease frequently occurring in elderly people. Ischemic brain disease can be subdivided into thrombosis, em-bolism, transient ischemic attack, and lacune. Ischemic cerebrovascular disease is mainly caused by thrombus and embolism. There is a pathological abnormality in the blood vessel supplying blood flow.
대뇌에 일시적인 뇌허혈이 유발되는 경우, 뇌조직에 산소와 포도당의 공급이 차단되어 신경세포에서는 ATP감소, 부종(edema)이 발생하며, 결국 뇌의 광범위한 손상이 유발된다. 신경세포의 사멸은 뇌허혈이 있은 후 상당한 시간 경과 후에 나타나는데, 이를 지연성 신경세포사(delayed neuronal death)라고 한다. 지연성 신경세포사는 몽골리안 저빌(Mongolian gerbil)을 이용한 일과성 전뇌 허혈모델(transient forebrain ischemic model)을 통한 실험에서 살펴보면, 5분간 뇌허혈 유도 4일 후 해마(hippcampus)의 CA1 영역에서 신경세포사가 관찰되는 것으로 보고되고 있다(Kirino T, Sano K. Acta Neuropathol., 62, 201-208, 1984; Kirino T. Brain Res., 239, 57-69, 1982).When temporary cerebral ischemia is induced in the cerebral brain, the supply of oxygen and glucose to the brain tissue is blocked, resulting in ATP reduction and edema in the nerve cells, which in turn causes extensive brain damage. The death of neurons occurs after a significant period of time after cerebral ischemia, which is called delayed neuronal death. According to an experiment through a transient forebrain ischemic model using a Mongolian gerbil, neuronal cell death was observed in the CA1 region of the
뇌허혈에 의한 신경세포사 기전에는 크게 2가지가 있다. 하나는 뇌허혈에 의해서 세포 바깥에 과도한 글루타메이트(glutamate)가 축적되게 되며, 이러한 글루타메이트가 세포내로 유입되어 결국 과도한 세포내 칼슘의 축적으로 신경세포사가 유발되는 흥분성 신경세포사 기전(Kang TC et al., J. Neurocytol., 30, 945-955, 2001)이고, 다른 하나는 허혈-재관류 시에 갑작스러운 산소 공급으로 인해 생체내 라디칼의 증가로 인해 DNA 및 세포질에 손상을 입어 유발되는 산화성 신경세포사 기전이다(Won MH et al., Brain Res., 836, 70-78, 1999; Sun AY, Chen YM, J. Biomed. Sci., 5, 401-414, 1998; Flowers F, Zimmerman JJ. New Horiz., 6, 169-180, 1998).There are two main mechanisms of neuronal cell death caused by ischemia. One is the excitatory neuronal cell death mechanism in which excessive glutamate accumulates outside the cells by cerebral ischemia, and these glutamate flows into the cells and eventually causes neuronal cell death due to excessive intracellular calcium accumulation (Kang TC et al., J). Neurocytol., 30, 945-955, 2001), and the other is the mechanism of oxidative neuronal cell death caused by damage to DNA and cytoplasm due to an increase in radicals in vivo due to sudden oxygen supply during ischemia-reperfusion ( Won MH et al., Brain Res., 836, 70-78, 1999; Sun AY, Chen YM, J. Biomed.Sci., 5, 401-414, 1998; Flowers F, Zimmerman JJ.New Horiz., 6 , 169-180, 1998).
허혈성 뇌질환에서 임상적으로 사용 가능한 약물은 희소하고, 현재 유일하게 허가된 약물은 항혈전제(항응고제, 항혈소판제, 혈전용해제를 포함)이다. 티크로피딘(ticlopidine), 시로스타졸(cilostazole) 및 프로스타시크린(prostacycline)의 항혈소판제나 항응고제는 종종 두통, 심계항진 및 간에 부담을 주는 부작용이 있어 사용에 제한이 따른다. 더불어, FDA 공인 시판 중인 조직플라즈미겐활성자(tissue plasminogen activator)는 혈전용해제로 뇌허혈을 유발시키는 혈전을 녹여 빠른 산소 및 포도당의 공급을 유도하는 물질로서, 직접적으로 신경세포를 보호하는 것이 아니기 때문에 빠른 사용이 필요하다. 또한, 혈전용해제라는 특징 때문에 과량 사용 또는 자주 사용 시에는 혈관벽이 얇아져 결국 출혈성 뇌혈관 질환을 유발하게 되므로 사용에 주의가 요구된다. There are few clinically available drugs for ischemic brain disease, and currently the only approved drugs are antithrombotic agents (including anticoagulants, antiplatelet agents, and thrombolytic agents). Antiplatelet or anticoagulants of ticlopidine, cilostazole, and prostacycline often have side effects such as headache, cardiac hyperactivity, and liver burden, which limits their use. In addition, the FDA-approved commercial tissue plasminogen activator is a substance that induces rapid supply of oxygen and glucose by dissolving blood clots that induce cerebral ischemia by thrombolysis, so it does not protect nerve cells directly. Use is necessary. In addition, due to the characteristic of thrombolytic release, when used excessively or frequently, the blood vessel wall becomes thinner, which eventually leads to hemorrhagic cerebrovascular disease.
따라서, 새로운 허혈성 뇌질환의 치료제의 개발이 요구될 뿐만 아니라, 이를 대조적으로 확인 할 수 있는 허혈성 뇌질환을 유발시키는 물질 개발의 필요성이 대두되고 있다. 이에 따라, 상기 치료제 또는 유발제를 효과적이고 객관적으로 스크리닝할 수 있는 동물 모델의 필요성이 요구되고 있다.Therefore, not only is there a need to develop a new therapeutic agent for ischemic brain disease, but there is a need to develop a substance that induces ischemic brain disease, which can be confirmed in contrast. Accordingly, there is a need for an animal model capable of effectively and objectively screening the therapeutic agent or inducer.
본 발명의 목적은 허혈성 뇌질환 저항성 동물 모델을 제공할 수 있다. An object of the present invention is to provide an ischemic brain disease resistant animal model.
또한 본 발명의 목적은 허혈성 뇌질환 저항성 동물 모델 제조 방법을 제공할 수 있다.It is also an object of the present invention to provide a method for manufacturing an animal model resistant to ischemic brain disease.
상기 목적의 달성을 위해, 본 발명은 DAPK1(Death-associated protein kinase 1) 동형접합성((homozygote, DAPK1-/-) 마우스와 Pin1(peptidylprolyl cis/trans isomerase 1) 이형접합성(heterozygote, Pin1+/-) 마우스를 교배하여 DAPK1 이형접합성(DAPK1+/-) 및 Pin1 이형접합성(Pin1+/-)을 포함하는 마우스를 선발하는 단계; 및To achieve the above object, the present invention is DAPK1 (Death-associated protein kinase 1) homozygous ((homozygote, DAPK1 -/- ) mouse and Pin1 (peptidylprolyl cis / trans isomerase 1) heterozygosity (heterozygote, Pin1 +/-) ) Crossing the mouse to select a mouse comprising DAPK1 heterozygous (DAPK1 +/- ) and Pin1 heterozygous (Pin1 +/- ); and
상기 DAPK1 이형접합성(DAPK1+/-) 및 Pin1 이형접합성(Pin1+/-) 특징을 포함하는 마우스를 교배하여 DAPK1 동형접합성(DAPK-/-) 및 Pin1 동형접합성(Pin1-/-)을 포함하는 마우스를 선발하는 단계;를 포함하는 DAPK1 및 Pin1 유전자가 이중 결손된 허혈성 뇌질환 저항성 동물 모델을 제공한다.The DAPK1 heterozygous (DAPK1 +/- ) and Pin1 heterozygous (Pin1 +/- ) mice are crossed to include DAPK1 homozygous (DAPK -/- ) and Pin1 homozygous (Pin1 -/- ) It provides an ischemic brain disease-resistant animal model in which the DAPK1 and Pin1 genes including;
또한, 본 발명은 DAPK1 동형접합성((homozygote, DAPK1-/-) 마우스와 Pin1 이형접합성(heterozygote, Pin1+/-) 마우스를 교배하여 DAPK1 이형접합성(DAPK1+/-) 및 Pin1 이형접합성(Pin1+/-)을 포함하는 마우스를 선발하는 단계; 및In addition, the present invention crosses DAPK1 homozygous ((homozygote, DAPK1 -/- ) mice with Pin1 heterozygote (Pin1 +/- ) mice, and DAPK1 heterozygous (DAPK1 +/- ) and Pin1 heterozygous (Pin1 +) Selecting a mouse including /- ); and
상기 DAPK1 이형접합성(DAPK1+/-) 및 Pin1 이형접합성(Pin1+/-) 특징을 포함하는 마우스를 교배하여 DAPK1 동형접합성(DAPK-/-) 및 Pin1 동형접합성(Pin1-/-)을 포함하는 마우스를 선발하는 단계;를 포함하는 DAPK1 및 Pin1 유전자가 이중 결손된 허혈성 뇌질환 저항성 동물 모델 제조 방법을 제공한다.The DAPK1 heterozygous (DAPK1 +/- ) and Pin1 heterozygous (Pin1 +/- ) mice are crossed to include DAPK1 homozygous (DAPK -/- ) and Pin1 homozygous (Pin1 -/- ) It provides a method for manufacturing an animal model resistant to ischemic brain disease in which the DAPK1 and Pin1 genes including the step of selecting a mouse are double-deleted.
본 발명의 허혈성 뇌질환 저항성 동물 모델은 DAPK1 및 Pin1 유전자가 이중 결손되어 저항성을 나타내므로, 종래 허혈성 뇌질환 감수성 동물 모델들과 비교하여 허혈성 뇌질환과 관련된 시험 물질의 스크리닝에 유용하게 이용될 수 있고, 허혈성 뇌질환과 관련된 유전자 발현 연구에 참고용으로 이용될 수 있어, 관련된약학 분야에서 유용하게 이용될 수 있다.The ischemic brain disease resistant animal model of the present invention can be usefully used for screening test substances related to ischemic brain disease compared to conventional ischemic brain disease susceptible animal models because the DAPK1 and Pin1 genes are double-deficient and exhibit resistance. , Can be used as a reference in the study of gene expression related to ischemic brain disease, it can be usefully used in the related pharmaceutical field.
도 1은 본 발명의 동물 모델의 제조 방법을 나타내는 모식도이다.
도 2는 2차 교배 하여 획득한 DAPK1 및 Pin1 유전자 이중 결손 마우스의 유전형을 분석한 결과를 나타낸 도이다.
도 3은 야생형, DAPK1 및 Pin1 이중 결손(DAPK1/Pin1 DAPK1/Pin1 DKO), DAPK1 단일 결손(DAPK1 KO) 또는 Pin1 단일 결손(Pin1 KO) 마우스의 몸 길이를 확인한 결과를 나타낸 도이다.
도 4는 야생형, DAPK1 및 Pin1 이중 결손(DAPK1/Pin1 DAPK1/Pin1 DKO), DAPK1 단일 결손(DAPK1 KO) 또는 Pin1 단일 결손(Pin1 KO) 마우스의 체중을 확인한 결과를 나타낸 도이다.
도 5는 DAPK1 및 Pin1 이중 결손(DAPK1/Pin1 DAPK1/Pin1 DKO), DAPK1 단일 결손(DAPK1 KO) 또는 Pin1 단일 결손(Pin1 KO) 마우스에 국소적 허혈성 중대뇌동맥 폐색술(MCAo)을 수행한 후, DAPK1 및 Pin1의 억제제 처리에 따른 유전자의 발현 정도를 확인한 결과를 나타낸 도이다.1 is a schematic view showing a method for producing an animal model of the present invention.
Figure 2 is a diagram showing the results of analyzing the genotype of DAPK1 and Pin1 gene double-deficient mice obtained by the second cross.
3 is a diagram showing the results of confirming the body length of a wild-type, DAPK1 and Pin1 double deletion (DAPK1 / Pin1 DAPK1 / Pin1 DKO), DAPK1 single deletion (DAPK1 KO) or Pin1 single deletion (Pin1 KO) mouse.
Figure 4 is a diagram showing the results of confirming the weight of the wild-type, DAPK1 and Pin1 double deletion (DAPK1 / Pin1 DAPK1 / Pin1 DKO), DAPK1 single deletion (DAPK1 KO) or Pin1 single deletion (Pin1 KO) mice.
Figure 5 after performing a local ischemic middle cerebral artery occlusion (MCAo) in DAPK1 and Pin1 double deletion (DAPK1 / Pin1 DAPK1 / Pin1 DKO), DAPK1 single deletion (DAPK1 KO) or Pin1 single deletion (Pin1 KO) mice, It is a diagram showing the results of confirming the expression level of the gene according to the inhibitor treatment of DAPK1 and Pin1.
본 발명은 DAPK1(Death-associated protein kinase 1) 동형접합성((homozygote, DAPK1-/-) 마우스와 Pin1(peptidylprolyl cis/trans isomerase 1) 이형접합성(heterozygote, Pin1+/-) 마우스를 교배하여 DAPK1 이형접합성(DAPK1+/-) 및 Pin1 이형접합성(Pin1+/-)을 포함하는 마우스를 선발하는 단계; 및The present invention crosses DAPK1 (Death-associated protein kinase 1) homozygous ((homozygote, DAPK1 -/- ) mice and Pin1 (peptidylprolyl cis / trans isomerase 1) heterozygous (heterozygote, Pin1 +/- ) mice and crosses DAPK1 heterozygous Selecting a mouse comprising conjugation (DAPK1 +/- ) and Pin1 heterozygosity (Pin1 +/- ); and
상기 DAPK1 이형접합성(DAPK1+/-) 및 Pin1 이형접합성(Pin1+/-) 특징을 포함하는 마우스를 교배하여 DAPK1 동형접합성(DAPK-/-) 및 Pin1 동형접합성(Pin1-/-)을 포함하는 마우스를 선발하는 단계;를 포함하는 DAPK1 및 Pin1 유전자가 이중 결손된 허혈성 뇌질환 저항성 동물 모델을 제공한다.The DAPK1 heterozygous (DAPK1 +/- ) and Pin1 heterozygous (Pin1 +/- ) mice are crossed to include DAPK1 homozygous (DAPK -/- ) and Pin1 homozygous (Pin1 -/- ) It provides an ischemic brain disease-resistant animal model in which the DAPK1 and Pin1 genes including;
본 발명에서 용어, "DAPK1(Death-associated protein kinase 1)"는 칼슘/칼모듈린 의존성 인산화 효소이고, "Pin1(peptidylprolyl cis/trans isomerase 1)"는 이성질화 효소로서, 상기 DAPK1 및 Pin1는 산화적 스트레스에 반응하여 세포사멸 신호를 활성화시키는 중요한 조절인자로 작용한다. DAPK1 및 Pin1의 신호기작은 세포내 신호전달 체계를 구성하는 특정 단백질과 상호작용하여 기질단백질의 구조적 변화를 유도하거나 특정 모티프(motif)의 인산화를 통하여 기능적 변화를 야기한다.In the present invention, the term, "DAPK1 (Death-associated protein kinase 1)" is a calcium / calmodulin-dependent phosphorylation enzyme, "Pin1 (peptidylprolyl cis / trans isomerase 1)" isomerization enzyme, the DAPK1 and Pin1 is an oxidation It acts as an important regulator that activates apoptosis signals in response to enemy stress. The signaling mechanism of DAPK1 and Pin1 interacts with specific proteins that make up the intracellular signaling system to induce structural changes in matrix proteins or functional changes through phosphorylation of specific motifs.
상기 동물 모델은 허혈성 뇌질환에 저항성이고, 상기 저항성은 DAPK1 및 Pin1의 유전자 결손에 의하여 유발되는 것으로, 이에 제한되지 않는다.The animal model is resistant to ischemic brain disease, and the resistance is caused by gene deletion of DAPK1 and Pin1, but is not limited thereto.
상기 허혈성 뇌질환 동물 모델은 야생형과 비교하여 체중이 감소하고 및 몸 길이가 짧아진다.The ischemic brain disease animal model has a reduced body weight and a shorter body length compared to the wild type.
상기 허혈성 뇌질환은 중풍, 뇌졸중, 뇌일혈, 뇌경색, 두부손상, 알츠하이머, 혈관성 치매, 크로이츠펠트-야콥병, 혼수 및 쇼크 뇌손상으로 이루어진 군에서 선택된 1종 이상이나, DAPK1 및 Pin1의 유전자 결손에 의한 허혈성 뇌질환의 저항성 목적을 달성하기 위해서라면 이에 제한되지 않는다.The ischemic brain disease is one or more selected from the group consisting of stroke, stroke, stroke, cerebral infarction, head injury, Alzheimer's disease, vascular dementia, Creutzfeldt-Jakob disease, coma and shock brain injury, or ischemic due to gene defects in DAPK1 and Pin1 In order to achieve the purpose of resistance to brain disease, it is not limited thereto.
동물모델에 사용되는 동물은 인간을 제외한 다양한 포유류를 포함하며, 이로 제한하는 것은 아니나, 소, 돼지, 양, 토끼, 설취류가 사용될 수 있다. 한 구현예에서는 상기 동물은 쥣과 (murine)동물, 예를 들면 마우스이다.Animals used in the animal model include various mammals except humans, but are not limited thereto, and cows, pigs, sheep, rabbits, and odors can be used. In one embodiment, the animal is a murine animal, for example a mouse.
또한, 본 발명에 따른 동물모델의 제조를 위해 사용할 수 있는 실험용 마우스의 종류로는 이에 제한되지는 않으나, 계통별 분류상 폐쇄군(Closed Colony)에 속하는 ICR 또는 DDY; 근교계(Inbred)에 속하는 BALB/cA, C57BL/6N, C3H/HeN, DBA/2N 또는 CBA/N; 교잡군(hybrid)에 속하는 BDF1(C57BL/6 x DBA/2), CDF1(CBA/N x DBA/2) 또는 B6C3F1(C57BL/6 x C3H/HeN); 돌연변이계(Mutant)에 속하는 BALB/c-nu 또는 C,B-17SCID 등 실험용 마우스로 사용되고 있는 것이라면 모두 이용할 수 있다. 바람직하게는 c57BL/6 마우스를 사용할 수 있다. 상기 c57BL/6 마우스는 종종 "C57 블랙(black) 6" 또는 단순히 "블랙(black) 6"로도 불리는 근교계 실험용 마우스이다. 이 종은 사람의 질병 모델을 위한 유전자 변형 마우스의 "유전적 기반(genetic background)"으로 가장 널리 사용되고 있으며, 이들은 congenic 균주들의 이용가능성, 손쉬운 교배, 강건성(robustness), 및 유전자 변형 모델들과의 관계 등에서 장점을 갖는다. 특히 C57BL/6로부터의 면역 반응은 BALB/c와 같은 다른 근교계 종들과 차별화 될 수 있다. In addition, the type of the experimental mouse that can be used for the production of an animal model according to the present invention is not limited thereto, but ICR or DDY belonging to the closed colony according to the classification of each lineage; BALB / cA, C57BL / 6N, C3H / HeN, DBA / 2N or CBA / N belonging to the Inbred; BDF1 (C57BL / 6 x DBA / 2), CDF1 (CBA / N x DBA / 2) or B6C3F1 (C57BL / 6 x C3H / HeN) belonging to the hybrid group; Any one that is used as an experimental mouse such as BALB / c-nu or C, B-17SCID belonging to the mutant system can be used. Preferably, c57BL / 6 mice can be used. The c57BL / 6 mouse is a mouse for a lab experiment, often referred to as a “C57 black 6” or simply “black 6”. This species is most widely used as a "genetic background" of genetically modified mice for human disease models, which are available with congenic strains, easy mating, robustness, and genetically modified models. It has advantages in relations, etc. In particular, the immune response from C57BL / 6 can be differentiated from other subspecies species such as BALB / c.
따라서 본 발명은 상기와 같은 방법에 따라 허혈성 뇌질환 동물모델을 제조하는 방법을 제공할 수 있으며, 또한, 상기 방법에 의해 제조된 허혈성 뇌질환 동물모델을 제공할 수 있다.Therefore, the present invention can provide a method for manufacturing an ischemic brain disease animal model according to the above method, and also provides an ischemic brain disease animal model produced by the method.
본 발명의 방법에 따라 제조된 허혈성 뇌질환 동물모델은 통상적으로 사용되는 허혈성 뇌질환 동물모델에 비해 DAPK1 및 Pin1 유전자 또는 단백질의 발현이 저해되어 저항성이 증가된 특징이 있다.The ischemic brain disease animal model prepared according to the method of the present invention is characterized by increased resistance by inhibiting the expression of DAPK1 and Pin1 gene or protein compared to the commonly used ischemic brain disease animal model.
또한, 본 발명은 DAPK1 동형접합성((homozygote, DAPK1-/-) 마우스와 Pin1 이형접합성(heterozygote, Pin1+/-) 마우스를 교배하여 DAPK1 이형접합성(DAPK1+/-) 및 Pin1 이형접합성(Pin1+/-)을 포함하는 마우스를 선발하는 단계; 및In addition, the present invention crosses DAPK1 homozygous ((homozygote, DAPK1 -/- ) mice with Pin1 heterozygote (Pin1 +/- ) mice, and DAPK1 heterozygous (DAPK1 +/- ) and Pin1 heterozygous (Pin1 +) Selecting a mouse including /- ); and
상기 DAPK1 이형접합성(DAPK1+/-) 및 Pin1 이형접합성(Pin1+/-) 특징을 포함하는 마우스를 교배하여 DAPK1 동형접합성(DAPK-/-) 및 Pin1 동형접합성(Pin1-/-)을 포함하는 마우스를 선발하는 단계;를 포함하는 DAPK1 및 Pin1 유전자가 이중 결손된 허혈성 뇌질환 저항성 동물 모델 제조 방법을 제공한다.The DAPK1 heterozygous (DAPK1 +/- ) and Pin1 heterozygous (Pin1 +/- ) mice are crossed to include DAPK1 homozygous (DAPK -/- ) and Pin1 homozygous (Pin1 -/- ) It provides a method for manufacturing an animal model resistant to ischemic brain disease in which the DAPK1 and Pin1 genes including the step of selecting a mouse are double-deleted.
또한 본 발명은 (1) 제1항의 허혈성 뇌질환 동물 모델 및 야생형 동물에 시험 물질을 투여하는 단계; 및In addition, the present invention comprises the steps of (1) administering a test substance to the ischemic brain disease animal model and wild-type animal of claim 1; And
(2) 상기 야생형의 DAPK1 및 Pin1의 유전자 또는 단백질 발현이 제1항의 허혈성 뇌질환 동물 모델만큼 저하된 경우 허혈성 뇌질환 치료제로 선발하거나, 제1항의 허혈성 뇌질환 동물 모델의 DAPK1 및 Pin1의 유전자 또는 단백질 발현이 변화된 경우 허혈성 뇌질환 유발제로 선발하는 단계;를 포함하는 허혈성 뇌질환 유발제 또는 치료제의 스크리닝 방법을 제공한다.(2) When the expression of the wild type DAPK1 and Pin1 gene or protein is reduced by the animal model of ischemic brain disease of claim 1, it is selected as a therapeutic agent for ischemic brain disease, or the genes of DAPK1 and Pin1 of animal model of ischemic brain disease of claim 1, or It provides an ischemic brain disease-causing agent or a screening method of the therapeutic agent, including the step of selecting as an ischemic brain disease-causing agent when the protein expression is changed.
상기 허혈성 뇌질환은 중풍, 뇌졸중, 뇌일혈, 뇌경색, 두부손상, 알츠하이머, 혈관성 치매, 크로이츠펠트-야콥병, 혼수 및 쇼크 뇌손상으로 이루어진 군에서 선택된 1종 이상이나, 이에 제한되지 않는다.The ischemic brain disease is at least one selected from the group consisting of stroke, stroke, stroke, cerebral infarction, head injury, Alzheimer's disease, vascular dementia, Creutzfeldt-Jakob disease, coma and shock brain injury, but is not limited thereto.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다.The present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.
<실시예 1> DAPK1(Death-associated protein kinase 1) 또는Pin1(peptidylprolyl cis/trans isomerase 1)의 단일 또는 이중 결손 마우스 모델 제작<Example 1> DAPK1 (Death-associated protein kinase 1) or Pin1 (peptidylprolyl cis / trans isomerase 1) single or double defective mouse model production
도 1에 나타낸 바와 같이, DAPK1 및 Pin1 이중 결손(DAPK1/Pin1 Double Knock Out, DAPK1/Pin1 DKO) 마우스를 제작하기 위하여 DAPK1 (-/-) 암컷 마우스와 Pin1 (+/-) 수컷 마우스의 교배를 1차로 진행하였다. Pin1 (-/-) 마우스의 생식능력이 제한적이기 때문에, 1차 교배 시 Pin1 (-/-) 마우스가 아닌 Pin1 (+/-) 마우스를 이용하여 DAPK1 (-/-) 마우스와 교배를 진행하였다. 이 후, DAPK1(+/-)/Pin1(+/-) 1세대 (F1) 마우스를 확보하고 동일한 유전형 (DAPK1+/-Pin1+/-) 간의 2차교배를 통하여 2세대 (F2) 마우스를 획득하였다. 2세대 마우스의 Genomic DNA를 이용하여 유전형을 분석하였다. 2세대 마우스의 꼬리를 절단하여 genomic DNA를 분리한 후, DAPK1 또는 DAPK1 유전자에 특이적 프라이머를 이용하여 DAPK1 또는 Pin1의 야생형(WT)과 KO 타입에 대한 DNA-PCR을 수행하였다. 각 타입에 대한 프라이머 서열은 하기 표 1에 나타내었다. 그 결과를 도 2에 나타내었다.As shown in FIG. 1, in order to construct DAPK1 and Pin1 double knock out (DAPK1 / Pin1 Double Knock Out, DAPK1 / Pin1 DKO) mice, crossing between DAPK1 (-/-) female mice and Pin1 (+/-) male mice was performed. It proceeded to the 1st. Because the reproductive capacity of Pin1 (-/-) mice is limited, mating was performed with DAPK1 (-/-) mice using Pin1 (+/-) mice instead of Pin1 (-/-) mice during the first mating. . Afterwards, DAPK1 (+/-) / Pin1 (+/-) first generation (F1) mice were obtained and second generation (F2) mice were obtained through a second cross between the same genotype (DAPK1 +/- Pin1 +/-). . The genotype was analyzed using genomic DNA from a second generation mouse . After genomic DNA was isolated by cutting the tail of a second-generation mouse, DNA-PCR was performed for the wild type (WT) and KO type of DAPK1 or Pin1 using primers specific to the DAPK1 or DAPK1 gene. Primer sequences for each type are shown in Table 1 below. The results are shown in FIG. 2.
도 2에 나타낸 바와 같이, DAPK1와 Pin1 유전자가 모두 결손된 DAPK1-/-Pin1-/- 마우스를 선발하였다.As shown in Fig. 2, DAPK1-/-Pin1-/-mice in which both the DAPK1 and Pin1 genes were deleted were selected.
<실시예 2> DAPK1/Pin1 DKO 마우스의 표현형 (Phenotype) 및 성장패턴 분석<Example 2> DAPK1 / Pin1 DKO mouse phenotype and growth pattern analysis
상기 실시예 1에서 선발한 DAPK1 및 Pin1 이중 결손(DAPK1/Pin1 DAPK1/Pin1 DKO), DAPK1 단일 결손(DAPK1 KO) 또는 Pin1 단일 결손(Pin1 KO) 마우스의 표현형 및 성장 패턴을 분석하여 비교하였다.The phenotype and growth pattern of DAPK1 and Pin1 double defects (DAPK1 / Pin1 DAPK1 / Pin1 DKO), DAPK1 single defect (DAPK1 KO) or Pin1 single defect (Pin1 KO) mice selected in Example 1 were analyzed and compared.
구체적으로, Wild type, DAPK1 KO, Pin1 KO, DAPK1/Pin1 DKO의 7주령 암컷 마우스의 몸 길이(cm)를 측정하여 비교하였다. 몸 길이는 코에서부터 항문까지를 측정하였다. 그 결과를 도 3에 나타내었다. 또한, 6주부터 14주까지 야생형(C57BL/6 마우스), DAPK1 KO, Pin1 KO 및 DAPK1/Pin1 DKO 마우스의 체중을 측정하여 각 그룹의 평균을 비교 및 분석하였다. 그 결과를 도 4에 나타내었다.Specifically, the body length (cm) of 7-week-old female mice of Wild type, DAPK1 KO, Pin1 KO, and DAPK1 / Pin1 DKO was measured and compared. Body length was measured from the nose to the anus. The results are shown in FIG. 3. In addition, the weights of wild-type (C57BL / 6 mice), DAPK1 KO, Pin1 KO, and DAPK1 / Pin1 DKO mice were measured from 6 weeks to 14 weeks to compare and analyze the average of each group. The results are shown in FIG.
도 3에 나타낸 바와 같이, Pin1 KO과 DAPK1/Pin1 DKO 마우스의 Body length가 야생형과 DAPK1 KO 마우스에 비하여 상대적으로 감소되어 있음을 확인하였다.As shown in FIG. 3, it was confirmed that the body lengths of the Pin1 KO and DAPK1 / Pin1 DKO mice were relatively reduced compared to the wild-type and DAPK1 KO mice.
또한, 도 4에 나타낸 바와 같이, 야생형과 DAPK1 KO 마우스의 체중 간에는 차이가 없었으나, Pin1 KO과 DAPK1/Pin1 DKO 마우스는 야생형에 비하여 유의한 감소가 나타남을 확인하였다In addition, as shown in Figure 4, there was no difference between the weight of the wild-type and DAPK1 KO mice, but Pin1 KO and DAPK1 / Pin1 DKO mice showed a significant decrease compared to the wild-type.
따라서, 본 발명의 DAPK1/Pin1 DKO 마우스는 야생형과 비교하여 체중 및 몸 길이가 유의적으로 감소됨을 확인하였다. Therefore, it was confirmed that the DAPK1 / Pin1 DKO mouse of the present invention significantly reduced body weight and body length compared to wild type.
<실시예 3> 허혈성 뇌손상을 통한 DAPK1/Pin1 DKO 마우스의 유전적 발현 조절 확인 및 반응 활성 확인<Example 3> Confirmation of genetic expression control and reaction activity of DAPK1 / Pin1 DKO mice through ischemic brain injury
상기 실시예 1에서 선발한 DAPK1 및 Pin1 이중 결손(DAPK1/Pin1 DAPK1/Pin1 DKO), DAPK1 단일 결손(DAPK1 KO) 또는 Pin1 단일 결손(Pin1 KO) 마우스의 유전자 발현 양상을 확인하였다. RT-PCR을 이용하여 DAPK1 KO 마우스와 Pin1 KO 마우스의 뇌조직에서 해당 유전자의 결손을 확인하였다(도 5A). 이 후, 이 후, 국소적 허혈성 중대뇌동맥 폐색술(MCAo)을 수행하여, 허혈성 뇌손상을 일으킨 후, 1시간 후 및 6시간 후의 유전자 발현을 확인하였다. 그 결과를 도 5에 나타내었다.The gene expression pattern of the DAPK1 and Pin1 double deletion (DAPK1 / Pin1 DAPK1 / Pin1 DKO), DAPK1 single deletion (DAPK1 KO) or Pin1 single deletion (Pin1 KO) mice selected in Example 1 was confirmed. RT-PCR was used to confirm the deletion of the gene in the brain tissues of DAPK1 KO and Pin1 KO mice (FIG. 5A). Then, thereafter, local ischemic middle cerebral artery occlusion (MCAo) was performed, and after ischemic brain injury, gene expression was confirmed after 1 hour and 6 hours. The results are shown in FIG.
도 5에 나타낸 바와 같이, 정상상태 (MCAo 비수술군)의 DAPK1 KO 마우스 뇌조직에서 WT 마우스와 비교시, DAPK1 유전자 발현이 억제되고 Pin1 유전자의 발현증가를 확인하였다. Pin1 KO 마우스 뇌조직에서는 반대로 Pin1의 유전자 발현이 억제되고 DAPK1 유전자의 발현됨을 확인하였다.As shown in FIG. 5, DAPK1 gene expression was suppressed and the expression of the Pin1 gene was confirmed to be increased when compared to the WT mouse in the normal state (MCAo non-surgical group) DAPK1 KO mouse brain tissue. In the Pin1 KO mouse brain tissue, it was confirmed that the gene expression of Pin1 is inhibited and the expression of the DAPK1 gene is reversed.
또한, 허혈성 뇌손상을 유발한 WT 마우스의 뇌조직에서 Pin1의 발현 증가 (MCAo 수술 후 6시간)와 DAPK1의 발현증가 (MCAo 수술 후 1일)를 확인하였다. MCAo 수술 이후 DAPK1 KO 뇌조직에서 Pin1의 발현감소가 관찰되었고, Pin1 KO 뇌조직에서는 DAPK1의 발현이 증가됨을 확인하였다.In addition, increased expression of Pin1 (6 hours after MCAo surgery) and increased expression of DAPK1 (one day after MCAo surgery) were observed in brain tissue of WT mice that caused ischemic brain injury. After MCAo surgery, a decrease in the expression of Pin1 was observed in DAPK1 KO brain tissue, and it was confirmed that the expression of DAPK1 was increased in Pin1 KO brain tissue.
<110> inje university industry-academic cooperation foundation <120> Ischemic injury stroke resistance animal model and method for producing the same <130> PN1708-274 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sense primer of DAPK1 WT and KO <400> 1 gtccctccag ttgcagttag aatc 24 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> antisense primer of DAPK1 WT <400> 2 ctttcagagg tctgcggctt ggtgcatgag 30 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> antisense primer of DAPK1 KO <400> 3 aggatctcgt cgtgacccat ggcga 25 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer of Pin1 WT and KO <400> 4 ccgatcctgt tctgcaaact 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> antisense primer of Pin1 WT <400> 5 ggattagaag caagattcga ct 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> antisense primer of Pin1 KO <400> 6 ccacttgtgt agcgccaagt gc 22 <110> inje university industry-academic cooperation foundation <120> Ischemic injury stroke resistance animal model and method for producing the same <130> PN1708-274 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sense primer of DAPK1 WT and KO <400> 1 gtccctccag ttgcagttag aatc 24 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> antisense primer of DAPK1 WT <400> 2 ctttcagagg tctgcggctt ggtgcatgag 30 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> antisense primer of DAPK1 EN <400> 3 aggatctcgt cgtgacccat ggcga 25 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer of Pin1 WT and KO <400> 4 ccgatcctgt tctgcaaact 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> antisense primer of Pin1 WT <400> 5 ggattagaag caagattcga ct 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> antisense primer of Pin1 EN <400> 6 ccacttgtgt agcgccaagt gc 22
Claims (4)
상기 허혈성 뇌질환 저항성 동물 모델은 야생형과 비교하여 체중이 감소되고 몸 길이가 짧아진 것을 특징으로 하는 동물 모델. The method of claim 1,
The ischemic brain disease resistant animal model is an animal model characterized in that the weight is reduced and the body length is shorter compared to the wild type.
상기 허혈성 뇌질환은 중풍, 뇌졸중, 뇌일혈, 뇌경색, 두부손상, 알츠하이머, 혈관성 치매, 크로이츠펠트-야콥병, 혼수 및 쇼크 뇌손상으로 이루어진 군에서 선택된 1종 이상인 것을 특징으로 하는 동물 모델.The method of claim 1,
The ischemic brain disease is an animal model, characterized in that at least one selected from the group consisting of stroke, stroke, stroke, cerebral infarction, head injury, Alzheimer's disease, vascular dementia, Creutzfeldt-Jakob disease, coma and shock brain injury.
상기 DAPK1 이형접합성(DAPK1+/-) 및 Pin1 이형접합성(Pin1+/-)을 포함하는 마우스를 교배하여 DAPK1 동형접합성(DAPK-/-) 및 Pin1 동형접합성(Pin1-/-)을 포함하는 마우스를 선발하는 단계;를 포함하는 허혈성 뇌질환 저항성 동물 모델의 제조 방법.
DAPK1 heterozygous ((homozygote, DAPK1 -/- ) mouse and Pin1 heterozygote (Pin1 +/- ) mice are crossed to include DAPK1 heterozygous (DAPK1 +/- ) and Pin1 heterozygous (Pin1 +/- ) Selecting a mouse to be; and
The mice comprising the DAPK1 heterozygosity (DAPK1 +/- ) and the Pin1 heterozygosity (Pin1 +/- ) are crossed and the mice comprising DAPK1 homozygosity (DAPK -/- ) and Pin1 homozygosity (Pin1 -/- ) A method of manufacturing an animal model resistant to ischemic brain disease, comprising: selecting.
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