KR102208549B1 - Compositions for diagnosing, preventing or treating neutrophilic lung inflammatory diseases using G-CSF and IL-1β - Google Patents
Compositions for diagnosing, preventing or treating neutrophilic lung inflammatory diseases using G-CSF and IL-1β Download PDFInfo
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- KR102208549B1 KR102208549B1 KR1020190057637A KR20190057637A KR102208549B1 KR 102208549 B1 KR102208549 B1 KR 102208549B1 KR 1020190057637 A KR1020190057637 A KR 1020190057637A KR 20190057637 A KR20190057637 A KR 20190057637A KR 102208549 B1 KR102208549 B1 KR 102208549B1
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- neutrophilic
- csf
- asthma
- inflammatory disease
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Abstract
본 발명은 과립구집락자극인자(G-CSF) 및 인터루킨-1β(IL-1β)을 이용한 호중구성 폐 염증질환의 진단, 예방 또는 치료용 조성물에 관한 것이다. 본 발명에 따르면 G-CSF를 호중구성 폐 염증질환 진단용 바이오마커로 이용할 수 있으며, IL-1β는 호중구성 폐 염증질환의 치료 표적으로써 관련 분야에서 유용하게 활용될 수 있을 것으로 기대된다. The present invention relates to a composition for diagnosis, prevention or treatment of neutrophilic pulmonary inflammatory disease using granulocyte colony stimulating factor (G-CSF) and interleukin-1β (IL-1β). According to the present invention, G-CSF can be used as a biomarker for diagnosing neutrophilic pulmonary inflammatory disease, and IL-1β is expected to be useful in related fields as a therapeutic target for neutrophilic pulmonary inflammatory disease.
Description
본 발명은 과립구집락자극인자(G-CSF) 및 인터루킨-1β(IL-1β)를 이용한 호중구성 폐 염증질환의 진단, 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for diagnosis, prevention or treatment of neutrophilic pulmonary inflammatory disease using granulocyte colony stimulating factor (G-CSF) and interleukin-1β (IL-1β).
천식(Asthma)은 폐 속의 기관지가 아주 예민해진 상태로, 때때로 기관지가 좁아져서 숨이 차고 가랑가랑하는 숨소리가 들리면서 기침을 심하게 하는 증상을 나타내는 질환이다. 천식은 유전 및 환경적 요인이 합쳐져 생기는 대표적인 알레르기 질환으로, 공기가 흐르는 길인 기관지의 염증으로 기관지 점막이 부어오르고 기관지 근육이 경련을 일으키면서 기관지가 막혀서 숨이 차게 된다. 만성폐쇄성폐질환(chronic obstructive pulmonary disease; COPD)은 유해한 입자나 가스에 흡입에 의해 폐에 비정상적인 염증반응이 일어나면서 폐 기능이 저하되고 호흡곤란을 유발하게 되는 호흡기 질환이다. Asthma (Asthma) is a condition in which the bronchi in the lungs become very sensitive, and sometimes the bronchi are narrowed, causing shortness of breath and a crotch of breath, causing severe coughing. Asthma is a typical allergic disease caused by a combination of genetic and environmental factors. The bronchial mucosa is swollen due to inflammation of the bronchi, which is the path through which the air flows, and the bronchial muscles cause spasm, which leads to blockage of the bronchi and shortness of breath. Chronic obstructive pulmonary disease (COPD) is a respiratory disease in which an abnormal inflammatory reaction occurs in the lungs by inhalation of harmful particles or gases, resulting in decreased lung function and shortness of breath.
이러한 천식 및 COPD는 기도 또는 폐 조직에 침투하는 면역 세포의 유형 및 침윤 양상에 따라 호산구성(eosinophilic) 및 호중구성(neutrophilic)으로 세분화될 수 있다. 천식의 경우 중증도는 개개인에 따라 차이가 있으나 대부분 경증 또는 중등증을 보이는데, 1990년도에 들어 진행된 중증 불응성 천식 환자의 병리 연구에서 경증 천식에서 보이지 않았던 호중구성 염증의 존재가 알려졌다. 호중구성 천식은 천식 환자의 약 25%에서 나타난다고 알려져 있고, 전신 염증과도 높은 연관이 있으며 많은 경우 기존 천식 치료제인 스테로이드 제제에 저항성을 보인다고 보고되어 있다(J Leukoc Biol 98, 549-556 (2015)). 이러한 호중구성 천식 환자군은 전체 환자에서 차지하는 비율은 높지 않지만 전체 천식 치료에 발생하는 사회 경제적 비용의 약 50%를 차지할 정도로 치료 및 관리가 힘든 실정이다. 따라서 호중구성 천식의 면역학적 기전을 이해하고 이를 표적으로 하는 새로운 치료제 개발의 필요성이 대두되고 있으나, 아직까지 호중구성 천식의 발병 원인 및 메커니즘에 대해서는 잘 규명되어 있지 않은 실정이다. Such asthma and COPD can be subdivided into eosinophilic and neutrophilic, depending on the type and invasion pattern of immune cells that infiltrate the airway or lung tissue. In the case of asthma, the severity of asthma varies depending on the individual, but most of them show mild or moderate symptoms. In a pathology study of patients with severe refractory asthma in 1990, the presence of neutrophilic inflammation, which was not seen in mild asthma, was known. Neutrophilic asthma is known to occur in about 25% of asthma patients, is highly associated with systemic inflammation, and in many cases it has been reported to show resistance to conventional asthma treatment steroids (J Leukoc Biol 98, 549-556 (2015). )). The neutrophilic asthma patient group accounts for about 50% of the socio-economic costs incurred for the treatment of all asthma, although the proportion of all patients is not high. However, treatment and management are difficult. Therefore, there is a need to understand the immunological mechanism of neutrophilic asthma and develop a new therapeutic agent targeting it, but the causes and mechanisms of neutrophilic asthma have not been well elucidated.
이에, 본 발명자들은 호중구성 염증반응을 특징으로 하는 호중구성 천식을 포함한 호중구성 폐 염증질환을 효과적으로 진단 및 치료할 수 있는 물질을 발굴하기 위해 연구 노력한 결과, 호중구가 높게 존재하는 천식 모델에서 호중구 수준에 따라 G-CSF가 높게 존재하는 것을 확인하였으며, 인간 호중구성 천식 환자에서 IL-1β가 G-CSF의 생성을 유도하는 상관관계를 확인함으로써 호중구성 천식 동물모델에서 IL-1β 억제를 통한 항염증 효과를 확인하였는바, 이에 기초하여 본 발명을 완성하였다. Accordingly, the present inventors have tried to find a substance capable of effectively diagnosing and treating neutrophilic pulmonary inflammatory diseases, including neutrophilic asthma, characterized by a neutrophilic inflammatory response. Accordingly, it was confirmed that G-CSF is high, and by confirming the correlation that IL-1β induces the production of G-CSF in human neutrophilic asthma patients, anti-inflammatory effect through IL-1β inhibition in neutrophilic asthma animal model It was confirmed that the present invention was completed based on this.
이에, 본 발명은 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF) 유전자 또는 상기 유전자가 코딩하는 단백질을 포함하는, 호중구성 폐 염증질환 진단용 마커 조성물을 제공하는 것을 목적으로 한다. Accordingly, an object of the present invention is to provide a marker composition for diagnosing neutrophilic pulmonary inflammatory disease, comprising a granulocyte colony-stimulating factor (G-CSF) gene or a protein encoded by the gene.
또한, 본 발명은 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF) 유전자의 mRNA 또는 상기 유전자가 코딩하는 단백질 수준을 측정하는 제제를 포함하는, 호중구성 폐 염증질환 진단용 조성물 및 상기 조성물을 포함하는 호중구성 폐 염증질환 진단용 키트를 제공하는 것을 다른 목적으로 한다. In addition, the present invention provides a composition for diagnosing neutrophilic pulmonary inflammatory disease and the composition, including an agent for measuring the level of mRNA of a granulocyte colony-stimulating factor (G-CSF) gene or a protein encoded by the gene. Another object is to provide a kit for diagnosing neutrophilic pulmonary inflammatory disease, including.
또한, 본 발명은 피검체 유래의 생물학적 시료에서 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF) 유전자의 mRNA 또는 상기 유전자가 코딩하는 단백질 수준을 측정하는 단계를 포함하는, 호중구성 폐 염증질환의 진단을 위한 정보제공방법을 제공하는 것을 또 다른 목적으로 한다. In addition, the present invention comprises the step of measuring the level of the mRNA of a granulocyte colony-stimulating factor (G-CSF) gene or a protein encoded by the gene in a biological sample derived from a subject, neutrophilic lung inflammation Another object is to provide a method of providing information for diagnosis of diseases.
또한, 본 발명은 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF)의 억제제로써 인터루킨-1β(IL-1β) 억제제를 유효성분으로 포함하는, 호중구성 폐 염증질환의 예방 또는 치료용 약학적 조성물을 제공하는 것을 또 다른 목적으로 한다.In addition, the present invention contains an interleukin-1β (IL-1β) inhibitor as an active ingredient as an inhibitor of granulocyte colony-stimulating factor (G-CSF), a pharmaceutical for the prevention or treatment of neutrophilic pulmonary inflammatory disease Another object is to provide a suitable composition.
또한, 본 발명은 호중구성 폐 염증질환 치료물질의 스크리닝 방법을 제공하는 것을 또 다른 목적으로 한다.In addition, another object of the present invention is to provide a method for screening a substance for treating neutrophilic pulmonary inflammatory disease.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다. However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF) 유전자 또는 상기 유전자가 코딩하는 단백질을 포함하는, 호중구성 폐 염증질환 진단용 마커 조성물을 제공한다. In order to achieve the object of the present invention as described above, the present invention comprises a granulocyte colony-stimulating factor (G-CSF) gene or a protein encoded by the gene, a marker composition for diagnosing neutrophilic pulmonary inflammatory disease Provides.
본 발명의 일구현예로, 상기 G-CSF는 IL-1β에 의해 발현이 유도되는 것일 수 있다. In an embodiment of the present invention, the G-CSF may be induced by IL-1β.
본 발명의 다른 구현예로, 상기 G-CSF 유전자는 서열번호 1의 염기서열로 이루어진 것일 수 있다. In another embodiment of the present invention, the G-CSF gene may be composed of the nucleotide sequence of SEQ ID NO: 1.
본 발명의 또 다른 구현예로, 상기 G-CSF 유전자가 코딩하는 단백질은 서열번호 2의 아미노산 서열로 이루어진 것일 수 있다. In another embodiment of the present invention, the protein encoded by the G-CSF gene may consist of the amino acid sequence of SEQ ID NO: 2.
본 발명의 또 다른 구현예로, 상기 호중구성 폐 염증질환은 호중구성 천식(neutrophilic asthma), 호중구성 만성폐쇄성폐질환(neutrophilic chronic obstructive pulmonary disease), 특발성 폐섬유화증(idiopathic pulmonary fibrosis; IPF), 급성 폐 손상(acute lung injury; ALI) 및 급성 호흡곤란 증후군(acute respiratory distress syndrome; ARDS)으로 이루어진 군에서 선택되는 하나 이상인 것일 수 있다. In another embodiment of the present invention, the neutrophilic pulmonary inflammatory disease is neutrophilic asthma, neutrophilic chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis (IPF), It may be one or more selected from the group consisting of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS).
또한, 본 발명은 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF) 유전자의 mRNA 또는 상기 유전자가 코딩하는 단백질 수준을 측정하는 제제를 포함하는, 호중구성 폐 염증질환의 진단용 조성물을 제공한다. In addition, the present invention provides a composition for diagnosing neutrophilic pulmonary inflammatory disease, comprising an agent measuring the level of mRNA of a granulocyte colony-stimulating factor (G-CSF) gene or a protein encoded by the gene. .
본 발명의 일구현예로, 상기 mRNA 수준을 측정하는 제제는 유전자의 mRNA에 상보적으로 결합하는 센스 및 안티센스 프라이머, 또는 프로브일 수 있다. In one embodiment of the present invention, the agent for measuring the mRNA level may be a sense and antisense primer or probe that complementarily binds to the mRNA of a gene.
본 발명의 다른 구현예로, 상기 단백질 수준을 측정하는 제제는 유전자가 코딩하는 단백질에 특이적으로 결합하는 항체일 수 있다. In another embodiment of the present invention, the agent for measuring the protein level may be an antibody that specifically binds to a protein encoded by a gene.
또한, 본 발명은 상기 진단용 조성물을 포함하는, 호중구성 폐 염증질환 진단용 키트를 제공한다. In addition, the present invention provides a kit for diagnosing neutrophilic pulmonary inflammatory disease, including the diagnostic composition.
또한, 본 발명은 피검체 유래의 생물학적 시료에서 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF) 유전자의 mRNA 또는 상기 유전자가 코딩하는 단백질 수준을 측정하는 단계를 포함하는, 호중구성 폐 염증질환의 진단을 위한 정보제공방법을 제공한다. In addition, the present invention comprises the step of measuring the level of the mRNA of a granulocyte colony-stimulating factor (G-CSF) gene or a protein encoded by the gene in a biological sample derived from a subject, neutrophilic lung inflammation Provides information provision method for diagnosis of disease.
본 발명의 일구현예로, 상기 mRNA 수준은 중합효소연쇄반응(PCR), 역전사 중합효소연쇄반응(RT-PCR), 실시간 중합효소연쇄반응(Real-time PCR), RNase 보호 분석법(RNase protection assay; RPA), 마이크로어레이(microarray), 및 노던 블롯팅(northern blotting)으로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정될 수 있다. In one embodiment of the present invention, the mRNA level is polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay. ; RPA), microarray, and northern blotting (northern blotting) can be measured through one or more methods selected from the group consisting of.
본 발명의 다른 구현예로, 상기 단백질 발현수준은 웨스턴 블롯팅(western blotting), 방사선면역분석법(radioimmunoassay; RIA), 방사 면역 확산법(radioimmunodiffusion), 효소면역분석법(ELISA), 면역침강법(immunoprecipitation), 유세포분석법(flow cytometry), 면역형광염색법(immunofluorescence), 오우크테로니(ouchterlony), 보체 고정 분석법(complement fixation assay), 및 단백질 칩(protein chip)으로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정될 수 있다. In another embodiment of the present invention, the protein expression level is Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation (immunoprecipitation). , Flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and one or more methods selected from the group consisting of protein chips. It can be measured through.
또한, 본 발명은 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF)의 억제제로써 인터루킨-1β(IL-1β) 억제제를 유효성분으로 포함하는, 호중구성 폐 염증질환의 예방 또는 치료용 약학적 조성물을 제공한다. In addition, the present invention contains an interleukin-1β (IL-1β) inhibitor as an active ingredient as an inhibitor of granulocyte colony-stimulating factor (G-CSF), a pharmaceutical for the prevention or treatment of neutrophilic pulmonary inflammatory disease Provide the appropriate composition.
본 발명의 일구현예로, 상기 IL-1β 억제제는 상기 IL-1β 단백질에 특이적으로 결합하는 항-IL-1β 항체일 수 있다. In one embodiment of the present invention, the IL-1β inhibitor may be an anti-IL-1β antibody that specifically binds to the IL-1β protein.
또한, 본 발명은 하기의 단계를 포함하는, 호중구성 폐 염증질환 치료물질의 스크리닝 방법을 제공한다. In addition, the present invention provides a method for screening a neutrophilic pulmonary inflammatory disease treatment substance comprising the following steps.
(a) In vitro 상에서 폐 세포에 후보물질을 처리하는 단계;(a) treating the lung cells with the candidate substance in vitro ;
(b) 상기 폐 세포에서 인터루킨-1β(IL-1β)의 발현 또는 활성을 측정하는 단계; 및(b) measuring the expression or activity of interleukin-1β (IL-1β) in the lung cells; And
(c) 상기 폐 세포에서 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF)의 발현을 측정하는 단계; 및(c) measuring the expression of granulocyte colony-stimulating factor (G-CSF) in the lung cells; And
(d) 상기 IL-1β의 발현 또는 활성이 억제되는 것에 의하여 G-CSF의 발현을 감소시키는 물질을 호중구성 폐 염증질환의 치료물질로 선정하는 단계.(d) selecting a substance that reduces the expression of G-CSF by inhibiting the expression or activity of IL-1β as a therapeutic substance for neutrophilic pulmonary inflammatory disease.
또한, 본 발명은 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF)의 억제제로써 인터루킨-1β(IL-1β) 억제제를 유효성분으로 포함하는 약학적 조성물을 개체에 투여하는 단계를 포함하는, 호중구성 폐 염증질환의 예방 또는 치료방법을 제공한다. In addition, the present invention comprises the step of administering to an individual a pharmaceutical composition comprising an interleukin-1β (IL-1β) inhibitor as an active ingredient as an inhibitor of granulocyte colony-stimulating factor (G-CSF). , To provide a method of preventing or treating neutrophilic pulmonary inflammatory disease.
또한, 본 발명은 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF)의 억제제로써 인터루킨-1β(IL-1β) 억제제를 유효성분으로 포함하는 약학적 조성물의, 호중구성 폐 염증질환의 예방 또는 치료용도를 제공한다. In addition, the present invention prevents neutrophilic pulmonary inflammatory disease of a pharmaceutical composition comprising an interleukin-1β (IL-1β) inhibitor as an active ingredient as an inhibitor of granulocyte colony-stimulating factor (G-CSF) Or it provides therapeutic use.
본 발명자들은 다양한 유형의 천식 동물모델의 기관지폐포세척액 및 각 유형의 천식 환자유래 생물학적 시료를 이용하여 호중구성 천식 특이적 염증 물질로써 G-CSF를 발굴하였고 인간에서 G-CSF가 IL-1β에 의해 유도되는 것을 확인하였으며, 나아가 IL-1β의 억제를 통한 호중구성 염증반응의 현저한 감소를 확인하였는바, 본 발명에 따르면 G-CSF를 호중구성 폐 염증질환 진단용 바이오마커로 이용할 수 있으며, IL-1β는 호중구성 폐 염증질환의 치료 표적으로써 관련 분야에서 유용하게 활용될 수 있을 것으로 기대된다. The present inventors discovered G-CSF as a neutrophilic asthma-specific inflammatory substance using bronchoalveolar lavage fluid of various types of asthma animal models and biological samples derived from each type of asthma patient, and G-CSF is induced by IL-1β in humans. It was confirmed that it was induced, and furthermore, it was confirmed that a remarkable decrease in neutrophilic inflammatory response through inhibition of IL-1β was confirmed. According to the present invention, G-CSF can be used as a biomarker for diagnosis of neutrophilic pulmonary inflammatory disease, Is expected to be useful in related fields as a therapeutic target for neutrophilic pulmonary inflammatory disease.
도 1은 호중구성 천식 특이적 염증 물질로써 G-CSF를 발굴한 결과를 보여주는 것으로, 도 1a는 혼합형 천식(Alum/OVA), 호중구성 천식(LPS/OVA) 및 호산구성 천식(HDM)이 유도된 동물모델을 각각 제조한 후 각 모델의 기관지폐포세척액 내 세포 분포를 확인한 결과이고, 도 1b는 각 유형의 천식 동물모델유래 기관지폐포세척액에서 G-CSF 농도를 측정한 결과이며, 도 1c는 각각 기관지폐포세척액 내 호중구와 G-CSF 및 호산구와 G-CSF 간의 상관관계를 분석한 결과이다.
도 2는 각 유형의 천식 환자에서 G-CSF가 호중구성 천식 특이적 염증 물질임을 확인한 결과로서, 도 2a는 혼합형 천식(Mixed), 호중구성 천식(Neutrophilic), 호산구성 천식(Eosinophilic), 및 비세포성 천식(pauci granulocytic) 유형의 환자유래 객담에서 각각 G-CSF 및 IL-1β 수준을 보여주는 결과이며, 도 2b는 호중구성 천식 환자에서 객담 샘플 내 G-CSF 및 IL-1β의 상관관계를 분석한 결과이다.
도 3은 인간 폐세포주인 A549 및 기관지 상피세포주인 Beas2B를 IL-1β로 자극하고 24시간 후 세포 배양액 내 G-CSF의 농도를 측정한 결과이다.
도 4는 호중구성 천식 마우스 모델에 항-IL-1β 중화항체를 복강투여한 후 기관지폐포세척액 내 침윤된 세포 분포를 측정한 결과이다. Figure 1 shows the results of discovering G-CSF as a neutrophilic asthma-specific inflammatory substance, and Figure 1a is induction of mixed asthma (Alum/OVA), neutrophilic asthma (LPS/OVA) and eosinophilic asthma (HDM) After preparing each of the animal models, the distribution of cells in the bronchoalveolar lavage fluid of each model was confirmed, and FIG. 1B is a result of measuring the G-CSF concentration in the bronchoalveolar lavage fluid derived from each type of asthma animal model, and FIG. This is the result of analyzing the correlation between neutrophils and G-CSF and eosinophils and G-CSF in bronchoalveolar lavage fluid.
Figure 2 is a result of confirming that G-CSF is a neutrophilic asthma-specific inflammatory substance in each type of asthma patient, Figure 2a is a mixed asthma (Mixed), neutrophilic asthma (Neutrophilic), eosinophilic asthma (Eosinophilic), and Bise Results showing the levels of G-CSF and IL-1β, respectively, in sputum derived from a patient with pauci granulocytic type, and FIG. It is the result.
3 is a result of measuring the concentration of G-CSF in the cell culture after 24 hours after stimulation of the human lung cell line A549 and the bronchial epithelial cell line Beas2B with IL-1β.
4 is a result of measuring the distribution of infiltrated cells in the bronchoalveolar lavage fluid after intraperitoneal administration of an anti-IL-1β neutralizing antibody to a mouse model of neutrophil asthma.
본 발명자들은 호중구성 염증반응을 특징으로 하는 호중구성 폐 염증질환을 효과적으로 진단 및 치료할 수 있는 물질을 발굴하기 위해 연구 노력한 결과, 호중구가 높게 존재하는 천식 모델에서 호중구 수준에 따라 G-CSF가 높게 존재하는 것을 확인하였으며, 인간 호중구성 천식 환자에서 IL-1β가 G-CSF의 생성을 유도하는 상관관계를 확인함으로써 호중구성 천식 동물모델에서 IL-1β 억제를 통한 항염증 효과를 확인하였는바, 이에 기초하여 본 발명을 완성하였다. As a result of research efforts to discover substances capable of effectively diagnosing and treating neutrophilic pulmonary inflammatory diseases characterized by neutrophilic inflammatory reactions, the present inventors have found that G-CSF is high depending on the level of neutrophils in asthma models with high neutrophils. In human neutrophilic asthma patients, by confirming the correlation of IL-1β inducing the production of G-CSF, we confirmed the anti-inflammatory effect through IL-1β inhibition in neutrophilic asthma animal models. Thus, the present invention was completed.
이에, 본 발명은 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF) 유전자 또는 상기 유전자가 코딩하는 단백질을 포함하는 호중구성 폐 염증질환 진단용 마커 조성물을 제공한다.Accordingly, the present invention provides a marker composition for diagnosing neutrophilic pulmonary inflammatory disease comprising a granulocyte colony-stimulating factor (G-CSF) gene or a protein encoded by the gene.
본 발명자들은 일실시예에서, 각각 혼합형 천식, 호중구성 천식 및 호산구성 천식을 유발시킨 동물모델의 기관지폐포세척액 내 사이토카인 수준을 측정한 결과 호중구가 높게 존재하는 천식 모델에서 G-CSF의 농도가 높게 검출되었으며, 나아가 G-CSF가 호산구성 천식과는 상반되게 호중구성 천식과 양의 상관관계를 갖는 것을 확인하였다. 이에 더하여, 각 유형의 천식 환자유래 객담 샘플에서 역시 호중구가 높게 존재하는 천식에서만 G-CSF 수준이 높게 측정되었으며 이와 함께 IL-1β 역시 높은 수준으로 존재하는 것을 확인하였고, 호중구성 천식 환자에서 G-CSF와 IL-1β의 농도가 상관관계를 갖는 것을 확인하였다(실시예 2 참조). In one embodiment, the present inventors measured the cytokine level in the bronchoalveolar lavage fluid of an animal model that caused mixed asthma, neutrophilic asthma, and eosinophilic asthma, respectively, and the concentration of G-CSF in the asthma model with high neutrophils was Furthermore, it was confirmed that G-CSF had a positive correlation with neutrophilic asthma, contrary to eosinophilic asthma. In addition, in each type of asthma patient-derived sputum sample, the G-CSF level was also high in asthma with high neutrophils, and it was confirmed that IL-1β also existed at a high level. It was confirmed that the concentrations of CSF and IL-1β had a correlation (see Example 2).
본원발명의 다른 실시예에서는, 인간 유래 폐세포주 및 기관지 상피세포주를 이용해 IL-1β가 G-CSF의 생성을 유도한다는 상관관계를 확인하였다(실시예 3 참조).In another example of the present invention, a correlation was confirmed that IL-1β induces the production of G-CSF using human-derived lung cell lines and bronchial epithelial cell lines (see Example 3).
상기 실시예 결과를 통해 G-CSF를 이용하여 호중구성 폐 염증질환을 특이적으로 진단할 수 있음을 알 수 있었다.From the results of the above examples, it was found that neutrophilic pulmonary inflammatory disease can be specifically diagnosed using G-CSF.
본 발명에 있어서, 상기 G-CSF 유전자는 서열번호 1 또는 서열번호 3의 염기서열로 이루어진 것일 수 있고, 상기 G-CSF 유전자가 코딩하는 단백질은 서열번호 2 또는 서열번호 4의 아미노산 서열로 이루어진 것일 수 있으며, 보다 바람직하게는 서열번호 1의 염기서열 및 서열번호 2의 아미노산 서열로 이루어진 것일 수 있다. 이때, 상기 서열번호 1로 표시되는 염기서열 또는 서열번호 2로 표시되는 아미노산 서열과 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 서열을 포함할 수 있다. In the present invention, the G-CSF gene may be composed of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and the protein encoded by the G-CSF gene is composed of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 It may be, more preferably, it may be composed of the nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2. At this time, 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more of sequence homology with the nucleotide sequence represented by SEQ ID NO: 1 or the amino acid sequence represented by SEQ ID NO: 2 It may include a sequence having.
또한, 본 발명은 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF) 유전자의 mRNA 또는 상기 유전자가 코딩하는 단백질 수준을 측정하는 제제를 포함하는 호중구성 폐 염증질환 진단용 조성물을 제공한다. In addition, the present invention provides a composition for diagnosing neutrophilic pulmonary inflammatory disease, including an agent measuring the level of mRNA of a granulocyte colony-stimulating factor (G-CSF) gene or a protein encoded by the gene.
본 발명에서 대상으로 하는 질환인 “호중구성 폐 염증질환”은 호중구 침윤을 특징으로 하는 염증반응에 의해 유발되는 기도, 폐 관련 질환으로, 보다 바람직하게는 호중구성 천식(neutrophilic asthma), 호중구성 만성폐쇄성폐질환(neutrophilic chronic obstructive pulmonary disease), 특발성 폐섬유화증(idiopathic pulmonary fibrosis; IPF), 급성 폐 손상(acute lung injury; ALI), 및 급성 호흡곤란 증후군(acute respiratory distress syndrome; ARDS)으로 이루어진 군에서 선택되는 하나 이상일 수 있으나, 이에 제한되는 것은 아니다. The disease target in the present invention, "neutrophilic pulmonary inflammatory disease", is a disease related to airways and lungs caused by an inflammatory reaction characterized by neutrophil infiltration, more preferably neutrophilic asthma, neutrophilic chronic The group consisting of neutrophilic chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis (IPF), acute lung injury (ALI), and acute respiratory distress syndrome (ARDS). It may be one or more selected from, but is not limited thereto.
본 발명에서 사용되는 용어, “진단(diagnosis)”이란 넓은 의미로는 환자의 병의 실태를 모든 면에 걸쳐서 판단하는 것을 의미한다. 판단의 내용은 병명, 병인, 병형, 경중, 병상의 상세한 양태, 및 합병증의 유무 등이다. 본 발명에서 진단은 호중구성 폐 염증질환의 발병 여부 및 진행단계 수준 등을 판단하는 것이다. The term "diagnosis" used in the present invention means in a broad sense to judge the condition of a patient's disease in all aspects. The content of the judgment is the name of the disease, etiology, disease type, severity, detailed mode of the bed, and the presence or absence of complications. In the present invention, the diagnosis is to determine the onset of neutrophilic pulmonary inflammatory disease and the level of progression.
본 발명에 있어서, 상기 mRNA 수준을 측정하는 제제는 유전자의 mRNA에 상보적으로 결합하는 센스 및 안티센스 프라이머일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the agent for measuring the mRNA level may be a sense and antisense primer that complementarily binds to the mRNA of a gene, but is not limited thereto.
본 발명에서 사용되는 용어, "프라이머(Primer)"란 DNA 합성의 기시점이 되는 짧은 유전자 서열로서, 진단, DNA 시퀀싱 등에 이용할 목적으로 합성된 올리고뉴클레오티드를 의미한다. 상기 프라이머들은 통상적으로 15 내지 30 염기쌍의 길이로 합성하여 사용할 수 있으나, 사용 목적에 따라 달라질 수 있으며, 공지된 방법으로 메틸화, 캡화 등으로 변형시킬 수 있다.The term "primer" used in the present invention refers to a short gene sequence that serves as the starting point of DNA synthesis, and refers to an oligonucleotide synthesized for use in diagnosis, DNA sequencing, or the like. The primers are usually synthesized and used in a length of 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation or capping by a known method.
본 발명에서 사용되는 용어, "프로브(Probe)"란 효소 화학적인 분리정제 또는 합성과정을 거쳐 제작된 수 염기 내지 수백 염기 길이의 mRNA와 특이적으로 결합할 수 있는 핵산을 의미한다. 방사성 동위원소나 효소 등을 표지하여 mRNA의 존재 유무를 확인할 수 있으며, 공지된 방법으로 디자인하고 변형시켜 사용할 수 있다.As used herein, the term "probe" refers to a nucleic acid capable of specifically binding to an mRNA having a length of several to hundreds of bases produced through enzymatic chemical separation or purification or synthesis. The presence or absence of mRNA can be confirmed by labeling radioactive isotopes or enzymes, and it can be designed and modified by a known method.
본 발명에 있어서, 상기 단백질 수준을 측정하는 제제는 유전자가 코딩하는 단백질에 특이적으로 결합하는 항체일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the agent for measuring the protein level may be an antibody that specifically binds to a protein encoded by a gene, but is not limited thereto.
본 발명에서 사용되는 용어, "항체"는 면역학적으로 특정 항원과 반응성을 갖는 면역글로불린 분자를 포함하며, 단클론(monoclonal) 항체 및 다클론(polyclonal) 항체를 모두 포함한다. 또한, 상기 항체는 키메라성 항체(예를 들면, 인간화 뮤린 항체) 및 이종결합항체(예를 들면, 양특이성 항체)와 같은 유전공학에 의해 생산된 형태를 모두 포함한다.As used herein, the term "antibody" includes immunoglobulin molecules immunologically reactive with a specific antigen, and includes both monoclonal and polyclonal antibodies. In addition, the antibody includes all forms produced by genetic engineering such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies).
또한, 본 발명은 상기 진단용 조성물을 포함하는 호중구성 폐 염증질환 진단용 키트를 제공한다.In addition, the present invention provides a kit for diagnosing neutrophilic pulmonary inflammatory disease comprising the diagnostic composition.
본 발명의 호중구성 폐 염증질환 진단용 키트는 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성성분을 포함하는 조성물, 용액 또는 장치로 구성된다.The kit for diagnosing neutrophilic pulmonary inflammatory disease of the present invention consists of a composition, solution, or device containing one or more other components suitable for an analysis method.
또한, 본 발명은 피검체 유래의 생물학적 시료에서 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF) 유전자의 mRNA 또는 상기 유전자가 코딩하는 단백질 수준을 측정하는 단계를 포함하는, 호중구성 폐 염증질환의 진단을 위한 정보제공방법을 제공한다.In addition, the present invention comprises the step of measuring the level of the mRNA of a granulocyte colony-stimulating factor (G-CSF) gene or a protein encoded by the gene in a biological sample derived from a subject, neutrophilic lung inflammation Provides information provision method for diagnosis of disease.
본 발명에서 사용되는 용어 "호중구성 폐 염증질환의 진단을 위한 정보제공방법"은 진단을 위한 예비적 단계로써 호중구성 폐 염증질환의 진단을 위하여 필요한 객관적인 기초정보를 제공하는 것이며 의사의 임상학적 판단 또는 소견은 제외된다.The term "providing information for diagnosis of neutrophilic pulmonary inflammatory disease" as used in the present invention is a preliminary step for diagnosis, providing objective basic information necessary for diagnosis of neutrophilic pulmonary inflammatory disease, and clinical judgment of a doctor Or the findings are excluded.
본 발명에 있어서, 상기 mRNA 수준은 당업계에 알려진 통상적인 방법으로 중합효소연쇄반응(PCR), 역전사 중합효소연쇄반응(RT-PCR), 실시간 중합효소연쇄반응(Real-time PCR), RNase 보호 분석법(RNase protection assay; RPA), 마이크로어레이(microarray), 및 노던 블롯팅(northern blotting)으로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the mRNA level is polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection by conventional methods known in the art. It may be measured by one or more methods selected from the group consisting of RNase protection assay (RPA), microarray, and northern blotting, but is not limited thereto.
본 발명에 있어서, 상기 단백질 수준은 당업계에 알려진 통상적인 방법으로 웨스턴 블롯팅(western blotting), 방사선면역분석법(radioimmunoassay; RIA), 방사 면역 확산법(radioimmunodiffusion), 효소면역분석법(ELISA), 면역침강법(immunoprecipitation), 유세포분석법(flow cytometry), 면역형광염색법(immunofluorescence), 오우크테로니(ouchterlony), 보체 고정 분석법(complement fixation assay), 및 단백질 칩(protein chip)으로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정될 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the protein level is a conventional method known in the art, Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation. 1 selected from the group consisting of immunoprecipitation, flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and protein chip It can be measured through more than one species method, but is not limited thereto.
상기 피검체 유래의 생물학적 시료는 조직, 세포, 전혈, 혈액, 타액, 객담, 뇌척수액 및 뇨 등을 포함할 수 있으나, 이것으로 제한되는 것은 아니다. The biological sample derived from the subject may include tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, urine, etc., but is not limited thereto.
한편, 본 발명자들은 또 다른 실시예에서, 호중구성 천식 동물모델에 IL-1β 중화항체 투여하고 기관지폐포세척액에서 면역세포의 침윤정도를 측정한 결과, 호중구 침윤의 현저한 감소를 확인하였다(실시예 4 참조). On the other hand, in another embodiment, the present inventors, as a result of administering an IL-1β neutralizing antibody to an animal model of neutrophilic asthma, and measuring the degree of infiltration of immune cells in bronchoalveolar lavage, confirmed a remarkable decrease in neutrophil infiltration (Example 4 Reference).
이에, 본 발명의 다른 양태로서, 본 발명은 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF)의 억제제로써 인터루킨-1β(IL-1β) 억제제를 유효성분으로 포함하는, 호중구성 폐 염증질환의 예방 또는 치료용 약학적 조성물을 제공한다. Thus, as another aspect of the present invention, the present invention comprises an interleukin-1β (IL-1β) inhibitor as an active ingredient as an inhibitor of granulocyte colony-stimulating factor (G-CSF), neutrophilic lung inflammation It provides a pharmaceutical composition for preventing or treating diseases.
상기 IL-1β 억제제는 IL-1β 단백질에 특이적으로 결합하는 항-IL-1β 항체일 수 있으나, IL-1β의 발현 또는 활성을 특이적으로 억제할 수 있는 물질이라면 그 종류가 제한되지 않는다. The IL-1β inhibitor may be an anti-IL-1β antibody that specifically binds to the IL-1β protein, but the type is not limited as long as it is a substance capable of specifically inhibiting the expression or activity of IL-1β.
본 발명에 따른 상기 약학적 조성물은 상기 IL-1β 억제제를 유효성분으로 포함하며, 약학적으로 허용 가능한 담체를 더 포함할 수 있다. 상기 약학적으로 허용 가능한 담체는 제제시에 통상적으로 이용되는 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 사이클로덱스트린, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 리포좀 등을 포함하지만 이에 한정되지 않으며, 필요에 따라 항산화제, 완충액 등 다른 통상의 첨가제를 더 포함할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제, 윤활제 등을 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제화에 관해서는 레밍턴의 문헌에 개시되어 있는 방법을 이용하여 각 성분에 따라 바람직하게 제제화할 수 있다. 본 발명의 약학적 조성물은 제형에 특별한 제한은 없으나 주사제, 흡입제, 피부 외용제 등으로 제제화할 수 있다. The pharmaceutical composition according to the present invention includes the IL-1β inhibitor as an active ingredient, and may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in formulation, and includes, but is limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It is not, and other conventional additives such as antioxidants and buffers may be further included if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, and the like may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, it can be preferably formulated according to each component using a method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in its formulation, but may be formulated as an injection, an inhalant, an external preparation for skin, or the like.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenous, subcutaneous, intraperitoneal or topical application) according to a desired method, and the dosage is It depends on the degree, drug form, administration route and time, but may be appropriately selected by those skilled in the art.
본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서 “약학적으로 유효한 양”은 의학적 치료 또는 진단에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료 또는 진단하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 다른 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” refers to an amount sufficient to treat or diagnose a disease at a reasonable benefit/risk ratio applicable to medical treatment or diagnosis, and the effective dose level is the type of disease, severity, drug Activity, sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or administered in combination with another therapeutic agent, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person skilled in the art.
구체적으로 본 발명의 약학적 조성물의 유효량은 환자의 연령, 성별, 상태, 체중, 체내에 활성 성분의 흡수도, 불활성률 및 배설속도, 질병종류, 병용되는 약물에 따라 달라질 수 있으며, 일반적으로는 체중 1 ㎏ 당 0.001 내지 150 ㎎, 바람직하게는 0.01 내지 100 ㎎을 매일 또는 격일 투여하거나, 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라서 증감 될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient in the body, inactivation rate and excretion rate, the type of disease, and drugs used in combination. 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight may be administered daily or every other day, or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dosage amount does not limit the scope of the present invention in any way.
본 발명의 또 다른 양태로서, 본 발명은 하기의 단계를 포함하는, 호중구성 폐 염증질환 치료물질의 스크리닝 방법을 제공한다. As another aspect of the present invention, the present invention provides a method for screening a neutrophilic pulmonary inflammatory disease treatment substance comprising the following steps.
(a) In vitro 상에서 폐 세포에 후보물질을 처리하는 단계;(a) treating the lung cells with the candidate substance in vitro ;
(b) 상기 폐 세포에서 인터루킨-1β(IL-1β)의 발현 또는 활성을 측정하는 단계; 및(b) measuring the expression or activity of interleukin-1β (IL-1β) in the lung cells; And
(c) 상기 폐 세포에서 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF)의 발현을 측정하는 단계; 및(c) measuring the expression of granulocyte colony-stimulating factor (G-CSF) in the lung cells; And
(d) 상기 IL-1β의 발현 또는 활성이 억제되는 것에 의하여 G-CSF의 발현을 감소시키는 물질을 호중구성 폐 염증질환의 치료물질로 선정하는 단계.(d) selecting a substance that reduces the expression of G-CSF by inhibiting the expression or activity of IL-1β as a therapeutic substance for neutrophilic pulmonary inflammatory disease.
본 발명에 있어서, 상기 폐 세포는 보다 바람직하게 호중구성 폐 염증질환이 유발된 폐 조직유래 세포일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the lung cells may more preferably be lung tissue-derived cells caused by neutrophilic pulmonary inflammatory disease, but are not limited thereto.
상기 후보물질은 화합물, 미생물 배양액 또는 추출물, 천연물 추출물, 핵산, 및 펩타이드로 이루어진 군으로부터 선택되는 것일 수 있다. The candidate material may be selected from the group consisting of a compound, a microorganism culture solution or extract, a natural product extract, a nucleic acid, and a peptide.
본 발명의 또 다른 양태로서, 본 발명은 상기 약학적 조성물을 개체에 투여하는 단계를 포함하는, 호중구성 폐 염증질환의 예방 또는 치료방법을 제공한다.As another aspect of the present invention, the present invention provides a method for preventing or treating neutrophilic pulmonary inflammatory disease, comprising administering the pharmaceutical composition to an individual.
본 발명에서 “개체”란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는 인간 또는 비-인간인 영장류, 생쥐(mouse), 쥐(rat), 개, 고양이, 말 및 소 등의 포유류를 의미한다. In the present invention, “individual” refers to a subject in need of treatment of a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses and cattle. Means mammal.
본 발명의 또 다른 양태로서, 본 발명은 상기 약학적 조성물의 호중구성 폐 염증질환 예방 또는 치료용도를 제공한다. As another aspect of the present invention, the present invention provides the use of the pharmaceutical composition for preventing or treating neutrophilic pulmonary inflammatory disease.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.
[실시예][Example]
실시예 1. 실험준비 및 실험방법Example 1. Experiment preparation and experiment method
1-1. 실험동물1-1. Experimental animals
본 실시예에서는 생후 6주 내지 8주된 야생형 C57BL/6(B6, CD45.1 및 CD45.2) 마우스를 사용하였다. 모든 마우스는 포스텍(POSTECH)의 병원균이 없는 동물 시설에서 사육하였으며, 실험은 포스텍의 동물 지침에 따라 수행하였다.In this example, wild-type C57BL/6 (B6, CD45.1 and CD45.2) mice aged 6 to 8 weeks old were used. All mice were reared in a pathogen-free animal facility of POSTECH, and experiments were performed according to the animal guidelines of POSTECH.
1-2. 혼합형, 호중구성 및 호산구성 천식 동물모델 제조1-2. Manufacture of mixed, neutrophil and eosinophilic asthma animal models
각각 혼합형 천식, 호중구성 천식 및 호산구성 천식 동물모델을 확립하기 위해 하기와 같은 방법을 이용하였다. The following methods were used to establish animal models of mixed asthma, neutrophilic asthma, and eosinophilic asthma, respectively.
먼저 혼합형 천식이 유도된 동물모델을 제조하기 위해, 마우스에 2 mg의 alum hydroxid 및 75 ㎍의 오브알부민(ovalbumin; OVA)을 혼합하여 0 및 7일째에 각각 복강투여 하였고, 14일째에 OVA 50 ㎍을 1주일에 2회씩 2주 동안 비강으로 투여하였다. First, in order to prepare an animal model in which mixed asthma was induced, 2 mg of alum hydroxid and 75 µg of ovalbumin (OVA) were mixed and intraperitoneally administered on
호중구성 천식이 유도된 동물모델은 동일한 마우스에 대하여 멸균된 20 ㎕의 PBS에 10 ㎍ LPS와 75 ㎍의 OVA(sigma)을 넣어 혼합한 후 각각 0, 1, 2 및 7일째에 비강으로 투여하고, 이어서 4일째부터 시작하여 4주 동안 일주일에 2회씩 20 ㎕의 멸균 PBS에 50 ㎍의 OVA를 혼합하여 반복 투여하였다.The animal model in which neutrophilic asthma was induced was mixed with 10 µg LPS and 75 µg OVA (sigma) in 20 µl PBS sterilized for the same mouse, and then administered intranasally on
마지막으로 호산구성 천식이 유도된 동물모델은 동일한 마우스에 집먼지 진드기(House dust mite; HDM) 25 ㎍을 각각 0, 1, 2, 14, 15, 16, 17일째에 비강으로 투여하여 제조하였다. Finally, the animal model in which eosinophilic asthma was induced was prepared by nasal administration of 25 µg of house dust mite (HDM) to the same mouse at 0, 1, 2, 14, 15, 16, and 17 days, respectively.
한편, 모든 비강 내 투여는 케타민(ketamine)/자일라진(xylazine) 용액 120 ㎕를 복강 투여하여 마우스를 마취시킨 후 실시하였다. Meanwhile, all intranasal administrations were performed after anesthetizing mice by intraperitoneally administering 120 µl of a ketamine/xylazine solution.
각 유형의 천식이 유도된 마우스 모델을 제조한 후 각 마우스유래 기관지폐포세척액 내의 세포 분포를 확인한 결과, 도 1a에 나타낸 바와 같이 혼합형 천식 마우스에서는 호중구 및 호산구가 모두 유의하게 증가되었으며 호중구성 천식 및 호산구성 천식 마우스에서는 각각 호중구 및 호산구만이 유의적으로 증가된 것을 확인함으로써 각 유형의 천식 동물모델이 잘 확립된 것을 알 수 있었다. As a result of confirming the distribution of cells in each mouse-derived bronchoalveolar lavage fluid after preparing a mouse model in which each type of asthma was induced, as shown in Fig. 1a, both neutrophils and eosinophils were significantly increased in mixed asthma mice, and neutrophilic asthma and eosinophils. By confirming that only neutrophils and eosinophils were significantly increased in constitutive asthma mice, it was found that each type of asthma animal model was well established.
실시예 2. 호중구성 폐 염증질환 특이적 염증 물질 발굴Example 2. Discovery of inflammatory substances specific to neutrophilic pulmonary inflammatory disease
호중구성 폐 염증질환에 특이적인 염증 물질을 발굴하기 위하여, 본 발명자들은 이전 연구결과에 근거하여 먼저 상기 실시예 1-2의 방법으로 각각 혼합형 천식(Alum/OVA), 호중구성 천식(LPS/OVA) 및 호산구성 천식(HDM)이 유도된 마우스 모델을 확립한 다음, 각 마우스 모델에서 얻은 기관지폐포세척액에서 사이토카인 수준을 측정하였다. 그 결과, 도 1b에 나타낸 바와 같이 호중구가 높게 존재하는 천식 모델에서 G-CSF의 농도가 높게 검출된 것을 확인하였다(호중구성 천식 > 혼합형 천식 > 호산구성 천식). 나아가 상기 도 1b의 결과를 이용해 각각 기관지폐포세척액 내 호중구 및 G-CSF, 또는 호산구 및 G-CSF 간의 상관관계를 분석한 결과, 도 1c에서 볼 수 있는 바와 같이 G-CSF 농도가 호중구와는 양의 상관관계를, 호산구와는 음의 상관관계를 나타내는 것을 확인하였다. In order to discover an inflammatory substance specific for neutrophilic pulmonary inflammatory disease, the present inventors first conducted mixed asthma (Alum/OVA) and neutrophilic asthma (LPS/OVA) by the method of Example 1-2, based on previous research results. ) And eosinophilic asthma (HDM) induced mouse models were established, and then cytokine levels were measured in bronchoalveolar lavage fluid obtained from each mouse model. As a result, as shown in Fig. 1b, it was confirmed that the concentration of G-CSF was detected high in the asthma model with high neutrophils (neutrophilic asthma> mixed type asthma> eosinophilic asthma). Further, as a result of analyzing the correlation between neutrophils and G-CSF, or eosinophils and G-CSF in the bronchoalveolar lavage fluid, respectively, using the results of Fig. 1b, the concentration of G-CSF is the amount that is different from neutrophils as shown in Fig. It was confirmed that the correlation of was negatively correlated with eosinophils.
이에 더하여, 상기 결과를 검증하기 위해 각 유형의 천식 환자로부터 객담 샘플을 얻어 하기 실험을 진행하였다. 보다 구체적으로, 천식 환자의 객담 내 세포 분포를 바탕으로 하기와 같은 기준에 따라 각 유형의 천식 환자군을 분류하였다: 혼합형 천식(Mixed)-호중구 > 60%, 호산구 > 3%; 호중구성 천식(Neutrophilic)-호중구 > 60%, 호산구 < 3%; 호산구성 천식(Eosinophilic)-호중구 < 60%, 호산구 > 3%, 비세포성 천식(Pauci-Granulocyte)- 호중구 < 60%, 호산구 < 3%. 다음으로 각 유형의 천식 환자군 유래 객담 샘플에서 G-CSF의 농도를 측정하였다. 그 결과, 도 2a에 나타낸 바와 같이 호중구가 많이 존재하는 혼합형 및 호중구성 천식 환자에서 G-CSF가 높게 검출된 것을 확인하였다. 또한, 상기 각 유형의 천식 환자군 유래 객담 샘플에서 다른 사이토카인들의 농도를 측정한 결과 IL-1β의 농도가 혼합형 및 호중구성 천식 환자에서 유의하게 높게 검출되었다. 이러한 결과를 바탕으로 G-CSF와 IL-1β의 상관관계를 분석한 결과, 도 2b에 나타낸 바와 같이 양의 상관관계를 나타내는 것을 확인하였으며, 이로부터 사람의 경우 IL-1β가 G-CSF의 생성을 유도하는 물질일 것이라는 가능성을 확인하였다. In addition, in order to verify the above results, sputum samples were obtained from each type of asthma patient and the following experiment was conducted. More specifically, based on the distribution of cells in the sputum of asthmatic patients, each type of asthma patient group was classified according to the following criteria: mixed asthma (Mixed)-neutrophils> 60%, eosinophils> 3%; Neutrophilic-neutrophils> 60%, eosinophils <3%; Eosinophilic-neutrophils <60%, eosinophils> 3%, acellular asthma (Pauci-Granulocyte)-neutrophils <60%, eosinophils <3%. Next, the concentration of G-CSF was measured in sputum samples derived from each type of asthma patient group. As a result, as shown in Fig. 2a, it was confirmed that G-CSF was detected high in mixed-type and neutrophilic asthma patients with a large amount of neutrophils. In addition, as a result of measuring the concentration of different cytokines in sputum samples derived from each type of asthma patient group, the concentration of IL-1β was significantly higher in mixed-type and neutrophilic asthma patients. Based on these results, as a result of analyzing the correlation between G-CSF and IL-1β, it was confirmed that a positive correlation was shown as shown in FIG. 2B. From this, in humans, IL-1β produces G-CSF. It was confirmed that it would be a material that induces
실시예 3. 호중구성 천식 환자에서 IL-1β에 의한 G-CSF 생성 유도 확인Example 3. Confirmation of induction of G-CSF production by IL-1β in patients with neutrophilic asthma
상기 실시예 2의 결과를 통해 호중구성 천식 환자에서 G-CSF와 IL-1β가 양의 상관관계를 갖는 것을 확인하였는바, 이를 토대로 IL-1β가 G-CSF의 생성을 유도하는지 검증하고자 하였다. 이를 위해, 인간 폐세포주인 A549 및 인간 기관지 상피세포인 Beas2B를 배양한 후 1 x 105개 세포에 IL-1β를 10 ng/ml의 농도로 자극하고 24시간 동안 배양한 후 ELISA를 통해 배양액 내 G-CSF의 농도를 측정하였다. The results of Example 2 confirmed that G-CSF and IL-1β had a positive correlation in patients with neutrophilic asthma. Based on this, we tried to verify whether IL-1β induces the production of G-CSF. To this end, after culturing the human lung cell line A549 and the human bronchial epithelial cells Beas2B, IL-1β was stimulated at a concentration of 10 ng/ml in 1 x 10 5 cells, cultured for 24 hours, and then in the culture medium through ELISA. The concentration of G-CSF was measured.
그 결과, 도 3에 나타낸 바와 같이 상기 두 가지 세포주 모두에서 대조군(Media)에 비해 IL-1β로 자극한 경우 G-CSF의 농도가 현저히 증가한 것을 확인하였다. 이러한 결과를 통해 인간 호중구성 천식 환자에서 IL-1β가 G-CSF의 생성을 유도하는 것을 알 수 있었다. As a result, as shown in FIG. 3, it was confirmed that the concentration of G-CSF was significantly increased when the two cell lines were stimulated with IL-1β compared to the control (Media). These results showed that IL-1β induces the production of G-CSF in human neutrophilic asthma patients.
실시예 4. IL-1β 중화항체의 호중구성 천식 치료효능 확인Example 4. Confirmation of the efficacy of IL-1β neutralizing antibodies to treat neutrophilic asthma
본 발명자들은 이전 연구를 통해 마우스에서 IL-17 및 TNF-α가 G-CSF의 생성을 유도하며, IL-17 및 TNF-α의 동시 억제가 호중구성 천식에 대한 치료효과를 나타냄을 확인하였는바, 이러한 결과를 바탕으로 인간에서는 IL-1β가 G-CSF의 생성을 유도한다는 결과를 토대로 IL-1β를 억제하는 경우 호중구성 천식의 치료효과가 나타나는지 여부를 검증하고자 하였다. 이를 위해, 상기 실시예 1-2의 방법에 따라 제조된 호중구성 천식 마우스 모델에 항-IL-1β 중화항체를 1주에 2회씩 2주 동안 복강투여하였다. Through a previous study, the present inventors have confirmed that IL-17 and TNF-α induce the production of G-CSF in mice, and that simultaneous inhibition of IL-17 and TNF-α exhibits a therapeutic effect on neutrophilic asthma. , Based on these results, we tried to verify whether IL-1β induces the production of G-CSF in humans, and if IL-1β is suppressed, the therapeutic effect of neutrophilic asthma appears. To this end, the anti-IL-1β neutralizing antibody was administered intraperitoneally to the neutrophilic asthma mouse model prepared according to the method of Example 1-2, twice a week for 2 weeks.
이후 기관지폐포세척액 내의 세포 분포를 확인한 결과, 도 4에 나타낸 바와 같이 호중구성 천식을 유발시킨 마우스(LPS/OVA)의 경우에는 정상대조군 마우스(PBS)에 비해 호중구의 침윤이 현저히 증가한 반면, 항-IL-1β 중화항체를 투여한 마우스(α-IL-1b)의 경우에는 호중구의 침윤이 유의하게 감소한 것을 확인하였다. 이러한 결과는 호중구성 천식에서 IL-1β의 억제가 효과적인 항염증 효능을 나타내며 호중구성 천식에 대한 치료효능이 있음을 보여주는 것이다. Subsequently, as a result of confirming the distribution of cells in the bronchoalveolar lavage fluid, as shown in Fig. 4, in the case of mice (LPS/OVA) inducing neutrophilic asthma, the invasion of neutrophils significantly increased compared to the normal control mice (PBS), whereas anti- In the case of mice administered with IL-1β neutralizing antibody (α-IL-1b), it was confirmed that neutrophil infiltration was significantly reduced. These results show that the inhibition of IL-1β in neutrophilic asthma has an effective anti-inflammatory effect and has therapeutic efficacy for neutrophilic asthma.
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. The above-described description of the present invention is for illustration purposes only, and those of ordinary skill in the art to which the present invention pertains can understand that it is possible to easily transform it into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.
<110> POSTECH ACADEMY-INDUSTRY FOUNDATION Soonchunhyang University Industry Academy Cooperation Foundation <120> Compositions for diagnosing, preventing or treating neutrophilic lung inflammatory diseases using G-CSF and IL-1b <130> PD19-013 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 2453 <212> DNA <213> Homo sapiens_G-CSF <400> 1 agtcgtggcc ccaggtaatt tcctcccagg cctccatggg gttatgtata aaggcccccc 60 tagagctggg ccccaaaaca gcccggagcc tgcagcccag ccccacccag acccatggct 120 ggacctgcca cccagagccc catgaagctg atgggtgagt gtcttggccc aggatgggag 180 agccgcctgc cctggcatgg gagggaggct ggtgtgacag aggggctggg gatccccgtt 240 ctgggaatgg ggattaaagg cacccagtgt ccccgagagg gcctcaggtg gtagggaaca 300 gcatgtctcc tgagcccgct ctgtccccag ccctgcagct gctgctgtgg cacagtgcac 360 tctggacagt gcaggaagcc acccccctgg gccctgccag ctccctgccc cagagcttcc 420 tgctcaagtg cttagagcaa gtgaggaaga tccagggcga tggcgcagcg ctccaggaga 480 agctggtgag tgaggtgggt gagagggctg tggagggaag cccggtgggg agagctaagg 540 gggatggaac tgcagggcca acatcctctg gaagggacgt gggagaatat taggagcagt 600 ggagctgggg aaggctggga agggacttgg ggaggaggac cttggtgggg acagtgctcg 660 ggagggctgg ctgggatggg agtggaggca ttacattcag gagaaagggc aagggcccct 720 gtgagatcag agagtggggg tgcagggcag agaggaactg aacagcctgg caggacatgg 780 agggagggga aagaccagag agtcggggag gacccgggaa ggagcggcga cccggccatg 840 gcgagtctca ctcagcatcc ttccatcccc agtgtgccac ctacaagctg tgccaccccg 900 aggagctggt gctgctcgga cactctctgg gcatcccctg ggctcccctg agcagctgcc 960 ccagccaggc cctgcagctg gtgagtgtca ggaaaggata aggctaatga ggagggggaa 1020 ggagaggagg aacacccatg ggctccccca tgtctccagg ttccaagctg ggggcctgac 1080 gtatctcagg cagcaccccc taactcttcc gctctgtctc acaggcaggc tgcttgagcc 1140 aactccatag cggccttttc ctctaccagg ggctcctgca ggccctggaa gggatctccc 1200 ccgagttggg tcccaccttg gacacactgc agctggacgt cgccgacttt gccaccacca 1260 tctggcagca ggtgagcctt gttgggcagg gtggccaagg tcgtgctggc attctgggca 1320 ccacagccag gcctgtgtat gggccctgtc catgctgtca cccccagcat ttcctcattt 1380 gtaataacgc ccactcagaa gggcccaacc actgatcaca gctttccccc acagatggaa 1440 gaactgggaa tggcccctgc cctgcagccc acccagggtg ccatgccggc cttcgcctct 1500 gctttccagc gccgggcagg aggggtcctg gttgcctccc atctgcagag cttcctggag 1560 gtgtcgtacc gcgttctacg ccaccttgcc cagccctgag ccaagccctc cccatcccat 1620 gtatttatct ctatttaata tttatgtcta tttaagcctc atatttaaag acagggaaga 1680 gcagaacgga gccccaggcc tctgtgtcct tccctgcatt tctgagtttc attctcctgc 1740 ctgtagcagt gagaaaaagc tcctgtcctc ccatcccctg gactgggagg tagataggta 1800 aataccaagt atttattact atgactgctc cccagccctg gctctgcaat gggcactggg 1860 atgagccgct gtgagcccct ggtcctgagg gtccccacct gggacccttg agagtatcag 1920 gtctcccacg tgggagacaa gaaatccctg tttaatattt aaacagcagt gttccccatc 1980 tgggtccttg cacccctcac tctggcctca gccgactgca cagcggcccc tgcatcccct 2040 tggctgtgag gcccctggac aagcagaggt ggccagagct gggaggcatg gccctggggt 2100 cccacgaatt tgctggggaa tctcgttttt cttcttaaga cttttgggac atggtttgac 2160 tcccgaacat caccgacgcg tctcctgttt ttctgggtgg cctcgggaca cctgccctgc 2220 ccccacgagg gtcaggactg tgactctttt tagggccagg caggtgcctg gacatttgcc 2280 ttgctggacg gggactgggg atgtgggagg gagcagacag gaggaatcat gtcaggcctg 2340 tgtgtgaaag gaagctccac tgtcaccctc cacctcttca ccccccactc accagtgtcc 2400 cctccactgt cacattgtaa ctgaacttca ggataataaa gtgtttgcct cca 2453 <210> 2 <211> 207 <212> PRT <213> Homo sapiens_G-CSF <400> 2 Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala Leu Gln 1 5 10 15 Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln Glu Ala Thr Pro 20 25 30 Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu 35 40 45 Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys 50 55 60 Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 65 70 75 80 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 85 90 95 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 100 105 110 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 115 120 125 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 130 135 140 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 145 150 155 160 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 165 170 175 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 180 185 190 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 195 200 205 <210> 3 <211> 3607 <212> DNA <213> Mus musculus_G-CSF <400> 3 acttgagaat cacaaatgaa gggcagaggg ggctccccaa gcacgtctgc cagaggatgg 60 agagacagaa gcagtcttga tctgagcacc cattcttccc cagggtctga gttccttgct 120 atcccctcca gggtttcacc agccttgttg gacaactcgg atccagatcc aacaccctgc 180 agcgattcag gcctgggtgt ggctggaaga gaggaagaga gtttgagggg gggacaagac 240 gtcaaggaag gaacagagat tccccgattt cacaaaaact ttcgcaaaca gcttttcccc 300 aaccccctgc attgtcctga gctatcatca aatttgcata aatcctggga agttattact 360 aagcctgagt tgccccagcc ccaggtaatt tcctcccggg gccttgatgg ctttatgtat 420 aaaggccccc tggagctggg ccctggcaga gcccagagct gcagcccaga tcacccagaa 480 tccatggctc aactttctgc ccagaggcgc atgaagctaa tgggtgagtg ccctggttac 540 ctgccctggg tgtggaatgg agagaggccg gataacaagt gcctcagacc cgtctgtttg 600 ctcctagccc tgcagctgct gctgtggcaa agtgcactat ggtcaggacg agaggccgtt 660 cccctggtca ctgtcagcgc tctgccacca tccctgcctc tgccccgaag cttcctgctt 720 aagtccctgg agcaagtgag gaagatccag gccagcggct cggtgctgct ggagcagttg 780 gtgagtgggg ttgccatagg tgtggaggga aggacttggg gggggtagcg ggggaggcag 840 gagcacaaat gagaagaggg cagactagga acacacagca gttgacagtg tttgcccagt 900 gtgcacaaga gcctagatgg caccctcaga atactgacag aaacattgag gaagggaaca 960 tggggggaga agggcacagg aaagagaatg agtagagaca gcagtgggac ctctggatgg 1020 gacagtcctt agggtcacta ggaaggtagg gagctgagaa gccaggagga cctgagctgg 1080 acaaagagcc tggcccaggt gagcaccctc agtatccttc catctacagt gtgccaccta 1140 caagctgtgt caccccgagg agctggtgtt gctgggccac tctctgggga tcccgaaggc 1200 ttccctgagt ggctgctcta gccaggccct gcagcaggtg agtattttcg gaaaaaggag 1260 gaccaggagg agaaggggga ggagggggag gaggagaaag agggagagga gggggagaag 1320 ggggaggagg agggggagga ggaagaagag ggggaggagg gggaggagaa ggaggggaag 1380 aaggaggagg agggggagaa ggaaggacta catcctacta tttgggctcc tgaagagagc 1440 aagcatccca agatgtactg cctaaccctg ctatcttccc cagacacagt gcctaagcca 1500 gctccacagt gggctctgcc tctaccaagg tctcctgcag gctctatcgg gtatttcccc 1560 tgccctggcc cccaccttgg acttgcttca gctggatgtt gccaactttg ccaccaccat 1620 ctggcagcag gtgagtacca tctggagggg gtcatggcag cacacctggg caccctggaa 1680 gctctctata ttttcagagg cctgtttttt gggaagacac ccctcgcccc ctaccccgct 1740 tcctccaggc tgagagacag acagactctg tccagagaca gactgggttg gagtcttggt 1800 cctaccgtta acgtttaatg agccccacag gccaacttaa ccaacttccc agcacttcct 1860 cacctcaagg aaggactgat ccctgacttc atctcctccc cacagatgga aaacctaggg 1920 gtggccccta ctgtgcagcc cacacagagc gccatgccag ccttcacttc tgccttccag 1980 cgccgggcag gaggtgtcct ggccatttcg tacctgcagg gcttcctgga gacggctcgc 2040 cttgctctgc accacttggc ctagacctga gcagaaagcc ctttccagat agtttattta 2100 tctctattta atatttatgc atatttaagc ctactattta aagacaaaga cgagaaaatg 2160 gagctctaag cttctagatc attctctcca cttccgagtt ttgttctcct gcttagagca 2220 gagagagaag gctcttgtgt cctcctgtgg aggccaggga aggagatggg taaataccaa 2280 gtattgattc ctgctgctgc tccaggcacc cagttctgtg gcagtacccc caaaaaatca 2340 gtgagccctg ccgtgctgag gcaccatctc aggggggccc aggcagcatc tggtctccct 2400 tccgggggac aagacatccc tgtttaatat ttaaacagca gtgttcccaa actgggttct 2460 tatatccctt gctctggtca accaggttgc agggtttcct gtcctcacag gaacgaagtc 2520 cctaaagaaa cagtggcagc caggtttagc cccggaattg actggattcc ttttttaggg 2580 ccctgctggc ctggaagttg gagtgggggg cagaggaggc aggaggaagc ctgggggggg 2640 ggttggcatg gagggaggcc ttcccatcca ccctcaccct ccaccccacc tgtcactata 2700 gccaagcttg cggataataa agtgtggtgt tccagtacgt gttttttgtc ttccttcttg 2760 gttggctaat gcctggctgg gggctggtga gtggcgtggc gtggtaggag aggacagata 2820 aagaaggcac tgggaggagg tgggatgctc tagcttgccc cgacctcctc tgggtgccat 2880 tctctactaa tcgataggga cactaaaaat gcccagtgtc gctgccgcca agccacagct 2940 tcttgggaaa gaaaaaaaac caaaactgat ggatgataaa tcaatggaga gaggagggtt 3000 gggggcaggc tgagcacgtg cacaggcctg gtctcttcat cacctatgaa cagccctctg 3060 caaagctgcc cacccggcag aatgacaggc agaatgacat catgcctttt acagtttaat 3120 gtgattactg gatcagaact caggtcctgg gttggctggc tttcccccca acaaactgtg 3180 tccccacaat ctcctgcttg gctcactcct cagctgaaga agcctagaag gcctgaaggt 3240 cagctgcagg ctcccccgtg gctcagagct gcccaagagg ctaacctgtc acttccgccc 3300 acaccactga ctcctcaatg cctcccacca cctcccttcc cacagcctca caggaccctg 3360 cccctggact cgcagagtat ggaagacaga aaaagtcctc accctcaata ccaaataatc 3420 tcacaggaac acgtggtgaa ctctggtcca gagggtggag tgggacttaa aagaccgaga 3480 catcctgccc tgtacagtac agtgccgtgc ggggcctttc accagaacat cctgctttca 3540 gtggtggtgc cgctgctgag tccttaccat tgcagtggga ctgtgaacaa cggtcccact 3600 ctttctc 3607 <210> 4 <211> 208 <212> PRT <213> Mus musculus_G-CSF <400> 4 Met Ala Gln Leu Ser Ala Gln Arg Arg Met Lys Leu Met Ala Leu Gln 1 5 10 15 Leu Leu Leu Trp Gln Ser Ala Leu Trp Ser Gly Arg Glu Ala Val Pro 20 25 30 Leu Val Thr Val Ser Ala Leu Pro Pro Ser Leu Pro Leu Pro Arg Ser 35 40 45 Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Ala Ser Gly 50 55 60 Ser Val Leu Leu Glu Gln Leu Cys Ala Thr Tyr Lys Leu Cys His Pro 65 70 75 80 Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Lys Ala Ser 85 90 95 Leu Ser Gly Cys Ser Ser Gln Ala Leu Gln Gln Thr Gln Cys Leu Ser 100 105 110 Gln Leu His Ser Gly Leu Cys Leu Tyr Gln Gly Leu Leu Gln Ala Leu 115 120 125 Ser Gly Ile Ser Pro Ala Leu Ala Pro Thr Leu Asp Leu Leu Gln Leu 130 135 140 Asp Val Ala Asn Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Asn Leu 145 150 155 160 Gly Val Ala Pro Thr Val Gln Pro Thr Gln Ser Ala Met Pro Ala Phe 165 170 175 Thr Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Ala Ile Ser Tyr 180 185 190 Leu Gln Gly Phe Leu Glu Thr Ala Arg Leu Ala Leu His His Leu Ala 195 200 205 <110> POSTECH ACADEMY-INDUSTRY FOUNDATION Soonchunhyang University Industry Academy Cooperation Foundation <120> Compositions for diagnosing, preventing or treating neutrophilic lung inflammatory diseases using G-CSF and IL-1b <130> PD19-013 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 2453 <212> DNA <213> Homo sapiens_G-CSF <400> 1 agtcgtggcc ccaggtaatt tcctcccagg cctccatggg gttatgtata aaggcccccc 60 tagagctggg ccccaaaaca gcccggagcc tgcagcccag ccccacccag acccatggct 120 ggacctgcca cccagagccc catgaagctg atgggtgagt gtcttggccc aggatgggag 180 agccgcctgc cctggcatgg gagggaggct ggtgtgacag aggggctggg gatccccgtt 240 ctgggaatgg ggattaaagg cacccagtgt ccccgagagg gcctcaggtg gtagggaaca 300 gcatgtctcc tgagcccgct ctgtccccag ccctgcagct gctgctgtgg cacagtgcac 360 tctggacagt gcaggaagcc acccccctgg gccctgccag ctccctgccc cagagcttcc 420 tgctcaagtg cttagagcaa gtgaggaaga tccagggcga tggcgcagcg ctccaggaga 480 agctggtgag tgaggtgggt gagagggctg tggagggaag cccggtgggg agagctaagg 540 gggatggaac tgcagggcca acatcctctg gaagggacgt gggagaatat taggagcagt 600 ggagctgggg aaggctggga agggacttgg ggaggaggac cttggtgggg acagtgctcg 660 ggagggctgg ctgggatggg agtggaggca ttacattcag gagaaagggc aagggcccct 720 gtgagatcag agagtggggg tgcagggcag agaggaactg aacagcctgg caggacatgg 780 agggagggga aagaccagag agtcggggag gacccgggaa ggagcggcga cccggccatg 840 gcgagtctca ctcagcatcc ttccatcccc agtgtgccac ctacaagctg tgccaccccg 900 aggagctggt gctgctcgga cactctctgg gcatcccctg ggctcccctg agcagctgcc 960 ccagccaggc cctgcagctg gtgagtgtca ggaaaggata aggctaatga ggagggggaa 1020 ggagaggagg aacacccatg ggctccccca tgtctccagg ttccaagctg ggggcctgac 1080 gtatctcagg cagcaccccc taactcttcc gctctgtctc acaggcaggc tgcttgagcc 1140 aactccatag cggccttttc ctctaccagg ggctcctgca ggccctggaa gggatctccc 1200 ccgagttggg tcccaccttg gacacactgc agctggacgt cgccgacttt gccaccacca 1260 tctggcagca ggtgagcctt gttgggcagg gtggccaagg tcgtgctggc attctgggca 1320 ccacagccag gcctgtgtat gggccctgtc catgctgtca cccccagcat ttcctcattt 1380 gtaataacgc ccactcagaa gggcccaacc actgatcaca gctttccccc acagatggaa 1440 gaactgggaa tggcccctgc cctgcagccc acccagggtg ccatgccggc cttcgcctct 1500 gctttccagc gccgggcagg aggggtcctg gttgcctccc atctgcagag cttcctggag 1560 gtgtcgtacc gcgttctacg ccaccttgcc cagccctgag ccaagccctc cccatcccat 1620 gtatttatct ctatttaata tttatgtcta tttaagcctc atatttaaag acagggaaga 1680 gcagaacgga gccccaggcc tctgtgtcct tccctgcatt tctgagtttc attctcctgc 1740 ctgtagcagt gagaaaaagc tcctgtcctc ccatcccctg gactgggagg tagataggta 1800 aataccaagt atttattact atgactgctc cccagccctg gctctgcaat gggcactggg 1860 atgagccgct gtgagcccct ggtcctgagg gtccccacct gggacccttg agagtatcag 1920 gtctcccacg tgggagacaa gaaatccctg tttaatattt aaacagcagt gttccccatc 1980 tgggtccttg cacccctcac tctggcctca gccgactgca cagcggcccc tgcatcccct 2040 tggctgtgag gcccctggac aagcagaggt ggccagagct gggaggcatg gccctggggt 2100 cccacgaatt tgctggggaa tctcgttttt cttcttaaga cttttgggac atggtttgac 2160 tcccgaacat caccgacgcg tctcctgttt ttctgggtgg cctcgggaca cctgccctgc 2220 ccccacgagg gtcaggactg tgactctttt tagggccagg caggtgcctg gacatttgcc 2280 ttgctggacg gggactgggg atgtgggagg gagcagacag gaggaatcat gtcaggcctg 2340 tgtgtgaaag gaagctccac tgtcaccctc cacctcttca ccccccactc accagtgtcc 2400 cctccactgt cacattgtaa ctgaacttca ggataataaa gtgtttgcct cca 2453 <210> 2 <211> 207 <212> PRT <213> Homo sapiens_G-CSF <400> 2 Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala Leu Gln 1 5 10 15 Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln Glu Ala Thr Pro 20 25 30 Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu 35 40 45 Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys 50 55 60 Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 65 70 75 80 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 85 90 95 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 100 105 110 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 115 120 125 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 130 135 140 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 145 150 155 160 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 165 170 175 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 180 185 190 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 195 200 205 <210> 3 <211> 3607 <212> DNA <213> Mus musculus_G-CSF <400> 3 acttgagaat cacaaatgaa gggcagaggg ggctccccaa gcacgtctgc cagaggatgg 60 agagacagaa gcagtcttga tctgagcacc cattcttccc cagggtctga gttccttgct 120 atcccctcca gggtttcacc agccttgttg gacaactcgg atccagatcc aacaccctgc 180 agcgattcag gcctgggtgt ggctggaaga gaggaagaga gtttgagggg gggacaagac 240 gtcaaggaag gaacagagat tccccgattt cacaaaaact ttcgcaaaca gcttttcccc 300 aaccccctgc attgtcctga gctatcatca aatttgcata aatcctggga agttattact 360 aagcctgagt tgccccagcc ccaggtaatt tcctcccggg gccttgatgg ctttatgtat 420 aaaggccccc tggagctggg ccctggcaga gcccagagct gcagcccaga tcacccagaa 480 tccatggctc aactttctgc ccagaggcgc atgaagctaa tgggtgagtg ccctggttac 540 ctgccctggg tgtggaatgg agagaggccg gataacaagt gcctcagacc cgtctgtttg 600 ctcctagccc tgcagctgct gctgtggcaa agtgcactat ggtcaggacg agaggccgtt 660 cccctggtca ctgtcagcgc tctgccacca tccctgcctc tgccccgaag cttcctgctt 720 aagtccctgg agcaagtgag gaagatccag gccagcggct cggtgctgct ggagcagttg 780 gtgagtgggg ttgccatagg tgtggaggga aggacttggg gggggtagcg ggggaggcag 840 gagcacaaat gagaagaggg cagactagga acacacagca gttgacagtg tttgcccagt 900 gtgcacaaga gcctagatgg caccctcaga atactgacag aaacattgag gaagggaaca 960 tggggggaga agggcacagg aaagagaatg agtagagaca gcagtgggac ctctggatgg 1020 gacagtcctt agggtcacta ggaaggtagg gagctgagaa gccaggagga cctgagctgg 1080 acaaagagcc tggcccaggt gagcaccctc agtatccttc catctacagt gtgccaccta 1140 caagctgtgt caccccgagg agctggtgtt gctgggccac tctctgggga tcccgaaggc 1200 ttccctgagt ggctgctcta gccaggccct gcagcaggtg agtattttcg gaaaaaggag 1260 gaccaggagg agaaggggga ggagggggag gaggagaaag agggagagga gggggagaag 1320 ggggaggagg agggggagga ggaagaagag ggggaggagg gggaggagaa ggaggggaag 1380 aaggaggagg agggggagaa ggaaggacta catcctacta tttgggctcc tgaagagagc 1440 aagcatccca agatgtactg cctaaccctg ctatcttccc cagacacagt gcctaagcca 1500 gctccacagt gggctctgcc tctaccaagg tctcctgcag gctctatcgg gtatttcccc 1560 tgccctggcc cccaccttgg acttgcttca gctggatgtt gccaactttg ccaccaccat 1620 ctggcagcag gtgagtacca tctggagggg gtcatggcag cacacctggg caccctggaa 1680 gctctctata ttttcagagg cctgtttttt gggaagacac ccctcgcccc ctaccccgct 1740 tcctccaggc tgagagacag acagactctg tccagagaca gactgggttg gagtcttggt 1800 cctaccgtta acgtttaatg agccccacag gccaacttaa ccaacttccc agcacttcct 1860 cacctcaagg aaggactgat ccctgacttc atctcctccc cacagatgga aaacctaggg 1920 gtggccccta ctgtgcagcc cacacagagc gccatgccag ccttcacttc tgccttccag 1980 cgccgggcag gaggtgtcct ggccatttcg tacctgcagg gcttcctgga gacggctcgc 2040 cttgctctgc accacttggc ctagacctga gcagaaagcc ctttccagat agtttattta 2100 tctctattta atatttatgc atatttaagc ctactattta aagacaaaga cgagaaaatg 2160 gagctctaag cttctagatc attctctcca cttccgagtt ttgttctcct gcttagagca 2220 gagagagaag gctcttgtgt cctcctgtgg aggccaggga aggagatggg taaataccaa 2280 gtattgattc ctgctgctgc tccaggcacc cagttctgtg gcagtacccc caaaaaatca 2340 gtgagccctg ccgtgctgag gcaccatctc aggggggccc aggcagcatc tggtctccct 2400 tccgggggac aagacatccc tgtttaatat ttaaacagca gtgttcccaa actgggttct 2460 tatatccctt gctctggtca accaggttgc agggtttcct gtcctcacag gaacgaagtc 2520 cctaaagaaa cagtggcagc caggtttagc cccggaattg actggattcc ttttttaggg 2580 ccctgctggc ctggaagttg gagtgggggg cagaggaggc aggaggaagc ctgggggggg 2640 ggttggcatg gagggaggcc ttcccatcca ccctcaccct ccaccccacc tgtcactata 2700 gccaagcttg cggataataa agtgtggtgt tccagtacgt gttttttgtc ttccttcttg 2760 gttggctaat gcctggctgg gggctggtga gtggcgtggc gtggtaggag aggacagata 2820 aagaaggcac tgggaggagg tgggatgctc tagcttgccc cgacctcctc tgggtgccat 2880 tctctactaa tcgataggga cactaaaaat gcccagtgtc gctgccgcca agccacagct 2940 tcttgggaaa gaaaaaaaac caaaactgat ggatgataaa tcaatggaga gaggagggtt 3000 gggggcaggc tgagcacgtg cacaggcctg gtctcttcat cacctatgaa cagccctctg 3060 caaagctgcc cacccggcag aatgacaggc agaatgacat catgcctttt acagtttaat 3120 gtgattactg gatcagaact caggtcctgg gttggctggc tttcccccca acaaactgtg 3180 tccccacaat ctcctgcttg gctcactcct cagctgaaga agcctagaag gcctgaaggt 3240 cagctgcagg ctcccccgtg gctcagagct gcccaagagg ctaacctgtc acttccgccc 3300 acaccactga ctcctcaatg cctcccacca cctcccttcc cacagcctca caggaccctg 3360 cccctggact cgcagagtat ggaagacaga aaaagtcctc accctcaata ccaaataatc 3420 tcacaggaac acgtggtgaa ctctggtcca gagggtggag tgggacttaa aagaccgaga 3480 catcctgccc tgtacagtac agtgccgtgc ggggcctttc accagaacat cctgctttca 3540 gtggtggtgc cgctgctgag tccttaccat tgcagtggga ctgtgaacaa cggtcccact 3600 ctttctc 3607 <210> 4 <211> 208 <212> PRT <213> Mus musculus_G-CSF <400> 4 Met Ala Gln Leu Ser Ala Gln Arg Arg Met Lys Leu Met Ala Leu Gln 1 5 10 15 Leu Leu Leu Trp Gln Ser Ala Leu Trp Ser Gly Arg Glu Ala Val Pro 20 25 30 Leu Val Thr Val Ser Ala Leu Pro Pro Ser Leu Pro Leu Pro Arg Ser 35 40 45 Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Ala Ser Gly 50 55 60 Ser Val Leu Leu Glu Gln Leu Cys Ala Thr Tyr Lys Leu Cys His Pro 65 70 75 80 Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Lys Ala Ser 85 90 95 Leu Ser Gly Cys Ser Ser Gln Ala Leu Gln Gln Thr Gln Cys Leu Ser 100 105 110 Gln Leu His Ser Gly Leu Cys Leu Tyr Gln Gly Leu Leu Gln Ala Leu 115 120 125 Ser Gly Ile Ser Pro Ala Leu Ala Pro Thr Leu Asp Leu Leu Gln Leu 130 135 140 Asp Val Ala Asn Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Asn Leu 145 150 155 160 Gly Val Ala Pro Thr Val Gln Pro Thr Gln Ser Ala Met Pro Ala Phe 165 170 175 Thr Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Ala Ile Ser Tyr 180 185 190 Leu Gln Gly Phe Leu Glu Thr Ala Arg Leu Ala Leu His His Leu Ala 195 200 205
Claims (18)
상기 호중구성 폐 염증질환은 호중구성 천식(neutrophilic asthma)인 것을 특징으로 하는, 마커 조성물.
As a marker composition for diagnosing neutrophilic pulmonary inflammatory disease, comprising a granulocyte colony-stimulating factor (G-CSF) gene or a protein encoded by the gene,
The neutrophilic pulmonary inflammatory disease is characterized in that neutrophilic asthma (neutrophilic asthma), marker composition.
상기 G-CSF는 IL-1β에 의해 발현이 유도되는 것을 특징으로 하는, 마커 조성물.
The method of claim 1,
The G-CSF is characterized in that the expression is induced by IL-1β, a marker composition.
상기 G-CSF 유전자는 서열번호 1의 염기서열로 이루어진 것을 특징으로 하는, 마커 조성물.
The method of claim 1,
The G-CSF gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1, marker composition.
상기 G-CSF 유전자가 코딩하는 단백질은 서열번호 2의 아미노산 서열로 이루어진 것을 특징으로 하는, 마커 조성물.
The method of claim 1,
The protein encoded by the G-CSF gene is characterized in that consisting of the amino acid sequence of SEQ ID NO: 2, marker composition.
상기 호중구성 폐 염증질환은 호중구성 천식(neutrophilic asthma)인 것을 특징으로 하는, 진단용 조성물.
As a composition for diagnosing neutrophilic pulmonary inflammatory disease, comprising an agent for measuring the level of the mRNA of the granulocyte colony-stimulating factor (G-CSF) gene or the protein encoded by the gene,
The neutrophilic pulmonary inflammatory disease is characterized in that neutrophilic asthma (neutrophilic asthma), diagnostic composition.
상기 mRNA 수준을 측정하는 제제는 유전자의 mRNA에 상보적으로 결합하는 센스 및 안티센스 프라이머, 또는 프로브인 것을 특징으로 하는, 진단용 조성물.
The method of claim 6,
The agent for measuring the mRNA level, characterized in that the sense and antisense primers, or probes complementarily binding to the mRNA of the gene, diagnostic composition.
상기 단백질 수준을 측정하는 제제는 유전자가 코딩하는 단백질에 특이적으로 결합하는 항체인 것을 특징으로 하는, 진단용 조성물.
The method of claim 6,
The agent for measuring the protein level, characterized in that the antibody that specifically binds to the protein encoded by the gene, diagnostic composition.
상기 호중구성 폐 염증질환은 호중구성 천식(neutrophilic asthma)인 것을 특징으로 하는, 진단용 키트.
As a kit for diagnosing neutrophilic pulmonary inflammatory disease comprising the composition of claim 6,
The neutrophilic pulmonary inflammatory disease is characterized in that neutrophilic asthma (neutrophilic asthma), diagnostic kit.
상기 호중구성 폐 염증질환은 호중구성 천식(neutrophilic asthma)인 것을 특징으로 하는, 정보제공방법.
For diagnosis of neutrophilic pulmonary inflammatory disease, comprising the step of measuring the level of mRNA of a granulocyte colony-stimulating factor (G-CSF) gene or a protein encoded by the gene in a biological sample derived from a subject As a method of providing information,
The neutrophilic pulmonary inflammatory disease, characterized in that neutrophilic asthma (neutrophilic asthma), information providing method.
상기 mRNA 수준은 중합효소연쇄반응(PCR), 역전사 중합효소연쇄반응(RT-PCR), 실시간 중합효소연쇄반응(Real-time PCR), RNase 보호 분석법(RNase protection assay; RPA), 마이크로어레이(microarray) 및 노던 블롯팅(northern blotting)으로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정되는 것을 특징으로 하는, 정보제공방법.
The method of claim 11,
The mRNA levels are polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RPA), microarray. ) And Northern blotting (northern blotting).
상기 단백질 발현수준은 웨스턴 블롯팅(western blotting), 방사선면역분석법(radioimmunoassay; RIA), 방사 면역 확산법(radioimmunodiffusion), 효소면역분석법(ELISA), 면역침강법(immunoprecipitation), 유세포분석법(flow cytometry), 면역형광염색법(immunofluorescence), 오우크테로니(ouchterlony), 보체 고정 분석법(complement fixation assay) 및 단백질 칩(protein chip)으로 이루어진 군으로부터 선택되는 1종 이상의 방법을 통해 측정되는 것을 특징으로 하는, 정보제공방법.
The method of claim 11,
The protein expression level is Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation, flow cytometry, Information characterized in that it is measured through at least one method selected from the group consisting of immunofluorescence, ouchterlony, complement fixation assay, and protein chip. How to provide.
(b) 상기 폐 세포에서 인터루킨-1β(IL-1β)의 발현 또는 활성을 측정하는 단계;
(c) 상기 폐 세포에서 과립구집락자극인자(granulocyte colony-stimulating factor; G-CSF)의 발현을 측정하는 단계; 및
(d) 상기 IL-1β의 발현 또는 활성이 억제되는 것에 의하여 G-CSF의 발현을 감소시키는 물질을 호중구성 폐 염증질환의 치료물질로 선정하는 단계를 포함하는, 호중구성 폐 염증질환 치료물질의 스크리닝 방법으로서,
상기 호중구성 폐 염증질환은 호중구성 천식(neutrophilic asthma)인 것을 특징으로 하는, 스크리닝 방법.(a) treating the lung cells with the candidate substance in vitro;
(b) measuring the expression or activity of interleukin-1β (IL-1β) in the lung cells;
(c) measuring the expression of granulocyte colony-stimulating factor (G-CSF) in the lung cells; And
(d) selecting a substance that reduces the expression of G-CSF by inhibiting the expression or activity of IL-1β as a therapeutic substance for neutrophilic pulmonary inflammatory disease. As a screening method,
The neutrophilic pulmonary inflammatory disease is characterized in that neutrophilic asthma (neutrophilic asthma), screening method.
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| PCT/KR2020/006425 WO2020231221A1 (en) | 2019-05-16 | 2020-05-15 | COMPOSITION FOR DIAGNOSIS, PREVENTION, OR TREATMENT OF NEUTROPHILIC PULMONARY INFLAMMATORY DISEASES BY USING G-CSF AND IL-1β |
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| Title |
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| Evelyn Tsantikos et al., ‘Granulocyte-CSF links destructive inflammation and comorbidities in obstructive lung disease’, J Clin Invest., 2018, Vol. 128, pp 2406-2418. 1부.* |
| Jesus Banuelos et al., ‘Granulocyte colony-stimulating factor blockade enables dexamethasone to inhibit lipopolysaccharide-induced murine lung neutrophils’, PLoS ONE, 2017, Vol. 12, pp 1-16. 1부.* |
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