KR101390463B1 - Methods for Production of Chronic Obstructive Pulmonary Disease for Autoimmune Disease Animal Model - Google Patents
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Abstract
본 발명은 자가 면역 질환에 의한 만성 폐쇄성 폐 염증(chronic obstructive pulmonary disease; COPD) 동물 모델의 제조방법에 관한 것으로, 더욱 자세하게는 자기 단백질인 엘라스틴에 대한 면역관용이 무너진 만성 폐쇄성 폐 염증의 새로운 동물 모델 및 이의 제조방법에 관한 것이다.
본 발명에 의한 만성 폐쇄성 폐 염증 동물 모델에서 자가 면역 질환에 의해 만성 폐쇄성 폐 염증이 발병한다는 것을 확인하였으므로 만성 폐쇄성 폐 염증에 대한 근본적인 진단 및 치료제 개발에 용이하게 사용할 수 있다. The present invention relates to a method for producing a chronic obstructive pulmonary disease (COPD) animal model due to autoimmune diseases, and more particularly, to a new animal model of chronic obstructive pulmonary inflammation in which the immune tolerance to its own protein elastin is broken down. And it relates to a manufacturing method thereof.
Chronic obstructive pulmonary inflammation in the animal model according to the present invention has been confirmed that chronic obstructive pulmonary inflammation is caused by autoimmune diseases, so it can be easily used for the development of a fundamental diagnosis and treatment for chronic obstructive pulmonary inflammation.
Description
본 발명은 자가 면역 질환에 의한 만성 폐쇄성 폐 염증 동물 모델의 제조방법에 관한 것으로, 더욱 자세하게는 자기 단백질인 엘라스틴에 대한 면역관용이 무너진 만성 폐쇄성 폐 염증의 새로운 동물 모델 및 이의 제조방법에 관한 것이다.
The present invention relates to a method for producing a chronic obstructive pulmonary inflammation animal model due to autoimmune diseases, and more particularly to a new animal model of chronic obstructive pulmonary inflammation with a reduced immune tolerance to its own protein elastin.
만성 폐쇄성 폐 염증(chronic obstructive pulmonary disease; COPD)은 직/간접 흡연이나 카드뮴(Cd), 실리카(Si) 같은 환경 오염원을 직접 다루는 직업군에서 발생할 수 있고 드물게 a1-안티트립신 유전자의 이상에 의하여 발생할 수 있다. 만성 폐쇄성 폐 염증은 폐에 발생한 염증으로부터 조직을 보호하기 위해 생성된 점액질(mucus)이 과도하게 생성된 후 소실(clearance)에 문제가 발생하여 기도가 폐쇄되며, 폐포 세포(alveolar cell)의 파괴로 인하여 유발된 저산소증으로 사망에까지 이를 수 있는 치명적인 질환이다.Chronic obstructive pulmonary disease (COPD) can occur in occupational groups that deal directly or indirectly with smoking or directly address environmental pollutants such as cadmium (Cd) and silica (Si), and rarely due to abnormalities in the a1-antitrypsin gene. Can be. Chronic obstructive pulmonary inflammation is caused by excessive mucus produced to protect the tissue from inflammation in the lungs, and then causes problems with clearance, resulting in obstruction of the airways and the destruction of alveolar cells. It is a fatal disease caused by hypoxia caused by death.
만성 폐쇄성 폐 염증은 발생 조직과 메커니즘에 따라 만성 기관지염(chronic bronchitis) 및 폐기종(emphysema)으로 구분된다. 만성 기관지염의 가장 큰 특징은 기도 상피세포에서 과발현된 점액질이 제거되지 않고 계속해서 과발현되어 결과적으로 축적된 점액질로 인하여 기도가 폐쇄되는 것이 특징이며, 폐기종은 폐포 세포(alveolar cell)가 파괴되어 저산소증 또는 심장질환, 및 비가역적인 세기관지(bronchiole) 폐쇄성을 보이는 것이 특징이다. Chronic obstructive pulmonary inflammation is classified into chronic bronchitis and emphysema depending on the tissue and mechanism of development. The most characteristic of chronic bronchitis is that the overexpressed mucus in airway epithelial cells is not removed but is continuously overexpressed, resulting in obstruction of the airways due to the accumulated mucus. Emphysema is caused by the destruction of alveolar cells and Heart disease and irreversible bronchiole obstruction.
최근, 폐기종은 염증세포에 의하여 활성화된 엘라스타아제(elastase)에 의한 가수분해로 인하여 폐포 세포가 비가역적으로 파괴되며, 조직병리학적으로 CD8+, T 림프구, 대식세포(macrophage) 및 호중성 백혈구(neutrophil)가 증가하고, 자가 단백질인 엘라스틴 펩타이드에 대한 높은 자가 항체 세포가 검출된 연구결과가 발표되었다 (S. H. Lee et al., Nature Medicine , 13(5):567, 2007). 또한, 만성 폐쇄성 폐 염증 환자군에서 엘라스틴 펩타이드 자극에 의해 Th1 및 Th17에서 많은 양의 사이토카인이 분비되는 것을 확인하였으며, 만성 폐쇄성 폐 염증 환자군의 폐 조직에서 Th17 세포에 의한 면역반응이 증가하는 것을 보여 만성 폐쇄성 폐 염증은 자가 면역성 질환과 밀접한 관계가 있다는 것을 보여주고 있다 (M. Shan et al. Science Translational Medicine, 1(4):4ra10, 2009)Recently, emphysema is irreversible destruction of alveolar cells due to hydrolysis by elastase activated by inflammatory cells, histopathologically CD8 +, T lymphocytes, macrophage (macrophage) and neutrophils ( neutrophils were increased and high autoantibody cells were detected for the elastin peptide, a self protein (SH Lee et al.). al ., Nature Medicine , 13 (5): 567, 2007). In addition, it was confirmed that a large amount of cytokines were secreted in Th1 and Th17 by elastin peptide stimulation in the patients with chronic obstructive pulmonary inflammation, and the immune response by Th17 cells was increased in the lung tissues of the patients with chronic obstructive pulmonary inflammation. Obstructive pulmonary inflammation has been shown to be closely associated with autoimmune diseases (M. Shan et al. al . Science Translational Medicine, 1 (4): 4ra10, 2009)
이에 본 발명자들은, 만성 폐쇄성 폐 염증 질환이 자가 면역성 질환과 관련이 있다는 것을 확인하기 위해 자가 면역 기전에 의한 만성 폐쇄성 폐 염증(chronic obstructive pulmonary disease; COPD) 동물 모델을 제작하여 본 발명을 완성하게 되었다.
In order to confirm that chronic obstructive pulmonary inflammatory disease is associated with autoimmune diseases, the present inventors have completed the present invention by constructing a chronic obstructive pulmonary disease (COPD) animal model by an autoimmune mechanism. .
본 발명의 목적은 만성 폐쇄성 폐 염증 질환이 자가 면역성 질환과 관련이 있다는 것을 확인하기 위한 자가 면역 기전에 의한 만성 폐쇄성 폐 염증(chronic obstructive pulmonary disease; COPD) 동물 모델 및 이의 제조방법을 제공하는 데 있다.
It is an object of the present invention to provide a chronic obstructive pulmonary disease (COPD) animal model by autoimmune mechanism and a method for preparing the same to confirm that chronic obstructive pulmonary inflammatory disease is associated with an autoimmune disease. .
상기의 목적을 달성하기 위하여, 본 발명은 (a) 동물에 엘라스틴 펩타이드(elastin peptide)와 완전 프로인트 항원보강제(complete Freund's adjuvant; CFA)의 혼합물을 투여하여 엘라스틴에 대한 자기 면역관용(self-tolerance)을 파괴하는 단계; 및 (b) 상기 동물에 엘라스틴 펩타이드를 추가로 투여하여 엘라스틴의 자기면역에 의한 만성 폐쇄성 염증을 유도하는 단계를 포함하는 만성 폐쇄성 폐 염증(chronic obstructive pulmonary disease; COPD) 동물 모델의 제조방법을 제공한다. In order to achieve the above object, the present invention (a) self-tolerance against elastin by administering a mixture of elastin peptide and a complete Freund's adjuvant (CFA) to the animal Destroying; And (b) additionally administering an elastin peptide to the animal to induce chronic obstructive inflammation due to autoimmunity of elastin. .
본 발명은 또한, 상기 방법에 의해 제조되고, (ⅰ) 기도의 신전성(airway compliance) 증가; (ⅱ) 폐 구조의 비대화; (ⅲ) MMP9 및 MMP12의 mRNA 발현량 증가; (ⅳ) 혈청 중 IgG1 및 IgG2a의 농도 증가; (ⅴ) Th1 및 Th17에 특이적인 사이토카인인 IFN-γ 및 IL-17의 발현량 증가; 및 (ⅵ) 대식세포에 액포가 많이 생성 증가로 이루어진 특성 중에서 선택되는 하나 이상의 특성을 가지는 만성 폐쇄성 폐 염증 동물 모델을 제공한다. The present invention is also prepared by the above method, and (i) increases the airway compliance of the airways; (Ii) enlargement of the lung structure; (Iii) increased mRNA expression levels of MMP9 and MMP12; (Iii) increased concentrations of IgG1 and IgG2a in serum; (Iii) increased expression levels of IFN-γ and IL-17, cytokines specific for Th1 and Th17; And (iii) provides a chronic obstructive pulmonary inflammation animal model having one or more characteristics selected from the characteristics consisting of increased production of vacuoles in macrophages.
본 발명은 또한, 만성 폐쇄성 폐 염증 동물 모델에 만성 폐쇄성 폐 염증 치료제 또는 치료제 후보 물질을 투여하고 만성 폐쇄성 폐 염증 동물 모델의 폐 염증이 치유되는지 여부를 지표로 하여 상기 만성 폐쇄성 폐 염증 치료제를 스크리닝하는 방법을 제공한다.
The present invention also provides a method for screening the chronic obstructive pulmonary inflammation therapeutic agent by administering a chronic obstructive pulmonary inflammation therapeutic agent or a candidate agent to the chronic obstructive pulmonary inflammation animal model and indicating whether the lung inflammation of the chronic obstructive pulmonary inflammation animal model is cured. Provide a method.
본 발명에 의한 만성 폐쇄성 폐 염증(chronic obstructive pulmonary disease; COPD) 동물 모델에서 자가 면역 질환에 의해 만성 폐쇄성 폐 염증이 발병한다는 것을 확인하였으므로 만성 폐쇄성 폐 염증에 대한 근본적인 진단 및 치료제 개발에 용이하게 사용할 수 있다.
Chronic obstructive pulmonary disease (COPD) animal model according to the present invention has been confirmed that the development of chronic obstructive pulmonary inflammation by autoimmune disease, so it can be easily used for the development of a fundamental diagnosis and treatment for chronic obstructive pulmonary inflammation have.
도 1은 만성 폐쇄성 폐 염증 동물 모델의 기도의 신전성(airway compliance)의 변화를 나타낸 그림이다.
도 2는 만성 폐쇄성 폐 염증 동물 모델의 폐 조직의 병리결과를 나타낸 그림이다.
도 3은 만성 폐쇄성 폐 염증 동물 모델에서 엘라스틴 주입에 의한 MMP9 및 MMP12 mRNA의 증가를 나타낸 그래프이다.
도 4는 만성 폐쇄성 폐 염증 동물 모델에서 엘라스틴 특이적인 IgG1 및 IgG2a 항체의 양을 측정한 그래프이다.
도 5는 만성 폐쇄성 폐 염증 동물 모델에서 엘라스틴 자극에 의한 IFN-γ 및 IL-17의 생산 증가를 나타낸 그래프이다 (A; IFN-γ(+) CD4 T 세포, B; IL-17(+) CD4 T 세포, C; Vehicle 그의 IFN-γ 및 IL-17 생산 정도, D; 엘라스틴 처리 그룹의 IFN-γ 및 IL-17 생산 정도).
도 6은 만성 폐쇄성 폐 염증 동물 모델에서 엘라스틴 주입에 의한 대식세포의 변화를 나타낸 그림이다 (A; Vehicle 그룹, B; 엘라스틴 처리 그룹)1 is a diagram showing a change in the airway compliance (airway compliance) in the animal model of chronic obstructive pulmonary inflammation.
Figure 2 is a diagram showing the pathological results of lung tissue in chronic obstructive pulmonary inflammation animal model.
3 is a graph showing the increase of MMP9 and MMP12 mRNA by elastin injection in chronic obstructive pulmonary inflammation animal model.
4 is a graph measuring the amount of elastin-specific IgG1 and IgG2a antibodies in a chronic obstructive pulmonary inflammation animal model.
5 is a graph showing increased production of IFN-γ and IL-17 by elastin stimulation in a chronic obstructive pulmonary inflammation animal model (A; IFN-γ (+) CD4 T cells, B; IL-17 (+) CD4) T cells, C; Vehicle IFN-γ and IL-17 production levels, D; Elastin-treated group IFN-γ and IL-17 production levels).
Figure 6 is a diagram showing the change of macrophages by elastin injection in chronic obstructive pulmonary inflammation animal model (A; Vehicle group, B; Elastin treatment group)
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명은 일 관점에서, (a) 동물에 엘라스틴 펩타이드(elastin peptide)와 완전 프로인트 항원보강제(complete Freund's adjuvant; CFA)의 혼합물을 투여하여 엘라스틴에 대한 자기 면역관용(self-tolerance)을 파괴하는 단계; 및 (b) 상기 동물에 엘라스틴 펩타이드를 추가로 투여하여 엘라스틴의 자기면역에 의한 만성 폐쇄성 염증을 유도하는 단계를 포함하는 만성 폐쇄성 폐 염증(chronic obstructive pulmonary disease; COPD) 동물 모델의 제조방법에 관한 것이다. In one aspect, the present invention provides a method of destroying self-tolerance for elastin by administering a mixture of elastin peptide and complete Freund's adjuvant (CFA) to an animal. step; And (b) additionally administering an elastin peptide to the animal to induce chronic obstructive inflammation due to autoimmunity of elastin. 2. A method for producing a chronic obstructive pulmonary disease (COPD) animal model .
본 발명에 있어서, (a)단계의 투여는 동물의 둔부 또는 꼬리의 피하(subcutaneous)로 투여하는 것을 특징으로 할 수 있다. In the present invention, the administration of step (a) may be characterized in that the subcutaneous administration (subcutaneous) of the buttocks or tail of the animal.
본 발명에 있어서, (b)단계의 투여는 동물의 비강(intranasal)으로 투여하는 것을 특징으로 할 수 있다. In the present invention, the administration of step (b) may be characterized in that the administration to the animal nasal (intranasal).
본 발명에 있어서, (b)단계의 엘라스틴 펩타이드는 엘라스틴 펩타이드와 완전 프로인트 항원보강제(CFA)의 혼합물을 접종한 후 20일 내지 22일 후에 추가로 투여하는 것을 특징으로 할 수 있다. In the present invention, the elastin peptide of step (b) may be further administered 20 to 22 days after the inoculation of the mixture of the elastin peptide and the complete Freund's adjuvant (CFA).
상기 투여는 당업계에 공지된 다양한 방법을 통해 실시할 수 있으며, 경구 투여법, 피내 주사법, 피하 주사법, 근육내 주사법, 복강내 주사법 및 정맥내 주사법 등 실험조건에 따라 약물을 효율적으로 전달할 수 있는 적절한 방법을 선택하여 사용할 수 있다.The administration may be carried out through a variety of methods known in the art, and can efficiently deliver drugs according to experimental conditions such as oral administration, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection and intravenous injection. The appropriate method can be selected and used.
본 발명에 있어서, 상기 동물은 마우스인 것을 특징으로 할 수 있다. In the present invention, the animal may be characterized in that the mouse.
본 발명에서 이용되는 동물은 인간을 제외한 어떠한 동물도 가능하며, 바람직하게는 포유동물, 더욱 바람직하게는 설치류 동물이며, 가장 바람직하게는 마우스인 것을 특징으로 할 수 있다. The animal used in the present invention may be any animal except human, preferably a mammal, more preferably a rodent, and most preferably a mouse.
본 발명의 일 실시예에서, 자가 면역 기전에 의해 만성 폐쇄성 폐 염증이 발명되는지 확인하기 위한 동물모델을 확립하기 위하여, 자가 단백질은 엘라스틴을 면역 유도 물질인 완전프로인트항원보강제(complete Freund's adjuvant; CFA)와 함께 주입하여 자기 면역관용(self-tolerance)을 파기함으로써 만성 폐쇄성 폐 염증 모델을 확립하였다. In one embodiment of the present invention, in order to establish an animal model for identifying whether chronic obstructive pulmonary inflammation is invented by an autoimmune mechanism, the autologous protein may be selected from elastin as a complete Freund's adjuvant (CFA), an immune inducing substance. ) To establish a model of chronic obstructive pulmonary inflammation by injecting with) to destroy self-tolerance.
엘라스틴 펩타이드를 CFA와 함께 섞어 각 그룹별로 마우스의 꼬리에 피하 주입하였다. 그룹 I (아세트산, 50㎕), 그룹 II (4㎎/㎖ CFA 50㎕ + 아스트산 50㎕), 그룹 III (4㎎/㎖ CFA 50㎕ + 2㎎/㎖ 엘라스틴 펩타이드(bovine lung elastin in acetic acid) 50㎕)로 나누어 총 100㎕이 되도록 투여하였으며, 각 그룹당 4마리의 마우스를 사용하였다. 부스팅(boosting)을 위해서 상기 투여 3주 후에 그룹 I 및 II의 경우 PBS(50㎕)를 마우스의 비강을 통해 주입하였으며, 그룹 III는 엘라스틴 펩타이드(bovine neck ligament elastin peptide) 50㎕(1㎎/㎖)을 마우스의 비강을 통해 주입하였다. 투여 1 내지 2개월 후에 조직 검사 및 면역학적 특성을 분석하여 엘라스틴 분해에 의한 만성 폐쇄성 폐 염증을 발병 정도를 확인하였다. 양성 대조군으로는 엘라스테이즈(porcine pancreatic elastase: PPE)를 주입하였다. 아세트산은 엘라스틴을 녹이기 위한 용매로 사용하였다. Elastin peptides were mixed with CFA and injected subcutaneously into the tails of mice in each group. Group I (acetic acid, 50 μl), group II (4 μg / ml CFA 50 μl + astaxic acid 50 μl), group III (4 mg / ml CFA 50 μl + 2 mg / ml elastin peptide (bovine lung elastin in acetic acid) A total of 100 µl was used and 4 mice were used in each group. Three weeks after the administration, PBS (50 μl) was injected through the nasal cavity of mice for boosting, and group III 50 μl (1 mg / mL of bovine neck ligament elastin peptide). ) Was injected through the nasal cavity of mice. After 1 to 2 months of administration, histological and immunological characteristics were analyzed to confirm the incidence of chronic obstructive pulmonary inflammation due to elastin degradation. As a positive control, elastase (porcine pancreatic elastase (PPE)) was injected. Acetic acid was used as a solvent to dissolve elastin.
본 발명의 자가 면역 기전에 의한 동물모델은 각 그룹당 4마리의 마우스를 사용하여 상기와 같은 실험을 진행한 결과, 4마리 중 3마리에서 자가 면역 기전에 의한 만성 폐쇄성 폐 염증이 발명하는 것을 확인하여, 75%의 성공율을 보이는 것을 확인하였으며, 3번의 반복실험을 통해 일관된 결과를 얻었다. Animal model according to the autoimmune mechanism of the present invention was carried out as described above using four mice in each group, it was confirmed that chronic obstructive pulmonary inflammation by the autoimmune mechanism in three of four , 75% success rate was confirmed, and consistent results were obtained through three repeated experiments.
본 발명은 다른 관점에서, 상기 방법에 의해 제조되고, (ⅰ) 기도의 신전성(airway compliance) 증가; (ⅱ) 폐 구조의 비대화; (ⅲ) MMP9 및 MMP12의 mRNA 발현량 증가; (ⅳ) 혈청 중 IgG1 및 IgG2a의 농도 증가; (ⅴ) Th1 및 Th17에 특이적인 사이토카인인 IFN-γ 및 IL-17의 발현량 증가; 및 (ⅵ) 대식세포에 액포가 많이 생성 증가로 이루어진 특성 중에서 선택되는 하나 이상의 특성을 가지는 만성 폐쇄성 폐 염증 동물 모델에 관한 것이다. In another aspect, the invention is prepared by the above method, comprising: (i) increasing airway compliance of the airways; (Ii) enlargement of the lung structure; (Iii) increased mRNA expression levels of MMP9 and MMP12; (Iii) increased concentrations of IgG1 and IgG2a in serum; (Iii) increased expression levels of IFN-γ and IL-17, cytokines specific for Th1 and Th17; And (iii) an increase in the production of vacuoles in macrophages.
본 발명의 만성 폐쇄성 폐 염증 동물 모델의 만성 폐쇄성 폐 염증의 발병 여부를 확인하기 위하여 만성 폐쇄성 폐 염증의 증상 중에 하나인 기도의 신전성(airway compliance)를 확인하기 위하여 실험을 수행한 결과, 엘라스틴 펩타이드를 CFA와 함께 주입하여 자기 면역 관용(self-tolerance)을 파기함으로써 기도의 신전성(airway compliance)가 증가하는 것을 확인하였다 (도 1). 또한, 기도의 재형성/기종(airway remodeling/emphysematous)의 변화를 조직학적 검사를 통해 확인한 결과, 아세트산을 주입한 그룹 1에서는 정상적인 폐 구조를 보였으며(도 2A), CFA만 주입한 그룹 2에서는 폐의 구조가 약간 비대하게 변한 것을 관찰하였다 (도 2B). 엘라스틴과 CFA를 함께 주입한 그룹 3에서는 폐 구조가 확장되어 비대화된 것을 확인하였으며(도 2C), 폐 조직의 신전성(compliance) 증가는 엘라스틴 주입에 의한 폐 구조의 비대화에 기인함을 확인할 수 있었다. In order to confirm the development of chronic obstructive pulmonary inflammation in the animal model of chronic obstructive pulmonary inflammation of the present invention, an experiment was performed to confirm the airway compliance, one of the symptoms of chronic obstructive pulmonary inflammation, elastin peptide Was injected with CFA to destroy autoimmunity (self-tolerance) was confirmed to increase the airway (airway compliance) (Fig. 1). In addition, changes in airway remodeling / emphysematous were confirmed by histological examination and showed normal lung structure in
상기에서와 같이 폐 구조의 변화를 유발하는 엘라스틴 분해효소인 MMP-9(Matrix Metalloproteases-9) 및 MMP12의 mRNA 발현정도를 확인하기 위해 폐 조직을 분리하여 RT-PCR(real time PCR)을 수행한 결과, 아세트산을 주입한 군과 엘라스틴 펩타이드를 주입한 군을 비교하였을 때, 엘라스틴 펩타이드를 주입한 군에서 엘라스틴 분해효소인 MMP-9 및 MMP-12의 mRNA의 발현량이 증가하는 것을 확인하였다 (도 3).As described above, RT-PCR (real time PCR) was performed by separating lung tissues to determine the mRNA expression levels of elastin degrading enzymes MMP-9 (Matrix Metalloproteases-9) and MMP12, which cause changes in lung structure. As a result, when comparing the group injected with acetic acid and the group injected with elastin peptide, it was confirmed that the expression level of mRNA of elastin degrading enzymes MMP-9 and MMP-12 increased in the group injected with elastin peptide (FIG. 3). ).
본 발명의 만성 폐쇄성 폐 염증 동물 모델에서 엘라스틴에 대한 자가 면역반응을 살펴보기 위해, 마우스의 혈청에 존재하는 항-엘라스틴(anti-elastin)의 자가 항체를 modified ELISA을 이용하여 측정한 결과, 아세트산을 투여한 그룹 1 및 CFA를 투여한 그룹 2에서는 IgG1 및 IgG2a의 자가 항체의 발현이 증가하지 않았으며, 엘라스틴과 CFA를 동시에 투여한 그룹 3에서는 IgG1 및 IgG2a의 자가 항체의 발현양이 증가한 것을 확인하였다 (도 4). IgG2a는 도움 T세포(Th1)와 B세포의 상호작용을 통해 분비되는 것으로 알려져 있다.In order to examine the autoimmune response to elastin in the chronic obstructive pulmonary inflammation animal model of the present invention, anti-elastin autoantibodies in the serum of mice were measured by modified ELISA, and acetic acid was determined. The expression of IgG1 and IgG2a autoantibodies did not increase in
또한, 만성 폐쇄성 폐 염증 동물 모델에서 T세포의 면역반응을 살펴보기 위해 마우스의 폐 조직에 존재하는 T세포를 분리한 후, 엘라스틴 펩타이드로 T세포를 활성화시켜 사이토카인의 생성정도를 확인한 결과, 엘라스틴 펩타이드와 CFA를 동시에 투여한 그룹에서 Th1 및 Th17에 특이적인 사이토카인인 IFN-γ 및 IL-17이 증가하는 것을 확인하였다 (도 5).In addition, in order to examine the immune response of T cells in the chronic obstructive pulmonary inflammation animal model, T cells present in the lung tissue of mouse were isolated and activated with T cells with elastin peptide to confirm the level of cytokine production. It was confirmed that IFN-γ and IL-17, which are cytokines specific for Th1 and Th17, were increased in the group administered with peptide and CFA simultaneously (FIG. 5).
엘라스틴 펩타이드를 투여한 군의 염증반응 정도를 분석하기 위하여 마우스의 기도에 삽관한 후에 기관지 폐포 세척(Bronchoalveolar lavage)를 수행하여엘라스틴을 분해하는 효소인 MMP12를 분비한다고 알려져 있는 염증세포인 대식세포를 관찰한 결과, 대식세포에 액포가 많이 생성한 것을 확인하였다 (도 6).In order to analyze the inflammatory response of the elastin peptide group, bronchial alveolar lavage was performed after insertion into the airway of the mouse to observe macrophages, inflammatory cells known to secrete MMP12, an enzyme that degrades elastin. As a result, it was confirmed that many vacuoles were generated in the macrophages (FIG. 6).
본 발명의 만성 폐쇄성 폐 염증 동물 모델은 엘라스틴 펩타이드를 주입하여 만성 폐쇄성 폐 염증 증상이 나타나는 것을 기도의 신전성(airway compliance), 폐 병리 및 MMP9/12의 증가로 확인하였으며, 이러한 증상은 엘라스틴에 특이적인 Th1 및 Th17의 면역반응에 의해 일어나는 것을 사이토카인 생성으로 확인하였다. Chronic obstructive pulmonary inflammation animal model of the present invention was confirmed by the injection of elastin peptide, symptoms of chronic obstructive pulmonary inflammation by increasing airway compliance, lung pathology and MMP9 / 12, these symptoms are specific to elastin It was confirmed by cytokine production that was caused by the immune response of Th1 and Th17.
본 발명은 또한, 자가 면역 반응에 기반을 둔 항-엘라스틴 면역성을 이용해 만성 폐쇄성 폐 염증 동물 모델을 확립하여 만성 폐쇄성 폐 염증이 자가 면역성 기전으로 발병한다는 사실을 확인할 수 있었다. The present invention also established an animal model of chronic obstructive pulmonary inflammation using anti-elastin immunity based on an autoimmune response to confirm that chronic obstructive pulmonary inflammation develops as an autoimmune mechanism.
본 발명은 또 다른 관점에서, 만성 폐쇄성 폐 염증 동물 모델에 만성 폐쇄성 폐 염증 치료제 또는 치료제 후보 물질을 투여하고 만성 폐쇄성 폐 염증 동물 모델의 폐 염증이 치유되는지 여부를 지표로 하여 상기 만성 폐쇄성 폐 염증 치료제를 스크리닝하는 방법에 관한 것이다.
In another aspect, the present invention is directed to a chronic obstructive pulmonary inflammation animal model of chronic obstructive pulmonary inflammation therapeutic agent or therapeutic agent candidates, and the chronic obstructive pulmonary inflammation therapeutic agent as an indicator of whether the pulmonary inflammation of the chronic obstructive pulmonary inflammation animal model is cured. It relates to a method of screening.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
실시예Example 1: 자가 면역 기전에 의한 동물모델 확립 1: Establish animal model by autoimmune mechanism
자가 면역 기전에 의해 만성 폐쇄성 폐 염증이 발명되는지 확인하기 위한 동물모델을 확립하기 위하여, 자가 단백질은 엘라스틴을 면역 유도 물질인 완전 프로인트항원보강제(complete Freund's adjuvant; CFA)와 함께 주입하여 자기 면역관용(self-tolerance)을 파기함으로써 만성 폐쇄성 폐 염증 모델을 확립하였다. To establish an animal model to confirm whether chronic obstructive pulmonary inflammation is invented by autoimmune mechanisms, autologous proteins are injected with elastin with a complete Freund's adjuvant (CFA), an immune inducing substance, for autoimmune tolerance. By destroying self-tolerance, a chronic obstructive pulmonary inflammation model was established.
먼저, 엘라스틴 펩타이드를 CFA와 함께 섞어 각 그룹별로 8주령의 female wild type C57BL/6 마우스의 꼬리에 피하 주입하여 엘라스틴에 대한 면역관용을 파괴하였다. 그룹 I (아세트산, 50㎕), 그룹 II (4㎎/㎖ CFA 50㎕ + 아스트산 50㎕), 그룹 III (4㎎/㎖ CFA 50㎕ + 2㎎/㎖ 엘라스틴 펩타이드(bovine lung elastin in acetic acid) 50㎕)로 나누어 총 100㎕이 되도록 투여하였으며, 각 그룹당 4마리의 마우스를 사용하였다. 부스팅(boosting)을 위해서 상기 투여 3주 후에 그룹 I 및 II의 경우 PBS(50㎕)를 마우스의 비강을 통해 주입하였으며, 그룹 III는 엘라스틴 펩타이드(bovine neck ligament elastin peptide) 50㎕(1㎎/㎖)을 마우스의 비강을 통해 주입하였다. 투여 1 내지 2개월 후에 조직 검사 및 면역학적 특성을 분석하여 엘라스틴 분해에 의한 만성 폐쇄성 폐 염증을 발병 정도를 확인하였다. 양성 대조군으로는 엘라스테이즈(porcine pancreatic elastase: PPE)를 주입하였다. 아세트산은 엘라스틴을 녹이기 위한 용매로 사용하였다. First, elastin peptides were mixed with CFA and subcutaneously injected into the tails of 8-week-old female wild type C57BL / 6 mice in each group to destroy immune tolerance to elastin. Group I (acetic acid, 50 μl), group II (4 μg / ml CFA 50 μl + astaxic acid 50 μl), group III (4 mg / ml CFA 50 μl + 2 mg / ml elastin peptide (bovine lung elastin in acetic acid) A total of 100 µl was used and 4 mice were used in each group. Three weeks after the administration, PBS (50 μl) was injected through the nasal cavity of mice for boosting, and group III 50 μl (1 mg / mL of bovine neck ligament elastin peptide). ) Was injected through the nasal cavity of mice. After 1 to 2 months of administration, histological and immunological characteristics were analyzed to confirm the incidence of chronic obstructive pulmonary inflammation due to elastin degradation. As a positive control, elastase (porcine pancreatic elastase (PPE)) was injected. Acetic acid was used as a solvent to dissolve elastin.
자가 면역 기전에 의한 동물모델의 확립을 위해 각 그룹 당 4마리의 마우스를 사용하여 상기와 같은 실험을 진행한 결과, 4마리 중 3마리에서 자가 면역 기전에 의한 만성 폐쇄성 폐 염증이 발병하여 약 75%의 성공율을 보였으며, 3번의 반복실험을 통해 일관된 결과를 얻었다.
In order to establish an animal model by autoimmune mechanism, the experiment was conducted using four mice in each group. As a result, three out of four animals developed chronic obstructive pulmonary inflammation due to autoimmune mechanism. The success rate was% and consistent results were obtained through 3 replicates.
실시예Example 2: 만성 폐쇄성 폐 염증 동물 모델의 만성 폐쇄성 폐 염증 발병 확인 2: Confirmation of Chronic Obstructive Pulmonary Inflammation in Animal Model of Chronic Obstructive Pulmonary Inflammation
2-1 : 기도의 2-1: prayer 신전성Shrine (( airwayairway compliancecompliance ) 측정) Measure
만성 폐쇄성 폐 염증의 증상 중에 하나인 기도의 신전성(airway compliance)를 확인하기 위하여 flexiVent system(SCIREQ Inc, Montreal, Canada)을 이용하여 측정하였다.To confirm airway compliance, one of the symptoms of chronic obstructive pulmonary inflammation, it was measured using the flexiVent system (SCIREQ Inc, Montreal, Canada).
먼저 마우스에 펜토바르비탈 나트륨(pentobarbital sodium; anlim Pharma Co., Seoul, Korea) 60㎎/㎏을 주입하여 마취시킨 다음 20 게이지 캐뉼라(gauge cannula)를 이용하여 기관내삽관(intubation)을 하였다. 벤틸레이션(Ventilation)을 실시한 후 브롬화 판크로늄(pancuronium bromide) 1㎎/㎖을 투여하여 마우스를 마비시킨 다음 신전성(compliance)을 측정하였다. 벤틸레이션(Ventilation)은 분당호흡수(respiratory rate)는 150 breaths/min로 하였고, 일호흡용적(tidal volume)은 3 CmH2O의 호기말 양압 호흡(positive end expiratory pressure; PEEP)으로 하여 10㎎/㎏으로 조절하여 세팅하였다. First, anesthetized with pentobarbital sodium (pentobarbital sodium; anlim Pharma Co., Seoul, Korea) 60 mg / kg, followed by endotracheal intubation using a 20 gauge cannula. After ventilation, the mice were paralyzed by administering 1 mg / ml of pancuronium bromide and then measured for compliance. Ventilation rate was 150 breaths / min, and tidal volume was 10 mg with positive end expiratory pressure (PEEP) of 3 CmH 2 O. The setting was adjusted to / kg.
그 결과, 엘라스틴 펩타이드를 CFA와 함께 주입하여 자기 면역 관용(self-tolerance)을 파기함으로써 만성 폐쇄성 폐 염증의 증상 중에 하나인 기도의 신전성(airway compliance)이 증가하는 것을 확인하였다 (도 1).
As a result, it was confirmed that airway compliance, which is one of the symptoms of chronic obstructive pulmonary inflammation, was increased by injecting elastin peptide with CFA to destroy self-tolerance (FIG. 1).
2-1 : 폐 병리 조직 검사 2-1: Lung Pathology Histology
기도의 재형성/기종(airway remodeling/emphysematous)의 변화를 조직학적 검사를 통해 살펴보았다. 먼저 마우스의 기도에 삽관된 튜브에 실린지를 연결하고 50℃ 정도에 용해된 아가로오스(low melting agarose)를 약 1㎖ 볼륨으로 기도를 통해 폐 안으로 넣어 준 후, 아가로오스가 굳기까지 1시간 동안 방치한다. 그 후 조직을 H&E 염색을 통하여 폐 병리 변화를 관찰하였다. Changes in airway remodeling / emphysematous were examined by histological examination. First, the syringe is connected to the tube inserted into the airway of the mouse, and agarose (low melting agarose) dissolved at about 50 ° C. is introduced into the lungs through the airways at a volume of about 1 ml. Leave it for a while. The tissues were then examined for changes in lung pathology through H & E staining.
그 결과, 아세트산을 주입한 그룹 1에서는 정상적인 폐 구조를 보였으며(도 2A), CFA만 주입한 그룹 2에서는 폐의 구조가 약간 비대하게 변한 것을 관찰하였다 (도 2B). 엘라스틴과 CFA를 함께 주입한 그룹 3에서는 폐 구조가 확장되어 비대화된 것을 확인하였으며(도 2C), 폐 조직의 신전성(compliance) 증가는 엘라스틴 주입에 의한 폐 구조의 비대화에 기인함을 확인할 수 있었다.
As a result, in the
2-3 : 폐 조직 분리 및 2-3: lung tissue separation and RTRT -- RCRRCR 실시 practice
폐 구조의 변화를 유발하는 엘라스틴 분해효소인 MMP-9(Matrix Metalloproteases-9) 및 MMP12의 mRNA 발현정도를 확인하기 위해 폐 조직을 분리하여 RT-PCR(reverse transcription polymerase chain reaction)을 수행하였다.In order to check the mRNA expression levels of elastin degrading enzymes MMP-9 (Matrix Metalloproteases-9) and MMP12 that cause changes in lung structure, lung tissues were separated and RT-PCR (reverse transcription polymerase chain reaction) was performed.
마우스의 폐를 적출하여 total RNA RNeasy Mini kit (Qiagen, Chatsworth, CA)을 이용하여 mRNA를 분리 한 후, SuperScript one-step RT-PCR kit (Invitrogen, Carlsbad, CA)을 이용하여 RT-PCR을 실시하였다. RT-PCR은 SYBR Green detection 방법을 이용하여 MMP-9, MMP-12 및 GAPDH에 대한 mRNA의 발현 정도를 확인하였다.
MRNA was extracted from the lungs of the mouse using total RNA RNeasy Mini kit (Qiagen, Chatsworth, CA), and then RT-PCR was performed using the SuperScript one-step RT-PCR kit (Invitrogen, Carlsbad, CA). It was. RT-PCR confirmed the expression level of mRNA for MMP-9, MMP-12 and GAPDH using the SYBR Green detection method.
MMP-12
GAPDH
그 결과, 대조군(vehicle)인 아세트산을 주입한 군과 엘라스틴 펩타이드를 주입한 군을 비교하였을때, 엘라스틴 펩타이드를 주입한 군에서 엘라스틴 분해효소인 MMP-9 및 MMP-12의 mRNA의 발현량이 증가하는 것을 확인하였다 (도 3).
As a result, when comparing the group injected with acetic acid as a control group and the group injected with elastin peptide, the expression amount of mRNA of elastin degrading enzymes MMP-9 and MMP-12 increased in the group injected with elastin peptide. It was confirmed (Fig. 3).
실시예Example 4: 엘라스틴에 대한 자가 항체 4: autoantibodies against elastin 역가Potency 측정 Measure
엘라스틴에 대한 자가 면역반응을 살펴보기 위해, 생쥐의 혈청에 존재하는 엘라스틴 특이적인 IgG1 및 IgG2a 항체의 양을 modified ELISA을 이용하여 측정하였다. To examine the autoimmune response to elastin, the amount of elastin-specific IgG1 and IgG2a antibodies present in the serum of mice was measured using a modified ELISA.
먼저 PBS에 녹인 0.2㎎/㎖의 엘라스틴 펩타이드(bovine lung elastin 또는 mouse elastin peptide)를 8㎍/well이 되도록 각 웰 마다 40㎕씩 분주하여 37℃에서 2시간 배양하여 ELISA plate에 코팅하고 unbound 엘라스틴 펩타이드는 0.05% Tween이 포함된 PBS로 세척한 후, 0.2%의 I-block을 이용하여 37℃에서 2시간 동안 블로킹(blocking)을 실시하였다. 1:10 또는 1:50으로 희석한 혈청을 4℃에서 하룻밤 동안 방치한 후, biotinylated chicken anti-mouse IgG1 또는 IgG2a를 0.2㎍/㎖을 40㎕ (8ng/well)씩 첨가하여 37℃에서 2시간 동안 2차 배양을 실시하였다. 세척 후에 알칼라인 포스파테이즈(alkaline phosphatase)가 컨쥬게이션(conjugation)된 스트렙타아비딘(streptavidin)을 첨가하여(original stock에서 1:1000으로 희석) 상온에서 30분 동안 반응시킨 후, 70㎕의 5mM의 nitrophenyl phosphate substrate를 첨가하여 반응을 시키고, 40㎕의 0.5N NaOH를 첨가하여 반응을 정지 시킨 후, 405nm에서 ELISA 리더를 이용해 색 변화를 측정하였다. First, 40 μl of 0.2 mg / ml elastin peptide (bovine lung elastin or mouse elastin peptide) dissolved in PBS was dispensed into each well for 8 ug / well, incubated at 37 ° C. for 2 hours, and coated on an ELISA plate. After washing with PBS containing 0.05% Tween, blocking was performed for 2 hours at 37 ° C using 0.2% I-block. Serum diluted to 1:10 or 1:50 was allowed to stand overnight at 4 ° C, and then 0.2 µg / ml of biotinylated chicken anti-mouse IgG1 or IgG2a was added 40 µl (8ng / well) for 2 hours at 37 ° C. During the second culture. After washing, the mixture was reacted for 30 minutes at room temperature by adding streptavidin conjugated with alkaline phosphatase (conjugated 1: 1000 in the original stock), followed by 70 µl of 5 mM After adding nitrophenyl phosphate substrate, the reaction was stopped, and 40 µl of 0.5N NaOH was added to stop the reaction. Then, color change was measured using an ELISA reader at 405 nm.
그 결과, 아세트산을 투여한 그룹 1 및 CFA를 투여한 그룹 2에서는 IgG1 및 IgG2a의 자가 항체의 발현이 증가하지 않았으며, 엘라스틴과 CFA를 동시에 투여한 그룹 3에서는 IgG1 및 IgG2a의 자가 항체의 발현양이 증가한 것을 확인하였다 (도 4). IgG2a는 도움 T세포(Th1)와 B세포의 상호작용을 통해 분비되는 것으로 알려져 있음.
As a result, the expression of the IgG1 and IgG2a autoantibodies did not increase in the
실시예Example 5: 5: CD4CD4 ++ T 세포의 분리 및 엘라스틴 Isolation of T Cells and Elastin 펩타이드Peptides 자극에 의한 사이토카인 생성 분석 Analysis of cytokine production by stimulation
T세포의 면역반응을 살펴보기 위해 마우스의 폐 조직에 존재하는 T세포를 분리한 후, 엘라스틴 펩타이드로 T세포를 활성화시켜 사이토카인의 생성 정도를 확인하였다. In order to examine the immune response of the T cells, T cells present in the lung tissue of the mouse were isolated, and then activated the T cells with elastin peptides to confirm the level of cytokine production.
폐 조직을 적출하여 단일세포 현탁액(single cell suspension)을 준비하여 magnetic activated cell sorting(MACS)를 이용하여 CD4+ T세포를 분리하였다. 분리한 CD4+ T세포(2 × 105 cell)에 10㎍의 엘라스틴 펩타이드(mouse lung elastin peptide)를 처리하여 활성화시킨 후, 세포 내 사이토카인 염색(intracellular cytokine staining)을 실시하여 IFN-γ(APC) 및 IL-17(PE)의 생성 정도를 유세포분석기(Fluorescence-activated cell sorting; FACS)로 분석하였으며, 데이타 분석은 FlowJo 프로그램을 사용하였다. Lung tissue was extracted to prepare a single cell suspension, and CD4 + T cells were isolated using magnetic activated cell sorting (MACS). The isolated CD4 + T cells (2 × 10 5 cells) were activated by treatment with 10 ug of elastin peptide (mouse lung elastin peptide), followed by intracellular cytokine staining to perform IFN-γ (APC). ) And IL-17 (PE) production were analyzed by Fluorescence-activated cell sorting (FACS), and data analysis was performed using FlowJo program.
그 결과, 엘라스틴 펩타이드와 CFA를 동시에 투여한 그룹에서 Th1 및 Th17에 특이적인 사이토카인인 IFN-γ 및 IL-17이 증가하는 것을 확인하였다 (도 5).
As a result, it was confirmed that IFN-γ and IL-17, which are cytokines specific for Th1 and Th17, were increased in the group administered with the elastin peptide and CFA simultaneously (FIG. 5).
실시예Example 6: 기관지 폐포 세척 세포액( 6: bronchial alveolar lavage cell fluid ( BronchoalveolarBronchoalveolar lavagelavage ; ; BALBAL )의 조직학적 검사.Histological examination.
엘라스틴 펩타이드를 투여한 군의 염증반응 정도를 분석하기 위하여 마우스의 기도에 삽관한 후에 기관지 폐포 세척(Bronchoalveolar lavage)를 수행하였다. 기관지 폐포 세척액 200㎕를 사이토스핀(cytospin)을 이용하여 세척액에 세포를 모아준 후, 사이토스핀 슬라이드를 김자액(Giemsa)으로 염색하여 관찰하였다. Bronchoalveolar lavage was performed after intubation into the airways of mice to analyze the degree of inflammatory response in the elastin peptide group. After 200 μl of bronchoalveolar lavage fluid was collected in the wash solution by using cytospin, cytospin slides were observed by staining with Giezsa solution.
그 결과, 엘라스틴 펩타이드를 투여한 군에서 엘라스틴을 분해하는 효소인 MMP12를 분비한다고 알려져 있는 염증세포인 대식세포에 액포가 많이 생성한 것을 관찰하였다 (도 6).
As a result, in the group to which the elastin peptide was administered, it was observed that a large amount of vacuole was generated in macrophages, which are inflammatory cells known to secrete MMP12, an enzyme that degrades elastin (FIG. 6).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
서열목록 전자파일 첨부Attach an electronic file to a sequence list
Claims (7)
(a) 인간을 제외한 동물에 엘라스틴 펩타이드(elastin peptide)와 완전 프로인트 항원보강제(complete Freund's adjuvant; CFA)의 혼합물을 투여하여 엘라스틴에 대한 자기 면역관용(self-tolerance)을 파괴하는 단계; 및
(b) 상기 엘라스틴 펩타이드와 완전 프로인트 항원보강제(CFA)의 혼합물을 접종한 후 20일 내지 22일 후에 엘라스틴 펩타이드를 추가로 투여하여 엘라스틴의 자기면역에 의한 만성 폐쇄성 염증을 유도하는 단계.
Methods for preparing a chronic obstructive pulmonary disease (COPD) animal model comprising the following steps:
(a) administering a mixture of elastin peptides and complete Freund's adjuvant (CFA) to animals other than humans to destroy self-tolerance to elastin; And
(b) 20 to 22 days after inoculating the mixture of the elastin peptide and the complete Freund's adjuvant to induce chronic obstructive inflammation due to autoimmunity of elastin.
The method of claim 1, wherein the administration of step (a) is performed subcutaneously of the buttocks or tails of animals other than humans.
The method of claim 1, wherein the step (b) is administered to the nasal (intranasal) of animals other than humans.
The method of claim 1, wherein the animal except the human is a mouse.
Prepared by the method of any one of claims 1 to 3 and 5, and (i) increasing the airway compliance of the airways; (Ii) enlargement of lung tissue; (Iii) increased mRNA expression levels of MMP9 and MMP12; (Iii) increased concentrations of IgG1 and IgG2a in serum; (Iii) increased expression levels of IFN-γ and IL-17, cytokines specific for Th1 and Th17; And (iii) an increase in vacuole production of macrophages.
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